Ulrich Karl Laemmli

A rapid and convenient method for peptide mapping of proteins has been developed. The technique, which is especially suitable for analysis of proteins that have been isolated from gels containg sodium dodecyl sulfate, involves partial enzymatic proteolysis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by polyacrylamide gel electrophoresis. The pattern of peptide fragments produced is characteristic of the protein substrate and the proteolytic enzyme and is highly reproducible. Several common proteases have been used including chymotrypsin, Staphylococcus aureus protease, and papain.

Pulse-chase experiments in wild-type and mutant phage-infected cells provide evidence that the following particles called prohead I, II and III are successive precursors to the mature heads. The prohead I particles contain predominantly the precursor protein P23 and possibly P22 (mol. wt 31,000) and IP III (mol. wt 24,000) and have an s value of about 400 S. Concomitantly with the cleavage of most of P23 (mol. wt 55,000) to P23∗ (mol. wt 45,000), they are rapidly converted into prohead II particles which sediment with about 350 S. The prohead II particles contain, in addition to P23∗, the major constituents of the viral shella—a core consisting of proteins P22 and IP III. In cell lysates, prohead I and prohead II particles contain no DNA in a DNase-resistant form and are not bound to the replicative DNA. We cannot, however, positively rule out the possibility that these particles may have contained some DNA while in the cells. The prohead II particles are in turn converted into particles which sediment with about 550 S after DNase treatment (prohead III). During this conversion about 50% of normal DNA complement becomes packaged in a DNase-resistant form, and roughly 50% of the core proteins P22 and IP III are cleaved. In lysates the prohead III particles are attached to the replicative DNA. The prohead III particle appears to be the immediate precursor of the full mature head (1100 S). Cleavage of protein P22 to small polypeptides and conversion of IP III IP III∗ are completed at this time. No precursor proteins are found in the full heads. Studies with various mutant phage showed that the prohead II to III conversion is blocked by mutations in genes 16 and 17 and that the conversion of the prohead III particles to the mature heads is blocked by mutations in gene 49. Cleavage of the head proteins, however, occurs normally in these mutant-infected cells. We conclude that the cleavage of the major component of the viral shell, P23, into P23∗ precedes the DNA packaging event, whereas cleavage of the core proteins P22 and IP III appears to be intimately linked to the DNA packaging event. Models relating the cleavage processes to DNA encapsulation are discussed.

Data are presented for sequence-specific chromatin-loop organization in histone-depleted nuclei from Drosophila melanogaster Kc cells. We find one loop for each of the tandemly repeated histone gene clusters. The attachment site is localized in the A + T rich H1-H3 spacer on a 657 bp fragment. In the cluster of the hsp70 heat-shock genes, in both control and heat-shocked cells, we find two attachment sites in close proximity upstream of regulatory elements. The transcribed sequences are not associated with the nuclear scaffold in control or in heat-shocked cells. A family of attachment sites related by hybridization to those of the hsp70 genes was discovered.

<h2>Abstract</h2> We have previously shown that histone-depleted metaphase chromosomes can be isolated by treating purified HeLa chromosomes with dextran sulfate and heparin (Adolph, Cheng and Laemmli, 1977a). The chromosomes form fast-sedimenting complexes which are held together by a few nonhistone proteins. In this paper, we have studied the histone-depleted chromosomes in the electron microscope. Our results show that: the histone-depleted chromosomes consist of a scaffold or core, which has the shape characteristic of a metaphase chromosome, surrounded by a halo of DNA; the halo consists of many loops of DNA, each anchored in the scaffold at its base; most of the DNA exists in loops at least 10–30 μm long (30–90 kilobases). We also show that the same results can be obtained when the histones are removed from the chromosomes with 2 M NaCl instead of dextran sulfate. Moreover, the histone-depleted chromosomes are extraordinarily stable in 2 M NaCI, providing further evidence that they are held together by nonhistone proteins. These results suggest a scaffolding model for metaphase chromosome structure in which a backbone of nonhistone proteins is responsible for the basic shape of metaphase chromosomes, and the scaffold organizes the DNA into loops along its length.

We have identified the products of four of the six genes involved in bacteriophage T4 tail fibre assembly by sodium dodecyl sulphate-acrylamide gel electrophoresis of tail fibre mutant lysates and particles purified from them. Two large polypeptides, a 150,000 molecular weight species which is the product of gene 34 (P34), and a 120,000 molecular weight species which is the product of gene 37 (P37), are the major structural components of the fibres. Two smaller polypeptides, the products of genes 38 and 57, act in the conversion of the large structural polypeptide chains into morphological and antigenic half fibres. P38, molecular weight 26,000, does not appear to be a structural protein of the phage. In its absence, P37 is synthesized but remains unassembled. P57 plays a pleiotropic role in phage assembly: in its absence, P37 and P34 are both synthesized, but neither is assembled into fibres, and P12, a 60,000 molecular weight protein of the baseplate, is not incorporated into baseplates. The state of these unassembled polypeptide chains from 38- and 57- lysates can be distinguished from their state in wild-type lysates by two criteria: (a) they are soluble in sodium dodecyl sulphate at room temperature, whereas normal fibres and phages require heating for solubilization, and (b) they are concentrated in the low-speed pellet fractions of the lysates, suggesting that they are either aggregated, or bound to the cell envelope. A gene 36 amber mutation depressed the synthesis of P37 and a gene 37 amber mutation depressed the synthesis of P38, suggesting that these three genes are cotranscribed. These findings allow the formulation in greater detail of the early stages of the fibre assembly pathway.

A method, called “protein blotting,” for the detection of DNA-binding proteins is described. Proteins are separated on an SDS-polyacrylamide gel. The gel ia sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replicais used to detect DNA-binding proteins, RKA-binding proteins or histonebinding proteins by incubation of the filter with [32P]DNA, [125I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed.

We find DNA fragments attached to the nuclear scaffold (SARs) both 5' and 3' of three Drosophila genes, defining looped domains ranging from 4.5 to 13 kb. For the two-promoter-containing gene Adh (alcohol dehydrogenase), we find two upstream and two downstream SARs. For Sgs-4, the 5' SAR covers 866 bp immediately upstream of the transcript, and in the case of fushi tarazu, the 5' SAR is found on a small fragment 4.8 kb upstream of the start of transcription. These four upstream scaffold-attached fragments comap with enhancer-like regulatory sequences. Sequence analysis of five upstream SARs reveals clusters of sequences closely related to the cleavage consensus of topoisomerase II, several copies of a specific 10 bp A-rich sequence (AATAAATCAAA), and another 10 bp T-rich stretch.

The anchor-away (AA) technique depletes the nucleus of Saccharomyces cerevisiae of a protein of interest (the target) by conditional tethering to an abundant cytoplasmic protein (the anchor) by appropriate gene tagging and rapamycin-dependent heterodimerization. Taking advantage of the massive flow of ribosomal proteins through the nucleus during maturation, a protein of the large subunit was chosen as the anchor. Addition of rapamycin, due to formation of the ternary complex, composed of the anchor, rapamycin, and the target, then results in the rapid depletion of the target from the nucleus. All 43 tested genes displayed on rapamycin plates the expected defective growth phenotype. In addition, when examined functionally, specific mutant phenotypes were obtained within minutes. These are genes involved in protein import, RNA export, transcription, sister chromatid cohesion, and gene silencing. The AA technique is a powerful tool for nuclear biology to dissect the function of individual or gene pairs in synthetic, lethal situations.

Electron micrographs of thin sections of metaphase chromosomes isolated from HeLa cells provide new insight into the higher-order arrangement of the nucleoprotein fiber. Micrographs obtained from chromosomes swollen by chelation of the divalent cation are particularly revealing. Under these conditions, chromosomes swell in width by a factor of about 4 and the basic, thick nucleoprotein fiber (200–300 Å) relaxes to the thin fiber (100 Å), which is probably a linear array of nucleosomes. Cross sections show a central area from which the fibers emerge in a radial fashion, often forming loops which are 3–4 μm long. Chromosomes fixed in the presence of 1 mM MgCl2 are more compact, having an average chromatid diameter of about 1 μm, and consist of the thick (200–300 Å) fiber. Radial loops of about 0.6 μm can be observed frequently in these chromosomes, although the loops are more difficult to visualize due to the compactness of the structure and the material contaminating the periphery. Chromosomes isolated with the help of hexylene glycol are extremely compact (diameter about 0.6 μm) but quite free of cytoplasmic material. They consist of a 500 Å fiber that forms rather regular projections at the periphery. These projections appear to be loops of the thick fiber (200–300 Å), possibly shortened by twisting into a short supercoil. The chromatin loops observed in the intact chromosomes are thought to be structurally related to the DNA loops observed previously in the histone-depleted chromosomes ( Paulson and Laemmli, 1977 Paulson J.R. Laemmli U.K. Cell. 1977; 12: 817-828 Abstract Full Text PDF PubMed Scopus (782) Google Scholar ). In this paper, we discuss a model in which the nucleoprotein fiber is folded into loops which are arranged in the chromatid in radial fashion, in such a way that their bases become the central axis of the chromatid.

SC1 is a prominent, 170,000 Mr, non-histone protein of HeLa metaphase chromosomes. This protein binds DNA and was previously identified as one of the major structural components of the residual scaffold structure obtained by differential protein extraction from isolated chromosomes. The metaphase scaffold maintains chromosomal DNA in an organized, looped conformation. We have prepared a polyclonal antibody against the SC1 protein. Immunolocalization studies by both fluorescence and electron microscopy allowed identification of the scaffold structure in gently expanded chromosomes. The micrographs show an immunopositive reaction going through the kinetochore along a central, axial region that extends the length of each chromatid. Some micrographs of histone-depleted chromosomes provide evidence of the substructural organization of the scaffold; the scaffold appears to consist of an assembly of foci, which in places form a zig-zag or coiled arrangement. We present several lines of evidence that establish the identity of SC1 as topoisomerase II. Considering the enzymic nature of this protein, it is remarkable that it represents 1% to 2% of the total mitotic chromosomal protein. About 60% to 80% of topoisomerase II partitions into the scaffold structure as prepared from isolated chromosomes, and we find approximately three copies per average 70,000-base loop. This supports the proposed structural role of the scaffold in the organization of the mitotic chromosome. The dual enzymic and apparent structural function of topoisomerase II (SC1) and its location at or near the base of chromatin loops allows speculation as to its involvement in the long-range control of chromatin structure.

The DNA in nuclei and chromosomes is highly organized on several different levels, from winding of the helix around histones to the clustering of hundreds of kilobase pairs into the banding patterns of metaphase chromosomes. Recent studies on the organization of DNA into loops have begun to shed light on the functional significance of chromosomal organization.

One level of DNA organization in metaphase chromosomes is brought about by a scaffolding structure that is stabilized by metalloprotein interactions. Fast-sedimenting, histone-depleted structures (4000-7000 S), derived from metaphase chromosomes by extraction of the histones, are dissociated by metal chelators or by thiol reagents. The chromosomal (scaffolding) proteins responsible for constraining the DNA in this fast-sedimenting form are solubilized under the same conditions. Chromosomes isolated in a metal-depleted form, which generate slow-sedimenting, histone-depleted structures, can be specifically and reversibly stabilized by Cu2+, but not by Mn2+, Co2+, Zn2+ or Hg2+. Metal-depleted chromosomes can also be stabilized by Ca2+ (at 37 degrees C), but this effect is less specific than that of Cu2+. The scaffolding protein pattern that is reproducibly generated following treatment with Cu2+ is composed primarily of two high molecular weight proteins--Sc1 and Sc2 (170,000 and 135,000 daltons). The identification of this simple protein pattern has depended upon the development of new chromosome isolation methods that are highly effective in eliminating cytoskeletal contamination.

The role of topoisomerase II (topo II) in chromosome condensation was studied in a mitotic extract derived from Xenopus eggs by specific immunodepletion. HeLa nuclei, which have a high complement of endogenous topo II, are converted to mitotic chromosomes in the topo II-depleted extract equally well as in the control. Chicken erythrocyte nuclei, however, which have a very low content of topo II, do not convert to condensed chromosomes in the depleted extract, although their condensation is normal upon addition of purified topo II. Dosage experiments support the possible notion of a structural involvement of topo II in chromosome condensation. In the topo II-depleted extract the erythrocyte nuclei progress to precondensation chromosomes, which lack the nuclear membrane-lamina complex and consist of a cluster of swollen chromatids.

The product of gene 31 (P31) of bacteriophage T4 is required for the formation of the phage capsid and its related structures. In the absence of active P31, product P23, the major component of the phage capsid, aggregates into “lumps” which sediment with the cell envelope. Temperature-shift experiments with ts-mutants in gene 31 demonstrate that the P23 aggregates can be dissolved by activated P31 and the dissolved P23 is normal, in that it can be used for incorporation into active phage. It is possible that P31 acts catalytically. Two different ts-mutants in gene 31 produce two different temperature-sensitive proteins. One is irreversibly inactivated if produced at the restrictive temperature; but when synthesized at the permissive temperature, it becomes heat stable and remains functional at the restrictive temperature. The other is reversibly affected by temperature, activated following shift to permissive temperature and inactivated if restrictive temperature is established.

Using the highly AT-specific fluorochrome daunomycin, a longitudinal optical signal called AT queue, thought to arise from a line-up of the highly AT-rich scaffold-associated regions (SARs) by the scaffolding, was identified in native chromosomes. Fluorescence banding is proposed to result from a differential folding path of the AT queue during its progression from telomere to telomere. The AT queue is tightly coiled or folded in a Q band, the resulting transverse striations across the chromatid, which also represent Giemsa subbands, generating a bright AT-rich signal over the Q region. The R bands, in contrast, contain a more central (unfolded) AT queue, yielding an AT-dull signal over the R regions. The AT queue is identified by immunofluorescence against topoisomerase II (topo II) and HMG-I/Y as the scaffold of native chromosomes; the fluorescence signal from both proteins is akin to a detailed Q-type banding pattern. Native chromosomes appear assembled according to the loop-scaffold model.

Chromatin boundary activities (BAs) were identified in Saccharomyces cerevisiae by genetic screening. Such BAs bound to sites flanking a reporter gene establish a nonsilenced domain within the silent mating-type locus HML. Interestingly, various proteins involved in nuclear-cytoplasmic traffic, such as exportins Cse1p, Mex67p, and Los1p, exhibit a robust BA. Genetic studies, immunolocalization, live imaging, and chromatin immunoprecipitation experiments show that these transport proteins block spreading of heterochromatin by physical tethering of the HML locus to the Nup2p receptor of the nuclear pore complex. Genetic deletion of NUP2 abolishes the BA of all transport proteins, while direct targeting of Nup2p to the bracketing DNA elements restores activity. The data demonstrate that physical tethering of genomic loci to the NPC can dramatically alter their epigenetic activity.

High-molecular-weight DNA is known to collapse into very compact particles in a salt solution containing polymers like poly(ethylene oxide) [(EO)n] or polyacrylate. The biological relevance of this phenomenon is suggested by our recent finding that high concentrations of the highly acidic internal peptides found in the mature T4 bacteriophage head, as well as poly(glutamic acid) and poly(aspartic acid), can collapse DNA in a similar manner. The structure of DNAs collapsed by various methods has been studied with electron microscope. We find (EO)n collapses T4 or T7 bacteriophage DNA into compact particles only slightly larger than the size of the T4 and T7 head, respectively. In contrast, polylysine collapses DNA into different types of structures. Double-stranded DNA collapsed with (EO)n is cut by the single-strand specific Neurospora crassa endonuclease (EC 3.1.4.21) into small fragments. Extensive digestion only occurs above the critical concentration of polymer required for DNA collapse, demonstrating the (EO)n-collapsed DNA contains enzyme-vulnerable regions (probably at each fold), which are preferentially attacked. The size of the DNA fragments produced by limit-digestion with the nuclease ranges between 200 and 400 base pairs when DNA is collapsed by (EO)n. Only fragments of DNA which are larger than 600 base pairs are cut by the endonuclease in (EO)n-containing solution.

Research Article20 December 1989free access Preferential, cooperative binding of DNA topoisomerase II to scaffold-associated regions. Y. Adachi Y. Adachi University of Geneva, Department of Biochemistry, Switzerland. Search for more papers by this author E. Käs E. Käs University of Geneva, Department of Biochemistry, Switzerland. Search for more papers by this author U.K. Laemmli U.K. Laemmli University of Geneva, Department of Biochemistry, Switzerland. Search for more papers by this author Y. Adachi Y. Adachi University of Geneva, Department of Biochemistry, Switzerland. Search for more papers by this author E. Käs E. Käs University of Geneva, Department of Biochemistry, Switzerland. Search for more papers by this author U.K. Laemmli U.K. Laemmli University of Geneva, Department of Biochemistry, Switzerland. Search for more papers by this author Author Information Y. Adachi1, E. Käs1 and U.K. Laemmli1 1University of Geneva, Department of Biochemistry, Switzerland. The EMBO Journal (1989)8:3997-4006https://doi.org/10.1002/j.1460-2075.1989.tb08582.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info DNA elements termed scaffold-associated regions (SARs) are AT-rich stretches of several hundred base pairs which are known to bind specifically to nuclear or metaphase scaffolds and are proposed to specify the base of chromatin loops. SARs contain sequences homologous to the DNA topoisomerase II cleavage consensus and this enzyme is known to be the major structural component of the mitotic chromosome scaffold. We find that purified topoisomerase II preferentially binds and aggregates SAR-containing DNA. This interaction is highly cooperative and, with increasing concentrations of topoisomerase II, the protein titrates quantitatively first SAR-containing DNA and then non-SAR DNA. About one topoisomerase II dimer is bound per 200 bp of DNA. SARs exhibit a Circe effect; they promote in cis topoisomerase II-mediated double-strand cleavage in SAR-containing DNA fragments. The AT-rich SARs contain several oligo(dA).oligo(dT) tracts which determine their protein-binding specificity. Distamycin, which is known to interact highly selectively with runs of A.T base pairs, abolishes the specific interaction of SARs with topoisomerase II, and the homopolymer oligo(dA).oligo(dT) is, above a critical length of 240 bp, a highly specific artificial SAR. These results support the notion of an involvement of SARs and topoisomerase II in chromosome structure. Previous ArticleNext Article Volume 8Issue 131 December 1989In this issue RelatedDetailsLoading ...

It has been proposed that scaffold-associated regions are DNA elements that form the bases of chromatin loops in eukaryotic cells. Recent evidence supports a role for these elements as cis-acting ‘handlers’ of both structural and functional chromatin domains.

Article1 March 1986free access The organisation of chromatin loops: characterization of a scaffold attachment site S.M. Gasser S.M. Gasser Departments of Molecular Biology and Biochemistry, University of Geneva, 30, Quai Ernest Ansermet, 1211 Geneva 4, Switzerland Search for more papers by this author U.K. Laemmli U.K. Laemmli Departments of Molecular Biology and Biochemistry, University of Geneva, 30, Quai Ernest Ansermet, 1211 Geneva 4, Switzerland Search for more papers by this author S.M. Gasser S.M. Gasser Departments of Molecular Biology and Biochemistry, University of Geneva, 30, Quai Ernest Ansermet, 1211 Geneva 4, Switzerland Search for more papers by this author U.K. Laemmli U.K. Laemmli Departments of Molecular Biology and Biochemistry, University of Geneva, 30, Quai Ernest Ansermet, 1211 Geneva 4, Switzerland Search for more papers by this author Author Information S.M. Gasser1 and U.K. Laemmli1 1Departments of Molecular Biology and Biochemistry, University of Geneva, 30, Quai Ernest Ansermet, 1211 Geneva 4, Switzerland The EMBO Journal (1986)5:511-518https://doi.org/10.1002/j.1460-2075.1986.tb04240.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Previous experiments have identified a 657-bp restriction fragment in the non-transcribed region of the Drosophila histone gene cluster that is specifically associated with the histone-depleted nuclear scaffold. The remaining fragments of the 5-kb histone repeat were shown to be readily released from the scaffold; hence it was proposed that the tandemly repeated cluster of histone genes forms a series of 5-kb loops restrained by a nuclear substructure at the sites of attachment. Here we show that the attachment fragment is tightly associated with protease-sensitive material, whereas the solubilized fragments are relatively protein-free. Exonuclease III digestion has been used to map the location of protein complexes on the attachment fragment. We have defined two regions of ∼200 bp whose borders provide kinetic barriers to exonuclease III degradation. They are separated by a nucleaseaccessible region of ∼100 bp. The protected regions are sufficient to mediate association of the fragment with the histonedepleted nuclei. Sequence analysis reveals an enrichment for sequences closely related to the topoisomerase II cleavage consensus in these two domains. Previous ArticleNext Article Volume 5Issue 31 March 1986In this issue RelatedDetailsLoading ...

We have developed procedures for depositing intact mitotic chromosomes and isolated residual scaffolds on electron microscope grids at controlled and reproducible levels of compaction. The chromosomes were isolated using a recently developed aqueous method. Our study has addressed two different aspects of chromosome structure. First, we present a method for improved visualization of radial chromatin loops in undisrupted mitotic chromosomes. Second, we have visualized a nonhistone protein residual scaffold isolated from nuclease-digested chromosomes under conditions of low salt protein extraction. These scaffolds, which have an extremely simple protein composition, are the size of chromosomes, are fibrous in nature, and are found to retain differentiated regions that appear to derive from the kinetochores and the chromatid axis. When our standard preparation conditions were used, the scaffold appearance was found to be very reproducible. If the ionic conditions were varied, however, the scaffold appearance underwent dramatic changes. In the presence of millimolar concentrations of Mg++ or high concentrations of NaCl, the fibrous scaffold protein network was observed to undergo a lateral aggregation or assembly into a coarse meshlike structure. The alteration of scaffold structure was apparently reversible. This observation is consistent with a model in which the scaffolding network plays a dynamic role in chromosome condensation at mitosis.

In this paper, we show that HeLa metaphase chromosomes still possess a highly organized structure retaining the familiar metaphase morphology following removal of virtually all the histones and most of the nonhistone proteins. The structure is stabilized by a relatively small number of nonhistones, which we call scaffolding proteins. These results are based on a method which allows the removal of the histones, and most of the nonhistone proteins, by competition with polyanions such as dextran sulfate and heparin. The histone-depleted chromosomes sediment in sucrose gradients as a broad peak between 4000 to 7000S. These structures are dissociated by mild trypsin or chymotrypsin treatment, or by 4 M urea, but are stable in 2 M NaCl and insensitive to treatment with RNAase A. The histone-depleted chromosomes have a DNA to protein ratio of about 6:1; gel electrophoresis reveals the presence of about 30 nonhistone proteins and the virtual absence of histones. These experiments suggest that nonhistone proteins exist in metaphase chromosomes which maintain the DNA chain in a highly folded conformation. Structural studies support this conclusion. Analysis by fluorescence microscopy of histone-depleted chromosomes stained with ethidium bromide shows that each chromatid is still paired with its sister chromatid, and consists of a central structure surrounded by a halo of DNA. The length of the central structure in each chromatid is about 2–3 times longer than the chromatid length in the original chromosome.

Three somewhat different types of particle accumulate in cells infected with a phage carrying a mutation in gene 21 (in addition to the tubular variant (polyhead) of the head). The major type is the so-called τ-particle. These particles are very fragile, associated with the cell membrane, and have a sedimentation coefficient of about 420 S. They possess no DNA if isolated, and contain predominantly the precursor proteins P23, P24, P22 and the internal protein IP III, in addition to protein P20 and several proteins of unknown genetic origin. The remainder of the particles are partially or completely filled with DNA. The ratio of τ-particles to these partially or completely filled particles depends upon the particular mutant (in gene 21) phage used. In cells infected with a phage carrying the amber mutation (N90) in gene 21, about 10% of the precursor head protein P23 is cleaved to P23∗, and correspondingly about 10% of the particles are partially or completely filled with DNA. In cells infected with the temperature-sensitive mutant (N8) in gene 21, about 1% of the particles are fully or partially filled, and correspondingly about 1% of the P23 is cleaved to P23∗. In either case, the DNA-associated particles contain predominantly the cleaved proteins P23∗ and IP III∗, and have none of the P22 and IP III found in τ-particles. This observation, and the correlation of the amount of partially or completely filled particles with the extent of the cleavage of P23 in the lysates, strongly suggest that cleavage of the head proteins is required for DNA packaging to occur. The τ-particles have properties similar to the so-called prohead I particles which we have isolated as intermediates in wild-type head assembly (preceding paper). However, temperature shift-down experiments, using several different phage carrying temperature-sensitive mutations in gene 21, indicate that the bulk of the τ-particles cannot be used for normal phage production.

The physiological manifestation of conditional lethal mutations in the genes involved in the formation of the T4 head have been re-examined. If the product of genes 20 or 40 is defective, open-ended tubular structures (polyheads) are synthesized. Infection with mutants in gene 22 leads to the formation of polyheads, about 70% of which consist of two or more concentric layers (multilayered polyheads). Cells infected with mutants in gene 21 and 24 yield a mixed burst of capsid-like particles (τ-particles) and polyheads, many of which have hemispherical caps. High resolution micrographs of τ-particles reveal a substructure similar to that seen on micrographs of polyheads, in contrast to normal capsids on whose surface no detail can be visualized. Studies of the kinetics of formation of the head-related structures show that the absence or malfunctioning of a particular gene product results in an efficient use of the late phage proteins for the formation of either polyheads or τ-partieles. Furthermore, kinetic studies of multilayered polyhead formation suggest that single-layered polyheads can serve as a “core” onto which subsequent layers are added. The minimum number of head genes whose active product is needed to form the various head-related structures has been determined by examining infection by a series of double mutants. Multilayered polyheads produced when the product of gene 22 is defective need for their assembly the active product of only two of the seven head genes studied, namely, genes 23 and 31. Single-layered polyheads, however, which are produced in cells infected either with mutants in gene 20 or 40 need, in addition to genes 23 and 31, the product of gene 22. The so-called τ-particles appear if the genes 21 and 24 are mutated either singly or together and require for their formation the functional product of all the other head genes. These results suggest that some of the gene products have a shape-specifying role. Gene 22 is associated with the initiation process in capsid formation and genes 20 and 40 with the formation of the hemispherical cap. Genes 21 and 24 appear to determine the size and surface structure of the capsids thus rendering them susceptible to DNA integration. It is also possible that their primary function is to act as a condensing factor for phage DNA.

We have purified two proteins from Drosophila that bind to the scs' boundary element of the hsp70 domain at locus 87A7. Their palindromic binding sites (CGATA-TATCG) symmetrically abut the previously mapped hypersensitive site of scs'. We have cloned a cDNA for one of these proteins, BEAF-32 (boundary element-associated factor of 32 kDa). It encodes a novel protein that is bound to scs' but not scs in vivo. Immunostaining localizes BEAF to hundreds of interbands and many puff boundaries on polytene chromosomes, suggesting that a chromosomal domain consists of a band (or puff) and part of the flanking interbands. Enhancer blocking assays implicate the palindromic binding site in boundary function. The lack of enhancer blocking in transiently transfected cells suggests an involvement of chromatin, nuclear structure, or both in boundary function.

An experimental assay was developed to search for proteins capable of antagonizing histone H1-mediated general repression of transcription. T7 RNA polymerase templates containing an upstream scaffold-associated region (SAR) were highly selectively repressed by H1 relative to non-SAR control templates. This is due to the nucleation of H1 assembly into flanking DNA brought about by the numerous A-tracts (AT-rich sequences containing short homopolymeric runs of dA.dT base pairs) of the SAR. Partial, selective titration of these A-tracts by the high mobility group (HMG) protein HMG-I/Y led to the complete derepression of transcription from the SAR template by inducing the redistribution of H1 on to non-SAR templates. SARs are associated with many highly transcribed regulated genes where they may serve to facilitate the HMG-I/Y-mediated displacement of histone H1 in chromatin. Indeed, HMG-I/Y was found to be strongly enriched in the H1-depleted subfraction which can be isolated from chromatin.

Topoisomerase IIalpha (topoIIalpha) and 13S condensin are both required for mitotic chromosome assembly. Here we show that they constitute the two main components of the chromosomal scaffold on histone-depleted chromosomes. The structural stability and chromosomal shape of the scaffolding toward harsh extraction procedures are shown to be mediated by ATP or its nonhydrolyzable analogs, but not ADP. TopoIIalpha and 13S condensin components immunolocalize to a radially restricted, longitudinal scaffolding in native-like chromosomes. Double staining for topoIIalpha and condensin generates a barber pole appearance of the scaffolding, where topoIIalpha- and condensin-enriched "beads" alternate; this structure appears to be generated by two juxtaposed, or coiled, chains. Cell cycle studies establish that 13S condensin appears not to be involved in the assembly of prophase chromatids; they lack this complex but contain a topoIIalpha-defined (-mediated?) scaffolding. Condensin associates only during the pro- to metaphase transition. This two-step assembly process is proposed to generate the barber pole appearance of the native-like scaffolding.

Our previous work identified the inner basket of the NPC as a physical activation/protection station for force-tethered, epigenetically silenced genes. Here we show that a specific nucleopore-to-gene-promoter interaction (Nup-PI) is an early physiological event of gene activation. Nup-PI was discovered with chromatin endogenous cleavage (ChEC) experiments that mapped in vivo the genomic interaction sites of the nucleoporin Nup2p fused to microccocal nuclease (Nup2-MN). Strong Nup-PI, cleavage by Nup2-MN, is observed at the promoters of the GAL genes and at HXK1 upon activation of these genes with galactose. Nup-PI at the GAL locus requires Gal4p and the UASg and TATA box elements but not SAGA and active transcription. The physical, activation-dependent interaction of the GAL locus with the NPC basket was confirmed by imaging. Chromosome-wide ChEC studies indicated that Nup-PI occurs at numerous genes. The data identify the NPC basket as a new, integral participant in gene expression.

We have studied the three-dimensional folding of the scaffolding in histone H1-depleted chromosomes by immunofluorescence with an antibody specific for topoisomerase II. Two different types of decondensed chromosomes are observed. The majority of the chromosomes are expanded, and the central fluorescence signal is surrounded by a large halo of chromatin. A much smaller number of chromosomes are more compact in length; they contain a smaller halo of chromatin and their scaffolds are not extended but folded into a genuine, quite regular helical coil. This conclusion is based on a three-dimensional structural analysis by optical sectioning. The number of helical coils is related to chromosome length. Surprisingly, sister chromatids have predominantly opposite helical handedness; that is, they are related by mirror symmetry.

Two forms of histone-depleted HeLa nuclei have been characterized (Lebkowski & Laemmli, 1982). In the studies presented here, the proteins associated with both structures are identified. Type I structures, which are obtained by extraction of isolated nuclei with a buffer containing 2 m-NaCl or dextran sulfate/heparin, retain 10 to 15% of the total nuclear proteins. These proteins are represented by the three nuclear lamina proteins of 60,000 to 70,000 Mr and numerous high molecular weight species. Histone-extraction in the presence of β-mercaptoethanol leads to more expanded type II structures. Accompanying this further DNA unfolding, is a selective loss of certain proteins that are associated with type I structures. Present in type II structures are 3 to 5% of the total nuclear proteins, almost exclusively represented by the three nuclear lamina proteins and two minor proteins of 64,000 and 200,000 Mr. The proteins of both type I and type II histone-depleted nuclei are comparable to those that remain as residual nuclear structures after histone-depletion of nuclease-digested nuclei. These residual protein structures are termed type I and type II nuclear scaffolds. The selective removal of proteins by β-mercaptoethanol is pH-dependent and maximal at alkaline pH values. It is further demonstrated that type I structures are stabilized by CaCl2. Nuclei incubated with CaCl2 are resistant to extraction with β-mercaptoethanol. In addition, evidence is presented that confirms that β-mercaptoethanol acts to disrupt metalloprotein interactions important for the stabilization of type I structures. The metal chelator, 1,10-phenanthroline, causes solubilization of the same set of proteins as β-mercaptoethanol. DNA binding studies are presented which show that 12 proteins of type I and four proteins of type II scaffolds bind DNA in vitro. The three nuclear lamina proteins are the major DNA binding proteins of both type I and type II scaffolds.

How is the enormous length of DNA in a mitotic chromosome packaged into such a compact structure? Recently it has become clear that at least one level of packaging, the coiling of the DNA into the basic nucleohistone fiber, is accomplished by the histones. The nucleohistone fiber of interphase and mitotic chromosomes has been studied extensively by electron microscopy. Thin-section techniques and surface spreading (see, e.g., DuPraw 1970; Ris 1975) reveal a knobby, “thick” fiber of about 200–300-Å diameter. This thick fiber appears to unfold at low ionic strength, or by removal of proteins, to a beads-on-a-string conformation (Woodcock 1973; Olins and Olins 1974) in which the “beads” are nucleosomes containing two each of the four histones (as an octamer) and 140–200 base pairs of DNA coiled around a histone core (Kornberg 1974; Oudet et al. 1975; and papers in this volume). To account for the thick chromatin fiber, Finch...

The human immunodeficiency virus type 1 (HIV) Rev protein is thought to be involved in the export of unspliced or singly spliced viral mRNAs from the nucleus to the cytoplasm. This function is mediated by a sequence-specific interaction with a cis-acting RNA element, the Rev response element (RRE), present in these intron-containing RNAs. To identify possible host proteins involved in Rev function, we fractionated nuclear cell extracts with a Rev affinity column. A single, tightly associated Rev-binding protein was identified; this protein is the mammalian nucleolar protein B23. The interaction between HIV Rev and B23 is very specific, as it was observed in complex cell extracts. The complex is also very stable toward dissociation by high salt concentrations. Despite the stability of the Rev-B23 protein complex, the addition of RRE, but not control RNA, led to the displacement of B23 and the formation of a specific Rev-RRE complex. The mammalian nucleolar protein B23 or its amphibian counterpart No38 is believed to function as a shuttle receptor for the nuclear import of ribosomal proteins. B23 may also serve as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus to allow further rounds of export of RRE-containing viral RNAs.

We have identified two classes of in vivo topoisomerase II cleavage sites in the Drosophila histone gene repeat. One class co-localizes with DNase I-hypersensitive regions and another novel class maps to a subset of consecutive nucleosome linker sites in the scaffold-associated region (SAR) of the histone gene loop. Prominent topoisomerase II cleavage is also observed in one of the linker regions of the two nucleosomes spanning satellite III, a centromeric SAR-like DNA sequence with a repeat length of 359 bp. At the sequence level, in vivo topoisomerase II cleavage is highly site specific. Comparison of 10 nucleosome linker sites defines an in vivo cleavage sequence whose major characteristic is a prominent GC-rich core. These GC-rich cleavage sites are flanked by extensive arrays of oligo(dA).oligo(dT) tracts characteristic of SAR sequences. Treatment of cells with distamycin selectively enhances cleavage at nucleosome linker sites of the SAR and satellite regions, suggesting that AT-rich sequences flanking cleavage sites may be involved in determining topoisomerase II activity in the cell. These observations provide evidence for the association of topoisomerase II with SARS in vivo.

The long-range order of the DNA in interphase nuclei was investigated by sedimentation studies. From these studies, two discreet sedimentation forms have been characterized for histone-depleted HeLa nuclei. Type I structures, obtained by extraction of isolated nuclei with a buffer containing 2 m-NaCl, have an s value of 18,000 S. Histone-depletion with the polyanions dextran sulfate/heparin produces slightly slower type I structures of 15,000 S. When the thiols, β-mercaptoethanol or dithiothreitol, or the metal chelators, 1,10-phenanthroline or neocuproine, are included during histone extraction, the interphase DNA is further unfolded, generating type II structures of 8500 S. Both forms are destroyed by proteolytic agents, but are insensitive to RNAase A. Fluorescence studies of type I and type II structures show that both retain the spherical shape of nuclei, with type II structures having a more expanded DNA halo than that of type I. It is suggested that metalloprotein interactions are important in one level of the DNA folding of type I structures, since the chelators 1,10-phenanthroline and neocuproine bring about formation of type II structures. Metal-depleted nuclei, prepared in buffers containing β-mercaptoethanol, provide a means for identifying the metal involved. Such nuclei generate type II structures upon histone-depletion. Addition of as little as 1 × 10−6 m-CuSO4 or 1 × 10−6 m-CaCl2 to metal-depleted nuclei restores the capacity to generate type I structures. The restoration of the type I complex is reversible following exposure of nuclei to CuSO4, but is irreversible after CaCl2 treatment. These experiments suggest that one level of DNA compaction in histone-depleted nuclei is stabilized by either Cu or Ca.

We have recently shown that, after the histones and most of the nonhistone proteins are gently removed from HeLa metaphase chromosomes, the chromosomal DNA is still highly organized and relatively compact. The structure of these histone-depleted chromosomes is due to the presence of a number of nonhistone proteins that form a central scaffold that retains the approximate size and shape of intact chromosomes and to which the DNA is attached, predominantly forming loops. We now demonstrate that the protein scaffold may be isolated independently of the DNA by treating HeLa chromosomes with micrococcal nuclease before removing the histones.The chromosomal scaffolds may be isolated by sucrose density gradient centrifugation as a well-defined peak that is stable in 2 M sodium chloride, but is dissociated by treatment with proteases, 4 M urea, or 0.1% sodium dodecyl sulfate. Polyacrylamide gel electrophoresis reveals that the protein content of scaffold preparations is identical to that of histone-depleted chromosomes. Fluorescence microscopy of purified scaffolds in isolation buffer shows that the particles still possess the familiar chromosome morphology. When the scaffolds are examined in the electron microscope, a fibrous structure with the approximate size and shape of intact, paired chromatids is seen. Less than 0.1% of the chromosomal DNA and virtually no histones are associated with the purified scaffold structures.

Scaffold-associated regions (SARs) are A + T-rich DNA regions of several hundred base-pairs that are known to bind specifically to nuclear or metaphase scaffolds. Surprisingly, histone H1 specifically associates with SARs. Under conditions of high co-operativity, at input ratios of H1 to DNA up to 15% (w/w), histone H1 binds preferentially to those DNA molecules harboring a SAR, leaving the non-SAR fragments free. Our experiments identify SARs as cis-acting sequences that nucleate co-operative H1 assembly along the SAR into the flanking non-SAR DNA. Experiments with simple DNA polymers implicate homopolymeric oligo(dA).oligo(dT) tracts in preferential histone H1 assembly. The homopolymer oligo(dA).oligo(dT) is, above a critical length of 130 base-pairs, a highly specific nucleator of H1 assembly. SARs may control the conformation of chromatin domains via a regulated H1 assembly and set up the potential transcriptional repertoire of the cell.

We have previously identified a number of specific DNA fragments called SARs (scaffold-associated regions) that are associated with the nuclear scaffold and define the basis of DNA loops. We demonstrate that cloned DNA fragments containing SAR sequences bind to nuclear scaffolds in vitro with the same specificity as have genomic SAR fragments. This specific interaction is observed with the biochemically complex type I scaffolds. These scaffolds are composed of the nuclear lamina proteins and a set of other proteins that forms the internal network of these structures. So-called type II scaffolds, which are composed primarily of the lamina proteins and lack the proteins of the internal network, do not bind the SAR fragments at a detectable level. Competition experiments show that different SARs share common structural elements and can bind to the same sites on the nuclear scaffold, although with different affinities. Moreover, the SAR binding sites appear to be evolutionarily conserved, as all the Drosophila SARs also bind with identical specificity to nuclear scaffolds derived from rat liver nuclei. These Sar interaction studies were carried out with lithium 3,5-diiodosalicylate-extracted nuclei. Interestingly, scaffolds prepared by high-salt extraction also bind the genomic and exogenously added SAR fragments specifically. However, the endogenous transcribed sequences, as opposed to the same fragments added as purified DNA, associate randomly with these scaffolds.

Scaffold-associated regions (SARs) are A + T-rich sequences defined by their specific interaction with the nuclear scaffold. These sequences also direct highly specific binding to purified histone H1, and are characterized by the presence of oligo(dA).oligo(dT) tracts, which are a target for the drug distamyin, an antibiotic with a wide range of biological activities. The interaction of distamycin with SAR sequences results in the complete suppression of binding to either scaffolds or histone H1, suggesting that (dA.dT)n tracts play a direct role in mediating these specific interactions and that histone H1 and nuclear scaffold proteins may recognize a characteristic minor groove width or conformation. The effect of distamycin on these specific DNA-protein interactions in vitro suggests that binding of SARs to the nuclear scaffold and SAR-dependent nucleation of H1 assembly might be important targets of the drug in vivo.

We have examined the higher-order loop organization of DNA in interphase nuclei and metaphase chromosomes from Drosophila Kc cells, and we detect no changes in the distribution of scaffold-attached regions (SARs) between these two phases of the cell cycle. The SARs, previously defined from experiments with interphase nuclei, not only are bound to the metaphase scaffold when endogenous DNA is probed but also rebind specifically to metaphase scaffolds when added exogenously as cloned, end-labeled fragments. Since metaphase scaffolds have a simpler protein pattern than interphase nuclear scaffolds, and both have a similar binding capacity, it appears that the population of proteins required for the specific scaffold-DNA interaction is limited to those found in metaphase scaffolds. Surprisingly, metaphase scaffolds isolated from Drosophila Kc cells contain both the lamin protein and a pore-complex protein, glycoprotein (gp) 188. To study whether lamin contributes to the SAR-scaffold interaction, we have carried out comparative binding studies with scaffolds from HeLa metaphase chromosomes, which are free of lamina, and from HeLa interphase nuclei. All Drosophila SAR fragments tested bind with excellent specificity to HeLa interphase scaffolds, whereas a subset of them bind to HeLa metaphase scaffolds. The maintenance of the scaffold-DNA interaction in metaphase indicates that lamin proteins are not involved in the attachment site for at least a subset of Drosophila SARs. This evolutionary and cell-cycle conservation of scaffold binding sites is consistent with a fundamental role for these fragments in the organization of the genome into looped domains.

SARs are candidate DNA elements for defining the bases of chromatin loops and possibly for serving as cis elements of chromosome dynamics. SARs contain numerous A tracts, whose altered DNA structure is recognized by cooperatively interacting proteins such as topoisomerase II. We constructed multi-AT hook (MATH) proteins and demonstrate that they specifically bind the clustered A tracts of SARs in chromatin and chromosomes. They are also potent inhibitors of chromosome assembly in mitotic Xenopus extracts, demonstrating the importance of SARs in this process. Titration of SARs with MATH20 (20 hooks) blocks shape determination of chromatids but not chromatin condensation per se. SARs are also required for shape maintenance of chromosomes. If MATH20 is added after formation of chromatids, they collapse and are reshaped by an active, mitotic process into spherical chromatid balls.

<h2>Abstract</h2> To map the genomic interaction sites of chromatin proteins, two related methods were developed and experimentally explored in <i>Saccharomyces cerevisiae</i>. The ChIC method (chromatin immunocleavage) consists of tethering a fusion protein (pA-MN) consisting of micrococcal nuclease (MN) and staphylococcal protein A to specifically bound antibodies. The nuclease is kept inactive during the tethering process (no Ca²⁺). The ChEC method (chromatin endogenous cleavage) consists of expressing fusion proteins in vivo, where MN is C-terminally fused to the proteins of interest. The specifically tethered nucleases are activated with Ca²⁺ ions to locally introduce double-stranded DNA breaks. We demonstrate that ChIC and ChEC map proteins with a 100–200 bp resolution and excellent specificity. One version of the method is applicable to formaldehyde-fixed nuclei, another to native cells with comparable results. Among various model experiments, these methods were used to address the conformation of yeast telomeres.

We demonstrate by immunofluorescence that a 70-kD protein (P70) purified from Xenopus egg extracts is associated with subnuclear foci (about 200) which we propose to be an assembly of DNA pre-replication centers (preRCs). A cDNA encoding this protein reveals that P70 is the Xenopus homologue of replication protein A (RPA also called RF-A). RPA is know to be a cellular, three-subunit single-stranded DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. The punctated preRCs exist transiently; they form post-mitotically during the period of nuclear membrane breakdown and disappear during ongoing DNA replication. P70 is homogeneously associated with chromatin at the later stages of the S-phase and is displaced from chromatin post replication, so that P70 cannot be detected on mitotic chromosomes. Double-immunofluorescence studies using biotin-dUTP demonstrate that initiation of DNA synthesis is confined to preRCs, resulting in the punctated replication pattern observed previously by others. PreRCs form efficiently on decondensed chromatin in membrane-free egg extracts if ATP and divalent cations are present. Our results suggest that preRCs are composed of an assembly of a large number of pre-initiation replication complexes poised for initiation at discreet subnuclear regions prior to nuclear reconstruction and initiation of DNA synthesis.

RPA is a cellular, three-subunit, single-stranded (ss) DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. By immunodepletion and complementation, we have identified RPA as an essential factor for cellular DNA replication in Xenopus extracts. RPA assembles post-mitotically on the decondensing chromosomes into numerous subnuclear pre-replication centres (preRCs) which serve, upon formation of the nuclear membrane, as RCs for the initiation of DNA synthesis. By a variety of experiments including the use of isolated components, we demonstrate that an inactive cdc2-cyclin B kinase complex is essential to allow post-mitotic assembly of the preRCs. In contrast, the active cdk2-cyclin A kinase does not impede or facilitate the assembly of preRCs. Digestion analysis using the single-strand-specific P1 nuclease as well as competition experiments with ssDNA, reveal that replication-associated unwinding of the DNA, assisted by RPA, requires the formation of the nuclear membrane. The p21 cdk-interacting protein Cip1 appears to inhibit DNA replication prior to the unwinding DNA step, but after assembly of preRC and nuclear reconstruction.

Histone H1 preferentially and cooperatively binds scaffold-associated regions (SARs) in vitro via specific interactions with the numerous short A + T-rich tracts (A-tracts) contained in these sequences. Selective titration of A-tracts by the oligopeptide distamycin abolishes this interaction and results in a redistribution of H1. Similarly, treatment of intact cells and isolated nuclei with distamycin specifically enhances cleavage of internucleosomal linkers of SARs by topoisomerase II and restriction enzymes. The increased accessibility of these linkers is thought to result from the unfolding (or opening) of the chromatin fiber and to be due to a reduced occupancy by histone H1. Chromatin extraction and H1 assembly experiments support this view. We discuss a model whereby open, H1-depleted chromatin regions may be generated by titration of A-tracts by putative distamycin analogues; this local opening may spread to adjacent regions assuming highly cooperative H1-H1 interactions in chromatin.

We have studied the maturation of T4 polyheads, the aberrant tubular structures related to the capsid of T4 bacteriophage. Conditions have been found under which more than 95% of the major head protein (P23) undergoes the same cleavage that occurs during development of the normal capsid. The concomitant structural changes in the polyheads have been followed using electron microscope image filtering techniques. As a result of the cleavage, a radical transformation of the hexagonal lattice occurs, involving a 10-15% expansion in the lattice dimensions. However, a metastable intermediate state similar to the uncleaved structure has been observed immediately after cleavage of the protein subunits. Some kind of additional physical stimulus seems to be required to trigger the major structural change, which appears to be highly cooperative.

We have identified the structural proteins of phage T4 precursor tails. Complete tails, labeled with 14C-labeled amino acids, were isolated from cells infected with mutants blocked in head assembly. The proteins were characterized by sodium dodecyl sulfate-acrylamide gel electrophoresis and subsequent autoradiography. The complete tails are made up of at least fifteen different species of phage proteins. To identify the genes specifying these proteins we prepared 14C-labeled amino acid lysates made with amber mutants defective in each of the twenty-one genes involved in tail assembly. Comparison of the gel pattern of the amber mutant lysates with wild type lysates enabled us to identify the following gene products, with molecular weights in parentheses: P6 (85,000); P7 (140,000); P8 (46,000); P9 (34,000); P10 (88,000); P11 (26,000); P12 (55,000); P15 (35,000); P18 (80,000); P19 (21,000); P29 (77,000). These eleven species are all structural proteins of the tail. The genetically unidentified tail proteins have molecular weights of 42,000, 41,000, 40,000 and 35,000. They are likely to be the products of known phage genes which were not resolved in the crowded middle region of the whole lysate gel patterns. The major tail proteins are all synthesized during the late part of the phage growth cycle. The mobilities of the proteins derived from tails did not differ from the mobilities of the proteins when derived from the unassembled pools of subunits accumulating in mutant infected cells, or when derived from complete phage particles. The genes for at least seven of the structural proteins are contiguous on the genetic map. Genes for proteins needed in many copies seem to be clustered separ- ately from genes whose products are needed in only a few copies. Consideration of protein sizes and published mapping data on phage T4 also suggest that the phage structural proteins are, on the average, much larger than the non-structural proteins. The requirement that at least fifteen different species of proteins must come together in forming a phage tail emphasizes the complexity of this morphogenetic process.

The protein compositions of purified metaphase chromosomes, nuclei and their residual scaffold and matrix structures, are reported. The protein pattern of nuclei on sodium dodecyl sulphate/polyacrylamide gels is considerably more complex and rich in non-histone proteins than that of chromosomes. Nuclei contain about three to four times more non-histone proteins relative to their histones than chromosomes. Besides the protein components of the peripheral lamina, several protein bands are specific or at least highly enriched in nuclei. Conversely, two proteins X0 (33 X 10(3) Mr) and X1 (37 X 10(3) Mr) are highly enriched in the pattern of metaphase chromosomes. We have compared morphologically the previously defined nuclear matrices type I and II. The type I nuclear matrix is composed of the known lamina proteins, which form the peripheral lamina structure, and a complex series of proteins that form the internal network of the matrix as observed by electron microscopy. This internal network is stabilized similarly to the metaphase scaffolding by metalloprotein interaction. Both the scaffolding and the internal network of the matrix dissociate if thiols or certain metal chelators are used in the extraction buffer. Under these conditions the resulting nuclear structure, called matrix type II, appears empty in the electron microscope, with the exception of some residual nucleolar material. This latter material can be extracted from the internal network by exhaustive treatment of the nuclei with RNase before extraction with high salt. Immunoblotting and activity studies show RNA polymerase II to be tightly bound to the type I, but not to the type II matrix, or to the scaffolding structure. No polymerase II enzyme was detected in isolated metaphase chromosomes. Another nuclear enzyme, poly(ADP-ribose) polymerase is not bound to either of the residual nuclear matrices or to the scaffolding structures. The association of RNA polymerase with the internal network of the nuclear matrix is consistent with the idea that transcription occurs in close association with this structure.

Abstract The most recent developments in studies on the maturation of the head of bacteriophage T4 are described and discussed. The major features of the maturation steps of the head are the following: (a) The viral DNA is pulled into an empty head in a series of events. (b) Cleavage of two core proteins, P22 (MW = 31,000), to small fragments and the internal protein IPIII (MW = 23,000) to IPIII* (MW = 21,000) appears to be intimately linked to the DNA packaging event, whereas the cleavage of the major head protein of the viral coat, P23 (MW = 55,000), to P23* (MW = 45,000) precedes the DNA packaging event. The P22 core proteins appear to be the precursors of the well‐known, highly acidic internal peptides. We have tested the idea that these internal peptides collapse DNA by a repulsive interaction as various polymers like polyethylene oxide (PeO) and polyacrylate(PAA) do. We found that high concentrations of the internal peptides, polyaspartic acid, and polyglutamic acid, collapse DNA. This supports the idea that repulsive interactions with the internal peptides may collapse the DNA inside the head, and thus pull the DNA in. The structure of the DNA collapsed by PeO was studied with the electron microscope and contrasted with the structure of DNA collapsed by polylysine. We find PeO collapses T4 DNA into compact particles best described as a ball of string, of about the size of the T4 head. Two structures are seen in preparations of polylysine‐collapsed DNA. One has the shape of a donut and the DNA strand appears to be radially distributed as a spiral; the other is a stemlike structure in which the DNA is folded back and forth in a pleated structure. The aberrant tubular polyhead contains the precursor protein P23, P22, and the internal proteins IPIII and IPII. Addition of chloroform to a polyhead preparation extracts the proteins P22, IPIII, and IPII. This removes the inside material (core) seen in polyheads prior to the chloroform extraction, as judged by electron microscopy. We conclude that P22, IPIII, and IPII (and supposedly IPI) are the major structural constituents of the core of polyheads, while P23 is the major constituent of the outer coat. Structural studies reveal that the core of the polyhead is highly organized into a helical structure consisting of 4–6 helical chains wound about a hollow center of approximately 150 a diameter. Cleavage of the various head proteins occurs when partially purified polyheads are incubated at 37°C. In a 100 minute incubation, about 60–70% of P23 (MW = 55,000) is converted to P23* (MW = 45,000) and a significant conversion of IPIII (MW = 23,000) to IPIII* (MW = 21,000) is seen. The protein P22 (MW = 31,000) disappears during this incubation and is supposedly cleaved to small fragments. The in vitro products, P23* and IPIII*, have the same molecular weight as the in vivo products, suggesting that the protease cleavage is specific. However, several other protein fragments are generated during the in vitro cleavage reaction which have not been observed in vivo. Appropriate mutant studies reveal that the products of genes 21 and 22 are required for these in vitro cleavage reactions.

AbstractBoundary elements are thought to define the peripheries of chromatin domains and to restrict enhancer-promoter interactions to their target genes within their domains. We previously characterized a cDNA encoding the BEAF-32A protein (32A), which binds with high affinity to the scs′ boundary element from the Drosophila melanogaster 87A7 hsp70 locus. Here, we report a second protein, BEAF-32B, that differs from 32A only in its amino terminus. Unlike 32A, it has the same DNA binding specificity as the complete BEAF activity affinity purified from Drosophila. We characterize three domains in these proteins. Heterocomplex formation is mediated by their identical carboxy-terminal domains, and DNA binding is mediated by their unique amino-terminal domains. The identical middle domains of 32A and 32B are dispensable for the functions described here, although they may be important for boundary element function. 32A and 32B apparently form trimers, and the ratio of 32A to 32B varies at different loci on polytene chromosomes as judged by immunofluorescence. The scs′ element contains a high- and low-affinity binding site for BEAF. We observed that interaction with the low-affinity site is facilitated by binding to the high-affinity site some 200 bp distant.

Metaphase chromosome condensation is a dynamic process that must utilize cis elements to form and maintain the final structure. Likewise, cis elements must regulate the accessibility of chromatin domains to protein machines involved in processes such as transcription. Scaffold associated regions appear to play important roles in both of these dynamic processes.

Two minimal scaffold-associated regions (SARs) from Drosophila were tested in stably transformed cells for their effects on the expression of reporter genes. The expression of genes bounded by two SARs is consistently stimulated by about 20- to 40-fold, if the average of a pool of cell transformants is analyzed. However, analysis of individual, stable cell transformants demonstrates that flanking SAR elements do not confer position-independent expression on the reporter gene and that the extent of position-dependent variegation is similarly large with or without the flanking SAR elements. The SAR stimulation of expression is observed in stable but not in transiently transfected cell lines. The Drosophila scs and scs' boundary elements, which do not bind to the nuclear matrix in vitro, are only about one-tenth as active as SARs in stimulating expression in stable transformants. Interestingly, the SAR stimulatory effect can be blocked by a fragment containing CpG islands (approximately 70% GC), if positioned between the SAR and the enhancer. In contrast, when inserted in the same position, control fragments, such as the scs/scs' elements, do not interfere with SAR function.

DNA-binding pyrrole–imidazole compounds were synthesized that target different Drosophila melanogaster satellites. Compound P31 specifically binds the GAGAA satellite V, and P9 targets the AT-rich satellites I and III. Remarkably, these drugs, when fed to developing Drosophila flies, caused gain- or loss-of-function phenotypes. While polyamide P9 (not P31) suppressed PEV of white-mottled flies (increased gene expression), P31 (not P9) mediated three well-defined, homeotic transformations (loss-of-function) exclusively in brown-dominant flies. Both phenomena are explained at the molecular level by chromatin opening (increased accessibility) of the targeted DNA satellites. Chromatin opening of satellite III by P9 is proposed to suppress PEV of white-mottled flies, whereas chromatin opening of satellite V by P31 is proposed to create an inopportune “sink” for the GAGA factor (GAF).

We have mapped the DNA sequences bound to the nuclear scaffold along 320,000 base-pairs of a genetically well-defined region of the Drosophila chromosome. We have found that the domains delimited by the scaffold attachment regions are heterogeneous in size (ranging from 26,000 to 112,000 base-pairs in this interval), and that the attachment sites are within unique sequences as judged by blot hybridization. We also found that looped domains contain up to five, or even eight, unrelated genes including, in some cases, more than one transcribed gene. The loop organization unravelled here in cultured cells does not correspond to the banding pattern seen in salivary gland polytene chromosomes.

The structural proteins of the tubular, aberrant polyheads of bacteriophage T4 have been analyzed. The major structural constituents of polyheads are the precursor proteins P23, P22, and the internal proteins IPIII and IPII. Addition of chloroform to a polyhead preparation extracts three of these proteins (P22, IPIII, and IPII). Such treatment removes the inside material (core) seen in polyheads prior to the chloroform extraction as judged by electron microscopy. We conclude that P22. IPIII, and IPII (and supposedly IPI) are the major structural constituents of the core of polyheads, while P23 is the major constituent of the outer coat. Cleavage of the various head proteins occurs when partially purified polyheads are incubated at 37°. In a 100-min incubation, about 60%–70% of P23 (MW = 56,000) is converted to P23∗ (MW = 46,000) and a significant conversion of IPIII (MW = 23,500) to IPIII∗ (MW = 21,000) is seen. The protein P22 (MW = 31,000) disappears during this incubation and is supposedly cleaved to small fragments. The in vitro products, P23∗ and IPIII∗, have the same molecular weight as their in vivo product, suggesting that the cleavage is specific. However, several other protein fragments are generated during the in vitro cleavage reaction, which have not been observed in vivo. Appropriate studies with mutants reveal that the products of genes 21 and 22 are required for these in vitro cleavage reactions. The cleavage reaction is strongly inhibited by the addition of chloroform to the reaction mixture and is partially inhibited by the addition of TPCK (tosyl-phenylamine-chloromethylketone).

The kinetics of the assembly of polyheads produced by infecting Escherichia coli B with T4 amber mutants in gene 20 was measured and compared with the growth of wild type phage. The rates of production of polyheads and of phages were found to be about the same. The final yields in lysis-inhibited cells were approximately 600 phage equivalents per infected bacterium. The initial appearance of polyheads is delayed 15–20 min compared with wild type phage production, although it is not due to a reduced rate of protein synthesis in mutant-infected cells. In such cells an accumulation of precursor protein for polyhead is thus caused. This pool is about three times larger than the one measured during wild type infection. The delay is extended if the amount of subunits available for polyhead formation is reduced. We conclude that the initiation of polyhead assembly depends upon the subunit concentration. Polyhead assembly continues at the same rate for several minutes when protein synthesis is inhibited with chloramphenicol at different times. The maturable polyhead precursor was estimated by measuring the amount of polyheads assembled after adding the drug, and it was found that 25% of the total protein pool was converted into polyheads. Using a new technique for the observation of single cells with the electron microscope we found that polyheads are arranged in bundles oriented parallel to the long axis of the cell. The average length of polyheads is roughly the same at all times during their formation.

Boundary elements interfere with communication between enhancers and promoters, but only when interposed. Understanding this activity will require identifying the proteins involved. The boundary element-associated factor BEAF is one protein that is implicated in boundary element function. Three genomic fragments (scs', BE76 and BE28) containing BEAF binding sites function as boundary elements in transgenic Drosophila, suggesting that this is an intrinsic property of the numerous genomic regions to which BEAF binds. To characterize additional proteins that interact with boundary elements, we have isolated a protein that binds to two of these boundary elements (BE76 and BE28) and have identified it as the transcription factor DREF. We present evidence that BEAF and DREF compete for binding to overlapping binding sites, and that this competition occurs in vivo. DREF is believed to regulate genes whose products are involved in DNA replication and cell proliferation, suggesting that the activation of transcription predicted to result from the displacement of BEAF by DREF might be limited to certain rapidly proliferating tissues. This is the first suggestion that the activity of a subset of boundary elements might be regulated.

Boundary elements are thought to define the ends of functionally independent domains of genetic activity. An assay for boundary activity based on this concept measures the ability to insulate a bracketed, chromosomally integrated reporter gene from position effects. Despite their presumed importance, the few examples identified to date apparently do not share sequence motifs or DNA binding proteins. TheDrosophila protein BEAF binds the scs′ boundary element of the 87A7 hsp70 locus and roughly half of polytene chromosome interband loci. To see if these sites represent a class of boundary elements that have BEAF in common, we have isolated and studied several genomic BEAF binding sites as candidate boundary elements (cBEs). BEAF binds with high affinity to clustered, variably arranged CGATA motifs present in these cBEs. No other sequence homologies were found. Two cBEs were tested and found to confer position-independent expression on a mini-whitereporter gene in transgenic flies. Furthermore, point mutations in CGATA motifs that eliminate binding by BEAF also eliminate the ability to confer position-independent expression. Taken together, these findings suggest that clustered CGATA motifs are a hallmark of a BEAF-utilizing class of boundary elements found at many loci. This is the first example of a class of boundary elements that share a sequence motif and a binding protein.

Article15 June 2001free access Specific targeting of insect and vertebrate telomeres with pyrrole and imidazole polyamides Kazuhiro Maeshima Kazuhiro Maeshima Departments of Biochemistry and Molecular Biology, University of Geneva, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Samuel Janssen Samuel Janssen Departments of Biochemistry and Molecular Biology, University of Geneva, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Ulrich K. Laemmli Corresponding Author Ulrich K. Laemmli Departments of Biochemistry and Molecular Biology, University of Geneva, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Kazuhiro Maeshima Kazuhiro Maeshima Departments of Biochemistry and Molecular Biology, University of Geneva, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Samuel Janssen Samuel Janssen Departments of Biochemistry and Molecular Biology, University of Geneva, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Ulrich K. Laemmli Corresponding Author Ulrich K. Laemmli Departments of Biochemistry and Molecular Biology, University of Geneva, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Author Information Kazuhiro Maeshima1, Samuel Janssen1 and Ulrich K. Laemmli 1 1Departments of Biochemistry and Molecular Biology, University of Geneva, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland ‡K.Maeshima and S.Janssen contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:3218-3228https://doi.org/10.1093/emboj/20.12.3218 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info DNA minor groove-binding compounds (polyamides) that target insect and vertebrate telomeric repeats with high specificity were synthesized. Base pair recognition of these polyamides is based on the presence of the heterocyclic amino acids pyrrole and imidazole. One compound (TH52B) interacts uniquely and with excellent specificity (Kd = 0.12 nM) with two consecutive insect-type telomeric repeats (TTAGG). A related compound, TH59, displays high specificity (Kd = 0.5 nM) for tandem vertebrate (TTAGGG) and insect telomeric repeats. The high affinity and specificity of these compounds were achieved by bidentate binding of two flexibly linked DNA-binding moieties. Epifluorescence microscopy studies show that fluorescent derivatives of TH52B and TH59 stain insect or vertebrate telomeres of chromosomes and nuclei sharply. Importantly, the telomere-specific polyamide signals of HeLa chromosomes co-localize with the immunofluorescence signals of the telomere-binding protein TRF1. Our results demonstrate that telomere-specific compounds allow rapid estimation of relative telomere length. The insect-specific compound TH52 was shown to be incorporated rapidly into growing Sf9 cells, underlining the potential of these compounds for telomere biology and possibly human medicine. Introduction One of the striking features of eukaryotic chromosomes is their low gene density. Human chromosome 21 is especially gene poor; although it contains 33.55 Mbp, it harbors only an estimated 284 genes (Hattori et al., 2000). One particularly long non-genic DNA stretch of chromosome 21 encompasses 7 Mb and contains only one gene. This kind of gene-poor region embraces almost 10 Mb or one-third of chromosome 21. The functions (if any) of these and other non-genic regions are unknown, and genetic tools to dissect their roles are largely lacking. To overcome this experimental void, we propose that sequence-specific artificial proteins and/or small molecules may serve as tools. Upon binding to the non-genic DNA of interest, it is hoped that such molecules can perturb and possibly reveal the functions of the targeted DNA elements. Attempts at achieving the above goal led to the synthesis of an artificial protein termed MATH (for multi-AT hooks), which bound non-genic, AT-rich regions called SARs (scaffold-associated regions) with high specificity. SARs are candidate cis-acting elements of chromosome dynamics (for a review see Hart and Laemmli, 1998)). In support of such a role, we observed that MATH20 (an 80 kDa protein containing 20 AT hooks) specifically inhibited chromosome condensation in mitotic Xenopus extracts (Strick and Laemmli, 1995). Moreover, expression of MATH20 in Drosophila melanogaster was found to suppress the position effect variegation (PEV) phenotype of white-mottled (wm4) flies (Girard et al., 1998). We proposed that suppression of PEV is due to the binding of MATH20 to the SAR-like, centric satellite III, ensuring chromatin opening and a reduced spreading of its heterochromatic state toward the white gene. To extend this approach, we synthesized DNA-binding compounds (referred to as polyamides) composed of heterocylic amino acids (see below), which bind different D.melanogaster satellites with high specificity (Janssen et al., 2000b). Remarkably, these drugs, when fed to developing Drosophila, caused gain- or loss-of-function phenotypes with white-mottled or brown-dominant flies, respectively (Janssen et al., 2000a). Both phenomena are explained at the molecular level by chromatin opening (increased accessibility) of the targeted DNA satellites. The biological insights obtained suggested that satellite sequences are not passive evolutionary residues, but essential components of gene regulation circuits. Our observations suggest that sequence-specific artificial proteins and polyamides can serve as powerful and innovative tools for many different applications, thereby yielding important biological information. The data obtained with the aforementioned polyamides are based on recent important progress, which described the synthesis of such molecules (Geierstanger et al., 1994). Polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids can target many predetermined DNA sequences with high specificity (for a recent review see Wemmer, 2000). Specific recognition of the base pairs is based on the principle that these linear compounds can fold to adopt a U-shaped conformation (hairpins) in the minor groove due to the presence of a flexible ‘turn monomer’ (γ-aminobutyric acid). As a result, the heterocyclic pyrrole and imidazole rings form side-by-side pairs that are accommodated in the minor groove. An Im/Py pair targets a G–C base pair, while a Py/Im pair recognizes C–G. A Py/Py pair is partially degenerate and binds both A–T and T–A base pairs (White et al., 1997). A limitation of these compounds is that when these aromatic rings are coupled contiguously, it seems impossible to target stretches of >7 bp without seriously compromising binding specificity and affinity. However, studies demonstrated that longer sequences can be targeted by tethering DNA-binding moieties with flexible linker molecules (Herman et al., 1999; Janssen et al., 2000b). The in vivo drug experiments with D.melanogaster discussed above encouraged us to explore further the experimental potential of polyamides as tools and biological interference agents. The ends of chromosomes are capped by structures called telomeres. These subchromosomal structures are for several reasons ‘ideal’ and interesting polyamide targets: telomeres of most eukaryotes are defined by tandem short DNA repeats and encompass in humans a total length of ∼300 kb per haploid genome. Hence, telomeric repeats are considerably less abundant than those of DNA satellites (several megabases) and targeting these subchromosomal regions is therefore more challenging. This problem is compounded by the fact that vertebrate telomeric repeats (TTAGGG) contain three consecutive Gs, which are considered to be a difficult polyamide target. Telomeres are positioned conspicuously at the ends of chromosomes, hence, it should be possible to evaluate unequivocally the specificity of such polyamides using fluorescently tagged compounds and fluorescence microscopy. Telomeres are non-genic sequences whose structure and function are well studied (for a review see McEachern et al., 2000). This system allows a comparison of the biological effect obtained with polyamides and the effects obtained by genetic means. Last, but not least, telomere biology is often altered in cancer cells and is generally manifested by activation of telomerase (for reviews see Prescott and Blackburn, 1999; Oulton and Harrington, 2000). Although the relationship between telomere length, telomerase activity, senescence, and normal and neoplastic growth is a complex issue (Blackburn, 2000), telomere-specific polyamides may serve as new tools to address this issue and may lead to the development of agents that inhibit neoplastic growth. The most dominant telomeric repeat of vertebrates consists of hexameric TTAGGG repeats (Meyne et al., 1989). A related pentameric repeat (TTAGG) occurs at telomeres of many insect species (Sahara et al., 1999). We describe here the synthesis of polyamides that interact differentially with either insect or vertebrate telomeric repeats with a remarkable specificity. Results Targeting insect TTAGG telomeric repeats Two hairpin polyamides, H64 and H65, were designed to bind an insect TTAGG telomeric repeat. H64 and H65 differ by only one extra pyrrole at the C-terminus (Figure 1). The N-terminus of both hairpins carries two imidazole rings to accommodate the GG dinucleotide. Since a pyrrole ring C-terminal of imidazole (in hairpin polyamides) systematically shows a strong preference for GT over GA, the consensus sequence of H64 and H65 can thus be defined as WWGGTW (W = A or T base) and WGGTW, respectively. The structural basis for this is not fully understood (Kielkopf et al., 2000). Figure 1.Schematic structure of telomere compounds used in this study. N-methylpyrrole rings are represented by open circles. The flexible, hydrophilic linker (8-amino-3,6-dioxaoctanoic acid) is indicated by a boxed L. The C-terminal dimethylaminopropylamide (Dp) is represented by a plus sign. A diamond represents β-alanine. N-methylimidazole is represented by a black circle. The γ-turn monomer (R-2,4,-diaminobutyric acid) is indicated by a curved line. The solid wedge attached to the plus sign (in the γ-turn monomer) represents the amino substitution at C2. Chemical structures of the symbols used are shown at the bottom. Download figure Download PowerPoint Figure 2A shows a DNase I footprint obtained with H64 on a probe containing four tandem TTAGG repeats and four tandem TTAGGG repeats. Examination of this panel demonstrates that H64 (left) protects the TTAGG repeats at subnanomolar concentrations (Kapp <1 nM), but interacts less well with the TTAGGG units (Kapp >5 nM). Closer inspection of the H64 footprint data reveals that at higher concentrations a number of additional sequences become protected. The most prominent occurs at the sequence TTACGAAT (indicated). H64 interacts with this sequence similarly well as with the hexameric repeats (Kapp >50 nM). Unexpectedly, the smaller compound H65 displayed only very weak affinity for either sequence; only at a concentration >100 nM was some protection of insect repeats observed (not shown). Figure 2.DNase I footprinting experiments of the telomere-specific compounds. Ligand concentrations (in nM) are indicated at the top of each lane. The letter G refers to a G nucleotide cleavage reaction. (A) The monomeric hairpin H64 or its homodimer TH52A preferably binds TTAGG repeats (the bottom C-rich strand that contains AATCC is labeled) but also protects several mismatch sequences on the vector at higher concentrations. TH52A possesses increased affinity but unimproved specificity. (B) TH52B possesses excellent specificity for TTAGG repeats. Again, the bottom C-rich strand is shown. Unlike H64 and TH52A, high specificity is retained even at a concentration several hundred times that required for protection. The position of the band that is not protected in the CCTAA repeats by TH52B corresponds to the adenine base positioned on the linker. (C) Footprint of TH52B on the top G-rich strand of the same probe as in (A) and (B), showing that TH52B protects TTAGG repeats at subnanomolar concentrations. (D) Insertion of an unpaired imidazole in compound TH57 coupled to the linker generates binding preference for TTAGGG repeats over TTAGG repeats. The bottom C-rich strand is labeled. (E) When an imidazole is also inserted at the same position in the C-terminal hairpin (left), the resulting compound (TH59) has affinity similar to TH57 and binds TTAGG and TTAGGG repeats equally well. (F) The TH59 ‘monomer’ H60 shows lower specificity for telomeric repeats than the tandem hairpin TH59, but similar affinity. Download figure Download PowerPoint We previously demonstrated that dimerization of DNA-binding moieties with a flexible linker can result in an improved binding specificity (Janssen et al., 2000b). This enhanced specificity is thought to occur by an enlargement of binding site size and a discrimination against partial match sites. To improve specificity for TTAGG repeats, H64 was therefore converted into a homodimer, termed TH52A, which is expected to bind the 11 bp sequence TAGGTTAGGTT as depicted in Figure 1. However, surprisingly, although TH52A had a 10- to 20-fold increased affinity compared with H64 (Figure 2A), its specificity was unimpressive since it bound many sites along the probe and protected almost the entire probe (coating) at a concentration >2.5 nM. In an attempt to increase specificity further, we next synthesized a heterodimer termed TH52B by linking H64 and H65. This dimer is expected to target the 10 bp sequence AGGTTAGGTT (Figure 1). DNase I footprinting revealed a remarkably improved specificity of interaction for TH52B (Figure 2B). Whereas the affinity was not significantly increased as compared with H64, the tandem hairpin TH52B displayed much higher specificity for its target binding site (TTAGG repeats), since it is almost devoid of affinity for hexameric TTAGGG repeats (Kapp ∼500 nM). The equilibrium dissociation constant (Kd) for TH52B was determined at 0.12 nM by quantitative DNase I footprinting (Wade et al., 1993; Figure 2C). Most importantly, TH52B, as opposed to its monomer H64 and dimer TH52A, no longer interacted with the TTACGAAT mismatch (or any other sequence on the probe) even at the highest concentration of 500 nM (Figure 2B). These experiments demonstrate that linking DNA-binding moieties can dramatically improve the binding specificity of polyamides (see also Herman et al., 1999). They also illustrate the subtlety and apparent unpredictability of the DNA recognition rules, as illustrated by the difference in behavior between TH52A and TH52B; although these molecules are very similar, TH52B (lacking only one pyrrole ring) has a much higher specificity than TH52A. Behavior of the flexible linker The binding scheme for TH52B (Figure 1) suggests that the ethylene oxide linker of TH52B spans the central A (bold) of its 10 bp AGGTTAGGTT target. We carried out a number of initial footprinting studies to establish the ‘linker rules’ at this position. This central A was either deleted or replaced with any nucleotide. Footprint studies indicated that T and A are equally well tolerated at this position, but that replacement of this central A by a G reduced the affinity by ∼4-fold and that replacement by a C abolished binding altogether (not shown). Also poorly tolerated was a deletion of this central A or the insertion of any nucleotide 3′ of it. Thus, the ethylene oxide linker is best suited to bridge a single W nucleotide. To determine the effect of linker length, we synthesized two compounds with a shorter, aliphatic, methylene linker (β-alanine and 5-aminovaleric acid) and one tandem hairpin with a longer linker composed of two amphipathic 8-aminodioxaoctanoic acid units. The latter linker adds 18 interatomic bonds and has an amphipathic character due to the presence of the ethylene oxide units. This tandem hairpin showed binding affinity similar to its parent TH52B (containing nine interatomic bonds) but an increased affinity for its ‘mismatch’ sequence TTAGGG (increased from ∼500 to 10 nM) (not shown). The shorter β-alanine linker (adding four interatomic bonds) showed both lowered affinity and specificity. Affinity was lowered by 15- to 20-fold and it bound many mismatch sites on the probe, suggesting that this linker is too short to allow proper binding of both hairpins. The valeric acid-spaced tandem hairpin (adding six interatomic bonds) was similar to its parent TH52B in both affinity and specificity, suggesting that the original linker is longer than necessary but that there is no entropic penalty to be paid up to (at least) nine interatomic bonds. Targeting vertebrate TTAGGG telomeric repeats Recognition of the additional G of the vertebrate hexameric telomere repeat could, in principle, be obtained easily by the addition of an extra imidazole in each hairpin of the tandem hairpin TH52B. A related sequence containing three consecutive Gs (AGGGA) was targeted previously by a hairpin containing three consecutive imidazoles [ImImImPy-γ-PyPyPyPy-β-Dp; (Swalley et al., 1996), where γ represents the γ-aminobutyric acid turn monomer and Dp represents dimethylaminopropylamide]. We therefore synthesized a new tandem hairpin polyamide termed TH58 containing an additional imidazole in each hairpin (Figure 1). Surprisingly, subsequent binding analysis by DNase I footprinting showed that TH58 did not protect TTAGGG repeats (nor any other sequence) up to the highest concentration tested (50 nM; not shown). Hence, we considered an alternative drug design for the recognition of TTAGGG repeats by moving one of the three imidazoles to the opposite side of the hairpin and leaving it ‘unpaired’. Few data have been published about unpaired imidazoles (without the opposite pyrrole) and their sequence preference. In our experience, unpaired imidazoles recognize both G–C and C–G base pairs (Janssen et al., 2000a), which is in accordance with the central location of the guanine exocyclic amine group in the minor groove (White et al., 1997 and references therein). The structure of the compound in which this principle is applied in the N-terminal hairpin is schematized in Figure 1 (compound TH57). The binding behavior of TH57 was evaluated by DNase I footprinting. Figure 2D shows that binding of TH57 to TTAGGG repeats (the opposite strand, CCCTAA, is labeled) now occurs at low nanomolar concentrations (Kapp ∼3 nM). At higher concentrations, binding to TTAGG repeats is also observed (Kapp ∼10 nM). We next synthesized a tandem hairpin polyamide where both hairpins have the same design as the N-terminal hairpin of TH57 (with the third Im moved to the ‘bottom’). The schematic structure of this new compound (termed TH59) is shown in Figure 1. The chemical structure of TH59 and a speculative binding model is shown in Figure 3. DNase I footprint analysis of TH59 (Figure 2E) showed that this compound bound with slightly increased affinity to its target sequence TTAGGG, but cannot discriminate between the two different telomeric sequences. The equilibrium dissociation constant (Kd) for TH59 was determined by quantitative DNase I footprinting (Wade et al., 1993) at 0.51 nM. We also evaluated the binding behavior of the TH59 ‘monomer’ and observed that this compound (termed H60, see Figure 1) bound with similar affinity but lower specificity (see Figure 2F). Figure 3.Putative binding model of TH59. Proposed binding model for the complex of TH59 with 5′-AGGGTTAGGGTT-3′. Circles with two dots represent lone pairs of electrons on N3 of purines and O2 of pyrimidines at the edges of the bases. Circles containing an H represent the N2 hydrogen of guanine. Putative bifurcated hydrogen bonds to the amide NHs are illustrated by dashed lines. Download figure Download PowerPoint Staining telomeres with fluorescently tagged polyamides Using fluorescently tagged polyamides and epifluorescence microscopy, we previously highlighted the position of different DNA satellites in nuclei and chromosomes. This staining method provides specificity information for polyamides on chromatin rather than on naked DNA. Molecules TH52B and TH59 bind two telomeric repeats. Since the length of eukaryotic telomeres varies between a few and tens of kilobase pairs, one might ultimately expect a hundred to a few thousand molecules bound per telomere. Is it possible to observe this signal above the background? Insect telomeres. To obtain fluorescently tagged polyamides, a primary amine was introduced at the C-terminus, which was subsequently acylated using a commercially available N-hydroxysuccinimide active ester of Texas Red (Janssen et al., 2000b). Insect chromosomal material was then isolated from Sf9 cells, a cell line derived from the armyworm, Spodoptera frugiperda (Vaughn et al., 1977), which is expected to contain insect-type telomeric repeats (Okazaki et al., 1993). Classical metaphase chromosomal spreads were prepared from Sf9 cells and then double stained with 4′,6-diamidino-2-phenylindole (DAPI) and TH52B-T (T for Texas Red). Figure 4A shows in blue (DAPI) the metaphase chromosomes and in red striking foci, which represent the subchromosomal signals of TH52B-T. Karyotypes of Sf9 cells are very complex and poorly characterized, consisting of innumerable mini chromosomes. Generally, two foci are observed at each chromosomal end, suggesting that TH52B-T highlights telomeres as expected. Included in Figure 4A is a black and white inset showing the TH52B-T telomeric signal separately. Note that although generally low ‘background’ signal is observed along the chromosomal body, one can also observe some subtelomeric signals Figure 4.Staining of insect-type telomere repeats (TTAGG) with TH52B-T. Chromosomes or nuclei prepared from Sf9 and HeLa cells were co-stained with TH52B-T (red) and DAPI (blue). Note that TH52B-T sharply highlights red foci in Sf9 (B) but not HeLa nuclei (C). The two images (B and C) were obtained under identical conditions and are shown on an identical intensity scale. TH52B-T also stains the ends of chromosomes, as observed in metaphase spreads derived from Sf9 cells (A). A number of non-telomeric signals can also be noted (black and white inset). Scale bars represent 5 μm. Download figure Download PowerPoint Figure 4B shows a representative image of an Sf9 nucleus stained with DAPI and TH52B-T, which again yields sharp red foci. Interestingly, the DAPI signal of stained Sf9 nuclei shows an unusual ‘grape-like’ structure rather than displaying the generally homogeneous appearance of eukaryotic nuclei. Closer examination of these images reveals that the red TH52B-T foci are often abutting blue grape-like domains, perhaps representing interphase chromosomal territories. Importantly, in line with our footprint data, no red foci were observed when HeLa nuclei were stained with TH52B-T (Figure 4C). As mentioned above, the footprinting data indicated that TH59, in contrast to TH52B, bound both the penta- and hexameric telomeric repeats. Chromosomal staining studies confirmed this notion. We observed that TH59-T stains telomeres of Sf9 chromosomes, although its signal was less sharp than that obtained with TH52B-T owing to a higher general background (not shown). Vertebrate telomeres. We assessed the specificity of the telomere polyamide TH59-T by staining chromosomal material derived from a variety of vertebrate cells. These studies collectively demonstrated that TH59-T (not TH52B-T) specifically stained telomeres of all these cell lines. A series of micrographs is shown in Figure 5. All images were obtained by double staining chromosomes or nuclei with DAPI and TH59-T. Figure 5A impressively shows that TH59-T sharply highlights telomere foci in HeLa cell nuclei. Telomeres are also marked robustly by TH59-T in spreads of HeLa metaphase chromosomes (Figure 5D), on isolated Indian Muntjac (Figure 5B) and on Xenopus laevis (Figure 5C) chromosomes. Figure 5.Staining of vertebrate telomeric repeats (TTAGGG) with TH59-T and co-localization with TRF1. Chromosomal material prepared from vertebrate cell lines was stained with polyamide TH59-T (red) and DAPI (blue). Note that TH59-T sharply highlights red foci in HeLa cell nuclei (A) and metaphase chromosomes derived from Indian Muntjac (B), X.laevis (C) or HeLa cells (D). (E) TH59-T foci co-localize with green telomere spots revealed by indirect immunofluorescence with TRF1-specific antibodies. The black and white inset shows the green and red signals separately. Scale bars represent 5 μm. Download figure Download PowerPoint The staining results with TH52 and TH59 are impressive since the micrographs revealed high signal-to-noise ratios. This is particularly evident by inspection of the black and white inset in Figure 5D; note that TH59-T yields little general background signal along the chromosomal body. These staining data semi-quantitatively extend the footprint studies from naked DNA to chromatin. The signals of telomere-specific polyamides and those of a telomere-binding protein, TRF1, co-localize The footprint data and the conspicuous staining results obtained with polyamides TH52 and TH59 (or derivatives thereof) strongly suggest that these compounds are localized at telomeres. We proved this notion by immunofluorescence. Human telomeres contain two related TTAGGG repeat-binding factors, TRF1 and TRF2 (Broccoli et al., 1997). [Both proteins have a C-terminal Myb-like helix–turn–helix domain and a central domain involved in the formation of homodimers (O'Reilly et al., 1999).] TRF1 and TRF2 are located predominantly at chromosomal ends where they contribute to the maintenance of telomere structure. TRF1 and TRF2 should therefore co-localize with TH59-T. Figure 5E shows a HeLa cell nucleus stained with TH59-T (red) together with the immunofluorescence signal of TRF1 (green). Examination of this micrograph shows that TH59-T and anti-TRF1 signals co-localize perfectly since the resulting overlapping spots appear yellow. This conclusion is also apparent from inspection of the insets of Figure 5E displaying the TH59-T and anti-TRF1 signals separately, in black and white. Similar observations were also obtained by staining chromosomal spreads. In addition, the immunofluorescence signals of TRF2 also co-localized with TH59-T (data not shown). These observations establish that TH59-T marks the position of telomeres in nuclei and chromosomes. Rapid estimation of relative telomere length In telomere research, it is often of interest to estimate and follow telomere length quantitatively. Three protocols are currently used for this purpose. One of these depends on digestion of the genomic DNA using enzymes with restriction sites in subtelomeric DNA, and Southern blotting (van Steensel and de Lange, 1997). This rather time-consuming procedure provides an estimate of telomere lengths of all cells of a sample. Two related alternative procedures are based on in situ hybridization using either chromosomal spreads or permeabilized cells followed by epifluorescence microscopy (Zijlmans et al., 1997) or flow cytometry (Hultdin et al., 1998). The latter two protocols generally use fluorescently labeled peptide nucleic acid (PNA) complementary to the telomere repeats as probes. We propose that this laborious, cumbersome hybridization step can be replaced by a simple staining protocol using fluorescent telomere-specific polyamides. To examine this issue, metaphase chromosome spreads were prepared from human lymphocytes and two closely related HeLa subclones that differ in telomere lengths. HeLa1.2.11 (termed HeLa-L) has long (L) telomeres of ∼15–40 kb (Smogorzewska et al., 2000), whereas HeLaII (HeLa-S) has shorter (S) telomeres of 3–6.5 kb (Smogorzewska et al., 2000). Slides prepared with these cell lines were double stained with DAPI and TH59-T under identical conditions, and several optical sections of chromosomal spreads were recorded with an image restoration, wide field fluorescence microscope (DeltaVision). In order to analyze the maximum intensity of each telomere, the various optical sections were combined by a maximum brightness projection. Visual inspection of these projected images indicated that HeLa-L telomeres were labeled more intensively by TH59-T than the telomeres of lymphocytes or HeLa-S cells (Figure 6A and B; data not shown). Figure 6.Quantitative aspect of vertebrate telomere signals. Metaphase chromosome spreads were prepared from human lymphocytes and HeLa-L cells, a HeLa subclone with long telomeres. Subregions of metaphase spreads are shown derived from human lymphocytes (A) and HeLa-L cells (B). The two images (A and B) were obtained under identical conditions and are shown on the same intensity scale. For quantification, the telomere foci were contoured appropriately (indicated) and the total integrated signal intensities were then determined for entire spreads. The total telomere signal intensity distribution obtained for different chromosomal spreads is shown in Figure 7. The fractional amount of polyamide bound at telomeres relative to that encompassing the chromosomal body was determined. An example of this is shown for HeLa-L chromosomes (B, C and D). The telomere spots contoured in (B) were extracted to yield (D). The image lacking telomere spots was then contoured again using a lower threshol

There are few tools available for dissecting and elucidating the functions of DNA satellites and other nongenic DNA. To address this, we have explored the experimental potential of DNA sequence-specific drugs containing pyrrole and imidazole amino acids (polyamides). Compounds were synthesized that target different Drosophila melanogaster satellites. Dimeric oligopyrroles were shown to target the AT-rich satellites I, III, and SARs (scaffold associated regions). One polyamide (P31) specifically binds the GAGAA satellite V. Specificity of targeting was established by footprinting, epifluorescence of nuclei, and polytene chromosomes stained with fluorescent derivatives. These polyamides were shown to mediate satellite-specific chromatin opening of the chromatin fiber. Remarkably, certain polyamides induced defined gain or loss-of-function phenotypes when fed to Drosophila melanogaster.

Drosophila and mammalian proteins protect genes from heterochromatic repression in Saccharomyces cerevisiae by two different mechanisms. Factors termed genuine boundary activities (BAs) establish a structural, unidirectional bulwark against heterochromatin. In contrast, factors termed desilencing activities (DAs) act by the formation of a bidirectional, euchromatic island that blocks spreading of heterochromatin. The Drosophila boundary protein BEAF and, unexpectedly, the mammalian factor Sp1 exhibited a robust BA in yeast. In contrast, mammalian CTCF, Drosophila GAGA factor, yeast Gcn5p, and many mammalian transcription factors, although inactive as upregulators of nonsilenced genes, work as DAs. DAs but not BAs protect telomere-linked genes from silencing, presumably due to looping of telomeres and ensuing multidirectional silencing. The data demonstrate that "genetic autonomy" of chromatin domains is established by both passive and active mechanisms.

We have optimized procedures for the isolation of mitotic chromosomes from tissue culture cells. The first procedure is a rapid method for obtaining individual, structurally intact chromosomes suitable for analysis by electron microscopy. Further purification of these on Percoll gradients results in chromosomes free of cytoplasmic contamination, allowing biochemical characterization of the structural proteins and enzymatic activities intrinsic to mitotic chromosomes. A third procedure permits efficient, large-scale purification of chromosomes clustered together, referred to as a chromosomal cluster. The use of EDTA-containing polyamine buffers minimizes modifications of proteins and DNA during isolation and maintains the integrity of the chromosomal structure. The conditions which lead to the isolation of chromosomal clusters, as opposed to individual chromosomes, have been analyzed. Comparison of the gel patterns of proteins derived from individual chromosomes, as compared to clusters, identifies additional proteins in the latter pattern. These proteins could be involved in maintaining interchromosomal organization or positioning in the metaphase cell.

To structurally dissect mitotic chromosomes, we aim to position along the folded chromatin fiber proteins involved in long-range order, such as topoisomerase IIα (topoIIα) and condensin. Immuno-electron microscopy (EM) of thin-sectioned chromosomes is the method of choice toward this goal. A much-improved immunoprocedure that avoids problems associated with aldehyde fixation, such as chemical translinking and networking of chromatin fibers, is reported here. We show that ultraviolet irradiation of isolated nuclei or chromosomes facilitates high-level specific immunostaining, as established by fluorescence microscopy with a variety of antibodies and especially by immuno-EM. Ultrastructural localizations of topoIIα and condensin I component hBarren (hBar; hCAP-H) in mitotic chromosomes were studied by immuno-EM. We show that the micrographs of thin-sectioned chromosomes map topoIIα and hBar to the center of the chromosomal body where the chromatin fibers generally converge. This localization is defined by many clustered gold particles with only rare individual particles in the peripheral halo. The data obtained are consistent with the view that condensin and perhaps topoIIα tether chromatin to loops according to a scaffolding-type model.

Journal Article A simple method to renature DNA-binding proteins separated by SDS-polyacrylamide gel electrophoresis Get access Vincent Ossipow, Vincent Ossipow De`partement de Biologie Moleculaire, Sciences II, Universite de Geneve30, Quai Ernest Ansermet, CH-1211 Geneve-4, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar Ulrich K. Laemmlii, Ulrich K. Laemmlii De`partement de Biologie Moleculaire, Sciences II, Universite de Geneve30, Quai Ernest Ansermet, CH-1211 Geneve-4, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar Ueli Schibler Ueli Schibler * De`partement de Biologie Moleculaire, Sciences II, Universite de Geneve30, Quai Ernest Ansermet, CH-1211 Geneve-4, Switzerland * De`partement de Biologie Moleculaire, Sciences II, Universite de Geneve 30, Quai Ernest Ansermet, CH-1211 Geneve-4, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 21, Issue 25, 25 December 1993, Pages 6040–6041, https://doi.org/10.1093/nar/21.25.6040 Published: 25 December 1993 Article history Received: 13 October 1993 Revision received: 17 October 1993 Accepted: 17 October 1993 Published: 25 December 1993

We have been able to map specific DNA fragments at the bases of chromatin loops with the help of a novel extraction procedure by using lithium-3',5'-diiodosalicylate. One such scaffold-attached region (SAR) is found in the non-transcribed spacer in each repeat of the histone gene cluster, on a 657 base pair (b.p.) restriction fragment. Exonuclease III digestion has localized two protein-binding domains on the SAR of the histone cluster. Each covers approximately 200 b.p. and they are separated by a nuclease-accessible region of about 100 b.p. These domains are rich in sequences closely related to the topoisomerase II cleavage consensus. We have studied the scaffold association of three developmentally regulated genes of Drosophila melanogaster: alcohol dehydrogenase (Adh), the homoeotic gene fushi tarazu (ftz) and Sgs-4, a gene encoding one of the glue proteins secreted by third-instar larvae. We find regions attached to the nuclear scaffold (SARS) both 5' and 3' of all three genes, defining small domains ranging from 4.5 to 13 kilobases. In the case of Adh, a gene with two promoters, we find two upstream and two downstream SARS. Those 5' of the gene co-map with regulatory regions for the adult and the larval transcripts, respectively. For Sgs-4, the 5' SAR covers 866 b.p. immediately upstream of the transcript, and encompasses the 200 b.p. regulatory region defined by two deletion mutants that produce little or no Sgs-4 protein. In ftz the 5' SAR is found 4.8 kilobases upstream of the start of transcription within a 2.5 kilobase element required for a high level of ftz expression in the early embryo. Sequence analysis of five upstream SARS reveals clusters of sequences closely related to the cleavage consensus of topoisomerase II. In addition, they contain multiple copies of two sequence motifs: a specific 10 b.p. A-rich sequence, and another 10 b.p. T-rich stretch. In conclusion, the intimate association of the SAR with the upstream/enhancer elements, the presence of clustered sequences highly homologous to the topoisomerase II cleavage consensus, and the localization of topoisomerase II in the scaffold, suggest a structure-function relation between chromosome organization and gene expression.

Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding protein that is essential for general DNA metabolism. RPA consists of three subunits (70, 33 and 14 kDa). We have identified by two-hybrid screening a novel Xenopus protein called XRIPalpha that interacts with the ssDNA-binding domain of the largest subunit of RPA. XRIPalpha homologues are found in human and in Drosophila but not in yeast. XRIPalpha is complexed with RPA in Xenopus egg extracts together with another 90 kDa protein that was identified as importin beta. We have demonstrated that XRIPalpha, but not importin alpha, is required for nuclear import of RPA. Immunodepletion of XRIPalpha from the egg extracts blocks nuclear import of RPA but not that of nucleoplasmin, a classical import substrate. RPA import can be restored by addition of recombinant XRIPalpha. Conversely, depletion of importin alpha blocks import of nucleoplasmin but not that of RPA. GST-XRIPalpha pull-down assay shows that XRIPalpha interacts directly with recombinant importin beta as well as with RPA in vitro. Finally, RPA import can be reconstituted from the recombinant proteins. We propose that XRIPalpha plays the role of importin alpha in the RPA import scheme: XRIPalpha serves as an adaptor to link RPA to importin beta.

Article1 April 1998free access In vivo analysis of scaffold-associated regions in Drosophila: a synthetic high-affinity SAR binding protein suppresses position effect variegation Franck Girard Franck Girard Present address: CNRS ERS155, 1919 route de Mende, 34033 Montpellier, France Search for more papers by this author Bruno Bello Bruno Bello Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, France Search for more papers by this author Ulrich K. Laemmli Ulrich K. Laemmli Department of Biochemistry and Molecular Biology, 30 Quai Ernest Ansermet Sciences II, 1211 Genève, Switzerland Search for more papers by this author Walter J. Gehring Corresponding Author Walter J. Gehring Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, France Search for more papers by this author Franck Girard Franck Girard Present address: CNRS ERS155, 1919 route de Mende, 34033 Montpellier, France Search for more papers by this author Bruno Bello Bruno Bello Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, France Search for more papers by this author Ulrich K. Laemmli Ulrich K. Laemmli Department of Biochemistry and Molecular Biology, 30 Quai Ernest Ansermet Sciences II, 1211 Genève, Switzerland Search for more papers by this author Walter J. Gehring Corresponding Author Walter J. Gehring Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, France Search for more papers by this author Author Information Franck Girard2, Bruno Bello1, Ulrich K. Laemmli3 and Walter J. Gehring 1 1Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, France 2Present address: CNRS ERS155, 1919 route de Mende, 34033 Montpellier, France 3Department of Biochemistry and Molecular Biology, 30 Quai Ernest Ansermet Sciences II, 1211 Genève, Switzerland *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:2079-2085https://doi.org/10.1093/emboj/17.7.2079 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Scaffold-associated regions (SARs) were studied in Drosophila melanogaster by expressing a synthetic, high-affinity SAR-binding protein called MATH (multi-AT-hook), which consists of reiterated AT-hook peptide motifs; each motif is known to recognize a wide variety of short AT-rich sequences. MATH proteins were expressed specifically in the larval eye imaginal discs by means of the tetracycline-regulated transactivation system and tested for their effect on position effect variegation (PEV). MATH20, a highly potent SAR ligand consisting of 20 AT-hooks, was found to suppress whitemottled 4 variegation. This suppression required MATH20 expression at an early larval developmental stage. Our data suggest an involvement of the high AT-rich SARs in higher order chromatin structure and gene expression. Introduction Scaffold-associated regions (SARs), also called matrix attachment regions or MARs, are operationally defined as DNA sequences that specifically associate with the nuclear scaffold or matrix and possibly define the bases of chromatin loops (Mirkovitch et al., 1984; Cockerill and Garrard, 1986; Gasser and Laemmli, 1986). SARs are very AT-rich regions of several hundred base pairs in length, that are possibly best described as being composed of numerous, irregularly spaced A tracts (short AT-rich sequences containing homopolymeric runs) (reviewed in Laemmli et al., 1992). Proteins that specifically bind to SARs do not appear to interact with a precise base sequence but rather recognize certain structural features of non-B DNA, such as narrow minor grooves, DNA bends and a propensity to unwind (Bode et al., 1992; Käs et al., 1993). SARs play roles in both chromosome condensation and gene expression. A recent report identified SARs as cis-elements of chromosome dynamics (Strick and Laemmli, 1995), while numerous publications demonstrated that SARs, in a flanking position, can strongly stimulate the expression of various heterologous reporter genes in different biological systems (Laemmli et al., 1992). The SAR-mediated stimulation of transgene expression is not observed in transient assays, but only after stable integration of the test constructs into the genome (Klehr et al., 1991; Poljak et al., 1994). Hence, these cis-acting elements may exert their effect via chromatin structure, since transiently transfected DNAs are known to be poorly organized into nucleosomes. A recent model attempts to explain the general stimulatory effect of SARs on transcription by proposing that SARs facilitate the displacement of histone H1 (a process referred to as chromatin opening) through mutually exclusive interactions with proteins of similar DNA-binding specificity, such as the high mobility group protein HMG-I/Y (Käs et al., 1993). HMG-I/Y contains three short DNA-binding domains, called AT-hooks, that bind the minor groove of A tracts similarly to the peptide antibiotic distamycin (Reeves and Nissen, 1990). Histone H1, HMG-I/Y and distamycin all bind selectively to the A tracts of SARs, and competition experiments between them have demonstrated that HMG-I/Y and distamycin are ‘dominant’: i.e. both can displace pre-bound histone H1 from an SAR template (Zhao et al., 1993). The observation that distamycin, added to cells, markedly stimulated cleavage at SARs by topoisomerase II in internucleosomal linker DNA but not at hypersensitive sites, lends in vivo support to this model; increased accessibility for cleavage presumably arises from the displacement of proteins, possibly histone H1 (Käs et al., 1993). The model is also consistent with elegant studies demonstrating that SARs are necessary to spread open chromatin from enhancers to gene promoters (Kirillov et al., 1996; Jenuwein et al., 1997). To study the role of SARs in chromosome condensation, synthetic multi-AT-hook proteins (MATH), consisting of numerous reiterated AT-hook peptide motifs derived from HMG-I/Y (Strick and Laemmli, 1995), were synthesized. Since the AT-rich SARs have enough adjacent binding sites to accommodate all the multiple, covalently linked AT-hooks, these HMG-I/Y derivatives bind SARs (both as DNA and chromatin) with exquisite specificity (Strick and Laemmli, 1995). Their effects on chromosome assembly were tested in Xenopus egg extracts capable of converting added nuclei to mitotic chromosomes in vitro. Remarkably, adding low levels of MATH20, a protein containing 20 hooks, inhibited the normal events of chromosome condensation, suggesting that SARs are cis-elements of mitotic chromosome dynamics (Strick and Laemmli, 1995). To address the importance of SARs in the fruit fly Drosophila melanogaster, we expressed MATH20 in the larval eye imaginal disc to test for an effect on position effect variegation (PEV). We found that regulated expression of MATH20 led to a suppression of PEV, suggesting an involvement of SARs or SAR-like AT-rich regions in long-range chromatin structure and gene regulation. Results Targeted expression of synthetic SAR-binding proteins in Drosophila With the aim of improving our understanding of SAR function(s) in vivo, we have overexpressed MATH20, the synthetic SAR-binding protein, in Drosophila by means of the tetracycline (tet)-regulated transactivation system (Tet system) (Bello et al., 1998). Our strategy consisted of targeting expression of these proteins specifically to the eye imaginal discs during larval development and scoring for effects on white variegation. The principle of the binary Tet system is depicted in Figure 1A. A fly strain expressing a tet-regulated transactivating protein [tTA, a fusion of the tet repressor DNA-binding domain and the VP16 transcriptional activation domain (Gossen and Bujard, 1992)] under the control of regulatory sequences [in our case, an eye-specific enhancer identified in the intronic sequences of the eyeless gene (Quiring et al., 1994)] is crossed to a strain containing the coding sequence for the gene of interest downstream of the tet operator sequences. The expression of this gene is then obtained in the progeny, in the same pattern as tTA is expressed, and can be tightly controlled by tet (Bello et al., 1998). As shown in Figure 1B–D, by monitoring tetO–LacZ reporter gene expression, the ey-tTA strain can be used to target protein expression in the third instar eye imaginal discs, primarily in the undifferentiated eye cells (Figure 1B), and progressively only in the differentiated cells posterior to the morphogenetic furrow (Figure 1C and D). Additionally, β-galactosidase activity is also observed in the eye imaginal discs during the first and second instars (data not shown). Tet, when added to the food at a concentration as low as 0.2 μg/ml, is able to completely silence the expression of the LacZ reporter gene, both in early (Figure 1E) and late third instar (Figure 1F). Figure 1.Protein overexpression by means of the tetracycline-regulated transactivation system in Drosophila. (A) Schematic representation of the Tet system applied to Drosophila (see text for details). In (B–F), β-galactosidase staining of eye-antennae imaginal discs from early (B and E), mid (C) and late third instar larvae (D and F), showing the activation of a tetO–LacZ reporter construct by the eyeless enhancer-tTA strain. In (E) and (F), larvae were maintained on medium containing tet 0.2 μg/ml, resulting in a complete inhibition of the tTA–induced LacZ expression. Download figure Download PowerPoint MATH20 suppresses PEV In Drosophila, PEV refers to the mosaic expression of a gene when chromosome rearrangements place it close to heterochromatin. This heterochromatin-mediated gene silencing is proposed to be a heritable, epigenetic event that involves no alteration in the DNA content. Silenced genes are believed to be packaged into a higher order chromatin structure, or alternatively might be localized to a special nuclear compartment that confers transcriptional repression (reviewed in Karpen, 1994; Elgin, 1996). A classical example of PEV is the white-mottled (wm4) inversion, in which the white gene necessary for the red pigmentation of the eye is juxtaposed close to heterochromatin of the X chromosome. The variegating phenotype of white-mottled is seen as numerous clones of red, wild-type cells in an otherwise white mutant background (Figure 2A). Since MATH proteins are very efficient in SAR binding, including satellite III found in the centromeric heterochromatin of the X chromosome (Strick and Laemmli, 1995, and below), we reasoned that overexpressing MATH proteins specifically in the eye imaginal discs of the developing larvae might modify white variegation, a process known to involve chromatin structure. Females of the ey-tTA strain in a wm4 background were crossed to males of the various independent tetO-MATH20 strains. After eclosion, males of the desired genotypes (which are heterozygous for both ey-tTA and tetO-MATH20 transgenes, and hemizygous for wm4) were kept for 5 days at 25°C and photographed. As a control, we used a line containing an empty tetO vector, showing a typical ‘salt and pepper’-like mosaic expression of the white gene (Figure 2A). Expressing MATH20 resulted in a significant derepression of the white gene, and a general loss of the variegating phenotype. This suppression of PEV was clearly visible in various independent MATH20 lines (Figure 2B–F), and was shown to be highly reproducible. Quantitative analysis of the red eye pigment levels for seven independent MATH20-expressing strains is shown in Figure 3A, with PEV suppressor ratios ranging from 1.6 to 2.8 when compared with the control line (two independent control lines gave identical results). In contrast, MATH11, a less potent SAR DNA-binding protein (Strick and Laemmli, 1995), revealed no PEV-modifying effects in four independent strains (Figure 3A, dashed bars). Figure 2.Eye-specific expression of MATH20 suppresses white variegation. (A–F) Females of the ey-tTA strain in a wm4h background were crossed to males of the various tetO-MATH20 strains. Progeny was grown on standard medium at 25°C. After adult eclosion, males of the desired genotype were kept for 5 days at 25°C before photography. Shown are photomicrographs of male heads of the following genotypes: (A) wm4h/Y, control tetO/+, ey-tTA/+; (B, E and F) wm4h/Y, tetO-MATH20/+, ey-tTA/+, with respectively strains 20-14, 20-2 and 20-19. (C and D) wm4h/Y, +/+, ey–tTA/tetO-MATH20, with respectively strains 20-4 and 20-6. (G–J) Inhibition of MATH20-induced PEV suppressor effect by tet treatment. Females of the ey-tTA strain in a wm4h background were crossed to males of the control tetO strain (G and H) or the tetO-MATH20-19 strain (I and J). Progeny was grown at 25°C, either on standard medium (G and I) or medium containing tet 0.2 μg/ml (H and J). Download figure Download PowerPoint Figure 3.Quantitation of MATH20-induced PEV suppression. (A) MATH20, but not MATH11, suppresses wm4 variegation. Females of the ey-tTA strain in a wm4h background were crossed to males of the control tetO strain or the various tetO-MATH11 (dashed bars) and tetO-MATH20 strains (shaded bars). Quantitation of the red eye pigment levels was done on groups of 40 male heads 5 days after eclosion, and repeated 4–6 times. Standard deviations are shown as thin lines above the histograms. (B) Inhibition of MATH20-induced PEV suppression by tet. Females of the ey-tTA strain in a wm4h background were crossed to males of the control tetO strain or three independent tetO-MATH20 strains. Progeny were grown at 25°C on either normal food (open bars) or food containing tet 0.2 μg/ml (black bars). Values are given as the ratio of the OD480 nm of the tetO–MATH20 lines to the OD480 nm of the empty tetO vector control line, and represent the average of three independent measurements. (C) Females of the ey-tTA strain in a wm4h background were crossed to males of the control tetO strain or tetO-MATH20-12 strain. Progeny were kept on normal or tet-containing food (0.2 μg/ml). Larvae were transferred to normal food at the indicated times after egg laying. The red eye pigment levels were measured in groups of 30 male heads, and repeated three times. Values are given as the average ratio of the OD480 nm of the tetO-MATH20-12 strain to the OD480 nm of the control strain. Download figure Download PowerPoint To test for the specificity of the MATH20-induced suppression of PEV, we made use of tet to silence the expression of the MATH20 transgene. Progeny of a cross between wm4, ey-tTA females and tetO-MATH20-19 or empty tetO were grown on either normal or tet-containing food, under exactly the same conditions of temperature and population density as before. While tet treatment has no effect on the white mosaic expression in the control line (Figure 2G–H), it clearly inhibits MATH20-induced suppression of PEV (compare Figure 2I and J). Quantitative analysis is given for three MATH20 strains (Figure 3B). Results are shown as the ratio of OD480 MATH20 to OD480 control: while modification of white variegation is observed in the absence of tet (Figure 3B, open bars), tet treatment, by maintaining silent the MATH20 transgene expression, leads to eye pigment levels very similar to those of the control (Figure 3B, black bars). We next made use of tet to examine whether suppression of PEV by MATH20 might require expression during an early developmental stage. For this purpose, 50 females of the wm4h, ey-tTA strain were crossed to males of the control or tetO-MATH20-12 strain. One hour egg collections were made, and kept on either normal or tet-containing food. Larvae were then transferred at regular intervals to normal food. In these conditions, the transgene is kept efficiently silent, but it is activated after shifting larvae off tet following a lag period of ∼12 h (Bello et al., 1998). As shown in Figure 3C, PEV suppression is still observed when larvae are exposed to tet up to 60 h after egg laying (AEL). If the gene is kept silenced longer (72–120 h), then no PEV suppression is observed. The eyeless enhancer activity and hence MATH20 expression can be detected throughout the larval stages and up through early pupal stages when differentiated eye cells develop from precursor cells. Since activation of the transgene, during the third instar period (72 h AEL), no longer reduced PEV, we conclude that MATH20 expression in the undifferentiated cells is required to achieve suppression of PEV. MATH20 suppresses cleavage by topoisomerase II in satellite III heterochromatin of chromosome X Is suppression of PEV by MATH20 mediated by interactions at SARs? The wm4 inversion juxtaposes the white gene to the heterochromatin of the X chromosome. Intriguingly, the predominant component of the heterochromatin is satellite III, also called the 1.688 or the 359 bp repeat satellite (Hsieh and Brutlag, 1979). Two or three repeats (718–1017 bp) of this satellite behave as fully fledged SARs in vitro; they preferentially bind nuclear scaffolds, topoisomerase II and HMG-I/Y (Käs and Laemmli, 1992; Karpen, 1994). Thus, the inverted white gene appears juxtaposed to a giant, reiterated SAR of ∼11 Mb. Topoisomerase II is one of the growing number of proteins associated with heterochromatin (Rattner et al., 1996). Although it is not known whether topoisomerase II is implicated in heterochromatin formation, it is one of the few proteins for which the site of interaction can be studied by stabilizing the so-called cleavage intermediate using cytotoxic drugs. Thus, this protein serves here as a convenient, heterochromatin-associated reporter protein which is expected to monitor the interaction of MATH20 in this chromatin. Moreover, topoisomerase II is known to be specifically enriched over satellite III heterochromatin, as revealed by microinjection of fluorescent topoisomerase II into Drosophila embryos (Denburg et al., 1996). This preferential interaction was also borne out by previous studies that demonstrated a major topoisomerase II cleavage site once per satellite III repeat; this 359 bp repeat contains two positioned nucleosomes, and the major topoisomerase II cleavage site occurs in one of two nucleosomal linker regions as depicted in Figure 4B. Figure 4.Specific suppression by MATH20 of topoisomerase II cleavage in satellite III of chromosome X. This figure demonstrates that MATH20 specifically inhibits topoisomerase II cleavage in satellite III, which is the predominant component of the heterochromatin of chromosome X. (A) Isolated Kc Drosophila nuclei were incubated in mitotic Xenopus egg extracts in the presence of different concentrations of MATH20 or HMG-I/Y. The topoisomerase II cleavage activity subsequently was monitored in satellite III by treating the extracts for 10 min with VM26. The DNA samples were displayed on a 1.2% agarose gel and the Southern blot hybridized with a satellite III repeat probe. VM26 and proteins were added as indicated at the top. The sample in lane 1 received no VM26 and no MATH20, and that in lane 2 received VM26 only. Samples in lanes 3–5 contained 80, 40 and 20 ng of MATH20, respectively. The sample in lane 6 contained 80 ng of HMG-I/Y. (B) The repeat structure of satellite III chromatin, which consists of two nucleosomes per 359 bp repeat unit. Topoisomerase II cleaves (arrow) once per unit, in every other nucleosomal linker region (Käs et al., 1993). Download figure Download PowerPoint Does MATH20 interfere with topoisomerase II cleavage in satellite III? We addressed this question using Xenopus egg extracts. These extracts are known to carry out faithfully many cellular processes. Indeed, we noted that the endogenous topoisomerase II of such extracts generated a cleavage ladder in satellite III repeats of Drosophila Kc nuclei that is indistinguishable from the one observed in cells (Figure 4A, lanes 1 and 2). Interestingly, this cleavage ladder is suppressed specifically in a dose-dependent manner by MATH20 (lanes 2–5). In contrast, no inhibition is observed by added HMG-I/Y (lane 6) which binds much more dispersively to the genome. In conclusion, MATH20 can interfere specifically with topoisomerase II cleavage in satellite III, strongly suggesting that the MATH20 protein encoded by the transgene expressed in flies is very likely to bind specifically the heterochromatin of chromosome X. Discussion The variegated phenotype of white mottled flies is the result of a large inversion in the X chromosome that places the white gene adjacent to centomeric heterochromatin. PEV is due to a stochastic inactivation of the white gene in some but not other cells at an early stage of eye development, followed by clonal maintenance through later stages. The resulting pattern of white gene expression is observed in the eye as patches of pigmentation. Morphologically, PEV is observed on polytene chromosomes as a spreading of heterochromatic structures into euchromatic genes (Elgin, 1996). Current evidence favors a model according to which silencing proteins of the centromeric heterochromatin spread into the juxtaposed euchromatic region by a cooperative assembly process. This spreading may be a consequence of the inversion by removing putative boundary elements that otherwise delimit heterochromatin (Locke et al., 1988). We have shown here that specific expression of the artificial high-affinity SAR-binding protein MATH20 in the developing eye imaginal discs results in suppression of white variegation. Using the tetracycline gene regulation system, we demonstrated that suppression of PEV requires the expression of MATH20 in the undifferentiated eye cells and is observed if this transgene is activated up to 60 h after egg laying. In contrast, no suppression is observed upon activation of MATH20 during the late third instar period. Interestingly, suppression of PEV by MATH20 was also observed for the BrownD variegating rearrangement (data not shown). We found no effect on whitevariegation by expression of MATH11; this protein with only 11 AT-hooks has a 7-fold lower binding affinity (KD = 18.2 pM) for SARs than MATH20 (2.6 pM). Consequently, proportionally higher amounts of protein were required to affect chromosome condensation in Xenopus extracts (Strick and Laemmli, 1995). Hence, it might be necessary to express MATH11 in the eye imaginal disc with a stronger promoter to achieve an effect on white variegation. The differential effect of MATH11 versus MATH20 underscores the notion that suppression of PEV is a specific phenomenon; it appears to be related to the binding strength of the effector. Adding MATH to a mitotic Xenopus extract led to the formation of abortive condensation products (Strick and Laemmli, 1995), and microinjection of MATH20 (but not HMG-I/Y) blocked HeLa cells in late G2-phase following passage through S–phase (R.Strick, R.Peperkok and U.K.Laemmli, in preparation). In agreement with this, we have observed that higher levels of embryonic and larval expression of either MATH20 or MATH11 led to lethality. Thus, suppression of PEV required tissue-specific expression of MATH20 at a low level that does not interfere with cell division. The inverted white gene is juxtaposed to a giant 11 Mb reiterated SAR in the form of satellite III repeats. MATH20 binds there with great specificity (Strick and Laemmli, 1995) and is able to interfere with the activity of topoisomerase II (Figure 4). This observation establishes proof of the principle that MATH20 can interact with or displace a pre-bound protein associated with heterochromatin. Although we cannot rule out other possible models, it is tempting to explain the effect of SAR on gene expression, chromatin opening and PEV by extending a model put forward by Laemmli and colleagues (Laemmli et al., 1992). In this model, certain proteins (called compacting proteins here) interact with SARs or certain AT-rich satellites cooperatively; this can lead to either chromatin folding, chromosome condensation (looping) or formation of heterochromatin. Conversely, other proteins such as HMG-I/Y and their monster derivatives, MATH, bind non-cooperatively to SARs and can displace the compacting proteins by disrupting their cooperative interactions, hence resulting in chromatin unpacking. These alternative chromatin states are governed by the binding strength and the relative local level of the chromatin packaging proteins versus those that undo it. Of importance for these considerations is the extent of SAR repetition at a given region. Since an assembly of cooperatively interacting proteins becomes energetically more favorable with increasing repeats, certain packaging proteins are expected to polymerize preferentially onto satellite III chromatin over individual SARs. In contrast, the non-cooperative MATH proteins would bind dispersively to single and reiterated SARs. Thus in a cell, while a single SAR in euchromatin may promote chromatin opening and stimulation of gene expression (Jenuwein et al., 1997), a reiterated SAR could result in silencing. It is easy to explain the effect of MATH20 on PEV by simple considerations based on the above model. The high affinity of MATH20 for SARs could allow it to bind to the satellite III region or other AT-rich regions, thus disrupting the cooperative interaction of the compacting proteins. This in turn would energetically disfavor the spreading of the polymerizing proteins into the flanking euchromatic region. As the probability of inactivating an adjacent euchromatic gene diminishes, one expects to observe suppression of PEV. It is impossible at present to obtain direct evidence for the model, and for a direct in vivo proof for MATH20 binding to satellite III. PEV appears to be a complex, poorly understood process, and the nature of the cis-DNA elements involved in heterochromatin formation has yet to be elucidated. The results reported here could also provide a clue about the nature of these cis-acting elements involved in PEV; the data suggest that this chromatin state might be mediated in part by proteins that interact with AT-rich repeats, such as those of satellite III. Materials and methods Fly strains The yw67c23 strain was used as recipient for injections. P-element-mediated germ line transformation was done using standard procedures (Spradling and Rubin, 1982a, b). For each construct, multiple independent lines were established, and the chromosomal location of the inserted transgene was determined by standard genetic analysis using balancer chromosomes. The whitemottled 4 inversion, In(1)wm4h strain, was used to score the effects of MATH proteins on white variegation. PEV-modifying effects were quantified by red eye pigment measurement (Ashburner, 1989), on groups of 40 male heads of the desired genotype, kept for 5 days at 25°C after eclosion. Flies were grown at 25°C on standard medium, unless specified otherwise in the text. Tetracycline (Sigma) was used at 0.2 μg/ml as described (Bello et al., 1998). Fly strains expressing tet-VP16 transactivator are described elsewhere (Bello et al., 1998). Plasmid constructs Further details of the cloning procedures are available upon request. MATH11 and MATH20 cDNAs (Strick and Laemmli, 1995) were inserted into pWTP (marked with miniwhite) (Bello et al., 1998) and/or pYTP (marked with yellow). pYTP was constructed by inserting a tet operator–P promoter–SV40 polyadenylation signal cassette from pWTP into pY vector, made by inserting the yellow gene from the YES vector (Patton et al., 1992) into Carnegie 4 (Rubin and Spradling, 1983). β-Galactosidase activity β-Gal stainings were done as described (Ashburner, 1989), on glutaraldehyde-fixed eye-antennal imaginal discs from early and late third instar larvae. Topoisomerase II cleavage assay Kc nuclei were isolated (Mirkovitch et al., 1984) and added to Xenopus egg extracts (Strick and Laemmli, 1995) to a final concentration of 10 000 nuclei/μl of extract, incubated at 21°C for 1 h and subsequently treated with 50 μM VM26 for 10 min. The reactions were stopped and isolated DNA samples were electrophoresed in a 1.2% agarose gel, electroblotted to nylon membranes and hybridized to satellite III repeat probes as described (Käs et al., 1993). Acknowledgements This paper is dedicated to Jean-Claude Cavadore, who passed away on March 19, 1997. We acknowledge T.Duvussel for technical assistance. This work was supported by grants from the Swiss National Fonds, the Kanton Basel-Stadt and Basel-Landschaft, the Human Frontier Science Program (to F.G), and the European Science Foundation (to B.B). References Ashburner M (1989) Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar Bello B, Resendez-Perez D and Gehring WJ (1998) Spatial and temporal targeting of gene expression in Drosophila by means of a tetracycline-dependent transactivator system. 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A structural study of the metaphase chromosome is a daunting activity with inadequate tools: The electron or optical microscopes are either too detailed or too coarse to elucidate the packaging mode of the chromatin fiber, and the biochemical approach falls easy prey to the criticism of destructive interference. To make matters worse, native chromosomes display few structural features, appearing as cylindrical structures quite homogeneously stuffed with a nucleoprotein fiber. It is necessary to try to elicit the structure from chromosomes; cytogeneticists have been doing this, albeit for a different purpose, using remarkably harsh methodologies.

We have analyzed the expression pattern of the D1 gene and the localization of its product, the AT hook-bearing nonhistone chromosomal protein D1, during Drosophila melanogaster development. D1 mRNAs and protein are maternally contributed, and the protein localizes to discrete foci on the chromosomes of early embryos. These foci correspond to 1.672- and 1.688-g/cm3 AT-rich satellite repeats found in the centromeric heterochromatin of the X and Y chromosomes and on chromosomes 3 and 4. D1 mRNA levels subsequently decrease throughout later development, followed by the accumulation of the D1 protein in adult gonads, where two distributions of D1 can be correlated to different states of gene activity. We show that the EP473 mutation, a P-element insertion upstream of D1 coding sequences, affects the expression of the D1 gene and results in an embryonic homozygous lethal phenotype correlated with the depletion of D1 protein during embryogenesis. Remarkably, decreased levels of D1 mRNA and protein in heterozygous flies lead to the suppression of position-effect variegation (PEV) of the white gene in the white-mottled (wm4h) X-chromosome inversion. Our results identify D1 as a DNA-binding protein of known sequence specificity implicated in PEV. D1 is the primary factor that binds the centromeric 1.688-g/cm3 satellite repeats which are likely involved in white-mottled variegation. We propose that the AT-hook D1 protein nucleates heterochromatin assembly by recruiting specialized transcriptional repressors and/or proteins involved in chromosome condensation.

Cytological silver-staining procedures reveal the presence of a "core" running along the chromatid axes of isolated HeLa mitotic chromosomes. In this communication we examine the relationship between this "core" and the nonhistone chromosome scaffolding, isolated and characterized in previous publications from this laboratory. When chromosomes on coverslips were subjected to the steps used for scaffold isolation in vitro and subsequently stained with silver, the characteristic "core" staining was unaffected. Control experiments suggested that the "core" does not contain large amounts of DNA. When scaffolds were isolated in vitro, centrifuged onto electron microscope grids, and stained with silver, they were found to stain selectively under conditions where specific "core" staining was observed in intact chromosomes. These results suggest that the nonhistone scaffolding is the principal target of the silver stain in chromosomes.

Article4 May 2006free access Displacement of D1, HP1 and topoisomerase II from satellite heterochromatin by a specific polyamide Roxane Blattes Roxane Blattes Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 CNRS-Université Paul Sabatier, Toulouse Cedex, France Search for more papers by this author Caroline Monod Caroline Monod Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 CNRS-Université Paul Sabatier, Toulouse Cedex, France Search for more papers by this author Guillaume Susbielle Guillaume Susbielle Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 CNRS-Université Paul Sabatier, Toulouse Cedex, France Search for more papers by this author Olivier Cuvier Olivier Cuvier Institut de Génétique Humaine, CNRS UPR 1142, Montpellier Cedex, France Search for more papers by this author Jian-hong Wu Jian-hong Wu Department of Biochemistry, Nanaline H Duke Building, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Tao-shih Hsieh Tao-shih Hsieh Department of Biochemistry, Nanaline H Duke Building, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Ulrich K Laemmli Ulrich K Laemmli Département de Biologie Moléculaire, Université de Genève, Sciences II, Geneva, Switzerland Search for more papers by this author Emmanuel Käs Corresponding Author Emmanuel Käs Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 CNRS-Université Paul Sabatier, Toulouse Cedex, France Search for more papers by this author Roxane Blattes Roxane Blattes Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 CNRS-Université Paul Sabatier, Toulouse Cedex, France Search for more papers by this author Caroline Monod Caroline Monod Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 CNRS-Université Paul Sabatier, Toulouse Cedex, France Search for more papers by this author Guillaume Susbielle Guillaume Susbielle Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 CNRS-Université Paul Sabatier, Toulouse Cedex, France Search for more papers by this author Olivier Cuvier Olivier Cuvier Institut de Génétique Humaine, CNRS UPR 1142, Montpellier Cedex, France Search for more papers by this author Jian-hong Wu Jian-hong Wu Department of Biochemistry, Nanaline H Duke Building, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Tao-shih Hsieh Tao-shih Hsieh Department of Biochemistry, Nanaline H Duke Building, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Ulrich K Laemmli Ulrich K Laemmli Département de Biologie Moléculaire, Université de Genève, Sciences II, Geneva, Switzerland Search for more papers by this author Emmanuel Käs Corresponding Author Emmanuel Käs Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 CNRS-Université Paul Sabatier, Toulouse Cedex, France Search for more papers by this author Author Information Roxane Blattes1,‡, Caroline Monod1,‡, Guillaume Susbielle1, Olivier Cuvier2, Jian-hong Wu3, Tao-shih Hsieh3, Ulrich K Laemmli4 and Emmanuel Käs 1 1Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 CNRS-Université Paul Sabatier, Toulouse Cedex, France 2Institut de Génétique Humaine, CNRS UPR 1142, Montpellier Cedex, France 3Department of Biochemistry, Nanaline H Duke Building, Duke University Medical Center, Durham, NC, USA 4Département de Biologie Moléculaire, Université de Genève, Sciences II, Geneva, Switzerland ‡These authors contributed equally to this work *Corresponding author. LBME, UMR5099, IBCG, 118 route de Narbonne, 31062 Toulouse Cedex 9, France. Tel.: +33 561 335959; Fax: +33 561 335886; E-mail: [email protected] The EMBO Journal (2006)25:2397-2408https://doi.org/10.1038/sj.emboj.7601125 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The functions of DNA satellites of centric heterochromatin are difficult to assess with classical molecular biology tools. Using a chemical approach, we demonstrate that synthetic polyamides that specifically target AT-rich satellite repeats of Drosophila melanogaster can be used to study the function of these sequences. The P9 polyamide, which binds the X-chromosome 1.688 g/cm3 satellite III (SAT III), displaces the D1 protein. This displacement in turn results in a selective loss of HP1 and topoisomerase II from SAT III, while these proteins remain bound to the adjacent rDNA repeats and to other regions not targeted by P9. Conversely, targeting of (AAGAG)n satellite V repeats by the P31 polyamide results in the displacement of HP1 from these sequences, indicating that HP1 interactions with chromatin are sensitive to DNA-binding ligands. P9 fed to larvae suppresses the position-effect variegation phenotype of white-mottled adult flies. We propose that this effect is due to displacement of the heterochromatin proteins D1, HP1 and topoisomerase II from SAT III, hence resulting in stochastic chromatin opening and desilencing of the nearby white gene. Introduction Polyamides are short polymers composed of aromatic pyrrole and imidazole amino acids that recognize specific DNA sequences with remarkable affinities (Dervan and Edelson, 2003). The use of polyamides provides an alternative approach to the study of DNA satellites (Janssen et al, 2000b), which, unlike regulatory sequences of genes, are difficult to study with classical molecular biology approaches. It was previously demonstrated that satellite-specific polyamides fed to developing Drosophila melanogaster can induce either gain or loss-of-function phenotypes in adult flies. One of these polyamides, P31, binds with great specificity the (AAGAG)n repeats of the Drosophila satellite V (SAT V) (Janssen et al, 2000b). P31 was found to induce well-defined homeotic transformations in a brown-dominant genetic background that are akin to mutations of the gene encoding GAGA factor (GAF) (Janssen et al, 2000a). The effect of P31 was proposed to be mediated by a redistribution of GAF from its euchromatin-binding sites, where this protein acts as a positive transcription activator to SAT V. The homeotic phenotype is then explained by a reduced availability of GAF for gene expression. In contrast, a gain-of-function phenotype was induced by the P9 polyamide. P9 specifically targets the AT-rich satellites SAT I (AATAT)n and, presumably, SAT III (density 1.688 g/cm3), which is composed of 359-base pairs (bp) repeats. It was observed that feeding P9 to white-mottled (wm4) larvae suppressed the position-effect variegation (PEV) phenotype in adult flies. The wm4 fly strain contains a chromosomal inversion that places the white gene near the centric heterochromatin of the X chromosome, which harbors the rDNA repeats and the enormous 11-Mbp SAT III array (Lohe et al, 1993). PEV is thought to arise through heterochromatin-mediated silencing of the juxtaposed white gene (for a review, see Huisinga et al, 2006). It was proposed that suppression of PEV by P9 (increased expression of the white gene) acts through chromatin opening of SAT III and a subsequent reduction of silencing effects on the nearby white gene. This paper aims to dissect the biological effect of P9 by examining the biochemical composition of SAT III using a combination of in vivo and in vitro experiments. D1 is an essential protein ubiquitously expressed throughout Drosophila development (Aulner et al, 2002) containing 10 copies of the AT hook motif, a DNA-binding domain that interacts with the minor groove of AT-rich DNA (Reeves and Nissen, 1990). D1 is associated with SAT I and SAT III repeats. Related to D1 is the artificial MATH20 protein that contains 20 AT hook motifs and, as D1, is expected to bind SAT I and SAT III. Overexpression of D1 enhances wm4 PEV (Aulner et al, 2002), while overexpression of MATH20 suppresses it (Girard et al, 1998). To explain these opposing effects, it was suggested that D1 increases, while MATH20 reduces, silencing effects mediated by SAT III (Monod et al, 2002). In contrast, overexpression of a D1 transgene carrying a deletion of its C-terminal domain (D1ΔE) suppresses wm4 PEV, an effect similar to that of MATH20. Because D1ΔE behaves as a dominant negative mutation of D1, we proposed that the C-terminal domain of D1 might serve to recruit proteins required for heterochromatin assembly to SAT I and SAT III. Heterochromatin protein 1 (HP1) is a well-characterized structural and functional component of heterochromatin. Binding of HP1 to heterochromatin occurs through the specific recognition of histone H3 methylated at the lysine 9 position by the chromo domain of HP1. Mutations in the gene encoding HP1 or in the genes that encode the histone deacetylase and methyl transferase activities required for the creation of an HP1-binding site suppress wm4 PEV (for a review, see Huisinga et al, 2006). The interplay between these proteins provides a rationale for the assembly and propagation of heterochromatin, which might involve other proteins as well. Topoisomerase II (topo II) might conceivably be implicated in wm4 PEV as it is prominently associated with centric heterochromatin and SAT III repeats, as demonstrated by in vivo cleavage (Käs and Laemmli, 1992) and localization studies (Swedlow et al, 1993). Topo II is a DNA topological handler that relaxes positively and negatively supercoiled DNA and mediates DNA strand passage during catenation/decatenation reactions. Previous studies established that topo II is required for the segregation and assembly of mitotic chromosomes (Adachi et al, 1991). Hence, this enzyme may perhaps play roles in heterochromatin and chromosome structure, in addition to its enzymatic function. One possibility is that D1 might help to recruit HP1 and topo II to AT-rich satellites and we explore here the mechanism whereby P9 exerts suppression of the wm4 phenotype. Using an immunofluorescence approach, we show that P9 mediates the displacement of D1 from SAT I and SAT III in vivo and in vitro. Moreover, P9 also specifically displaces HP1 and topo II from SAT III, while these proteins remain bound to other genomic sites that are not targeted by P9. Conversely, targeting SAT V repeats with the P31 polyamide results in the selective displacement of HP1 from these sites, supporting the hypothesis that HP1 association with chromatin involves a direct DNA-binding component. We propose that the suppression of the wm4 phenotype by P9 is mediated by displacement of D1, HP1 and topo II from SAT III. To refine our analysis, we studied the effect of topo II inhibitors on wm4 flies and we show that feeding VM26, a topo II poison, to wm4 flies also results in suppression of white-mottled PEV, supporting a structural and/or functional role for this enzyme in heterochromatin-mediated silencing. Results Specificity of DNA-binding polyamides for different Drosophila satellites Previous studies established that the satellite-specific polyamides, P9 and P31, fed to developing flies induce gain or loss-of-function phenotypes, respectively. The binding properties and the biological effects of these polyamides are recapitulated in Supplementary Table I and discussed below. P31: This compound is known to bind with impressive specificity the (AAGAG)n repeats of satellite V (SAT V) as determined by footprinting and fluorescence microscopy. P31TR, a derivative labeled with Texas red, was demonstrated to bind to the chromocenter of polytene chromosomes and to the 1.7-Mbp (AAGAG)n repeats inserted in the brown-dominant allele of the brown gene (Janssen et al, 2000b). This analysis was extended here to high resolution using mitotic chromosomes obtained from larval neuroblasts (Figure 1A). This panel localizes SAT V repeats to pericentric regions on chromosomes 2, 3 and 4 and to the very tip of the X chromosome. Figure 1A also includes a nucleus, where the signal is well confined to the SAT V repeats congregated in the chromocenter. Figure 1.Localization of satellites in diploid cells using fluorescent polyamides. (A–D) Brains dissected from female third-instar larvae were fixed and stained using fluorescent polyamides and counterstained with DAPI (gray). The properties of the drugs are described in Supplementary Table I. P31TR (A) detects satellite V, P9F (B, D) satellites III and I, Lex9F (inset in B) satellite I. SAT I, III and V maps are diagrammed in Figure 4G below. A sample double-stained with P9F and P31TR is shown in (C). The localization of satellite and rDNA repeats is shown for a magnified X chromosome double-stained with P9F and P31TR (D; c: centromere). Scale bars: 5 μm. Download figure Download PowerPoint P9: This oligopyrrole is specific for AT-rich satellites and, when fed to developing wm4 flies, suppresses the white-mottled PEV phenotype (Janssen et al, 2000a). The biological effect of P9 is most likely exerted through binding to the alpha-like satellite III (SAT III), the major satellite juxtaposed to the inverted white gene of wm4 flies. This notion was examined further here by microscopy of spread mitotic chromosomes stained with the fluorescein-labeled derivative P9F. Figure 1B shows that P9F strongly stains the large (11 Mbp) SAT III abutting the centromere of the X chromosome (see also the enlarged X chromosome in panel D). P9F also detects SAT I (AATAT)n repeats throughout chromosome 4 and related repeats on either side of the centromere of chromosome 3, as well as a short array of SAT I repeats near SAT V (red) on the X chromosomes (Figure 1D). The order of satellites from the tip of the X chromosome to the rDNA locus is SAT V, SAT I and SAT III, as shown in panel D. These assignments are based on staining with Lex9F (inset Figure 1B) and co-staining with P31TR and P9F (Figure 1C). Lex9F (see Supplementary Table I) is a dimeric oligopyrrole that prefers interaction with long (>W9) AT-rich runs. Hence, Lex9F serves to distinguish between the (AATAT)n repeats of SAT I and the shorter AT-runs of SAT III, which it does not bind (inset panel B). These results are in excellent agreement with previous satellite mapping data (Lohe et al, 1993) and are summarized in Figure 4G below. The main aim of this report is to elucidate the molecular basis of the gain-of-function phenotype mediated by P9. We proposed that P9 reduces the heterochromatin state of SAT III, presumably as a result of protein displacement. This proposal is explored here, using an experimental approach where polyamides serve two functions: (a) fluorescent derivatives are used to localize the DNA satellites in fixed material (Figure 1) in combination with immunostaining studies and (b) to act as ‘drugs’ in vivo and in vitro so as to affect the biochemical composition of heterochromatin. D1 binds SAT I and SAT III: P9-mediated displacement of D1 in vitro The D1 protein is associated with SAT I and SAT III repeats and overexpression of D1 enhances PEV of wm4 flies. Figure 2A shows that the immunosignal of D1 (red) colocalizes with the pattern of P9F staining (green, panel B and merged in panel C). Hence, D1 binds SAT I and SAT III repeats in interphase and metaphase chromatin. Figure 2.Uptake of oligopyrroles by whole embryos and larvae and displacement of D1. (A–C) Larval neuroblast nuclei and mitotic chromosomes stained with an antibody against D1 (red) and P9F (green) and counterstained with DAPI (gray). (D–F) Cellular blastoderm embryos were treated with 0, 10 or 50 μM P9, immunostained for D1 (red) and mounted in DAPI (gray). Treatment with 10 μM P9 results in a partial loss of the D1 signal, which is complete at 50 μM P9. (G–I) Third-instar larvae were fed colchicine (100 μg/ml) and P9 (0 or 100 μM) to assess displacement of D1 (red signal) from SAT I and SAT III repeats on mitotic chromosomes from eye imaginal disks. Arrowheads indicate the position of SAT III repeats, as determined by P9F staining (not shown). (J–M) The D1 signal in permeabilized larval brains treated with 0, 0.25, 1 or 2.5 μM P9, respectively. Merged DAPI (gray) and D1 (red) signals are shown. Arrowheads show the DAPI-bright SAT III array on the X chromosome. Scale bars: 5 μm. Download figure Download PowerPoint We explored whether the biological effect of P9 on PEV might arise by displacement of D1. This notion was first studied in vitro with gel retardation/competition experiments. Figure 3A shows that binding of purified D1 to a 359-bp SAT III monomer generates a ladder of retarded complexes reflecting increased binding of D1 (Figure 3A, lanes 1–4). In contrast to D1, addition of P9 (MW=962) resulted in faster migrating complexes (lanes 5–9), due to unbending of the curved SAT III fragment, although retardation was seen at the highest P9 concentration tested (500 nM, lane 10). Figure 3.The P9 oligopyrrole competes with D1 for binding to SAT III repeats in vitro. (A) A SAT III monomer was incubated with 2, 5 or 10 ng D1 (2.7, 6.75 and 13.5 nM, lanes 2–4) or P9 (25, 50, 100, 250 or 500 nM, lanes 6–10). No-protein controls are shown in lanes 1 and 5. (B) SAT III was incubated with 1, 5 or 10 ng D1 (1.35, 6.75 or 13.5 nM) in the presence of 0, 100, 250 or 500 nM P9 as shown above the gel. Lane 1 contained no protein. The displacement of D1 by the drug is almost complete at 500 nM P9 (lanes 11–13). (C) SAT III monomer DNA 3′-end labeled on the lower strand was incubated with 0 (lanes 1 and 6) or 50, 100, 250, 500 nM P9 (lanes 2–5) and digested with DNAse I. Solid bars denote regions protected from digestion at the lowest P9 concentrations used and correspond to the largest dA·dT tracts (W) of SAT III (W6/W7–W14, where the number denotes the number of dA·dT bp). Note that only W tracts that are clearly resolved on the gels shown are indicated. The arrowhead indicates a P9-induced hypersensitive site. Binding of the same concentrations of P9 to SAT III (D) was compared to that of D1 and MATH20 (E). Lanes 2–4 of panel (E) correspond to 2, 5 and 10 ng D1 (2.7, 6.75 and 13.5 nM), while lanes 5–7 contained 2.5, 1.5 and 0.5 ng MATH20 (1.5, 0.9 and 0.3 nM). Lanes 1 and 8 are no-protein controls. The solid line starting at the bottom of (D) indicates the SAT III region shown on these gels relative to (C). These results are summarized on the sequence of a SAT III monomer shown in (F). Filled boxes indicate the regions protected by binding of D1, MATH20 and P9. Scissors indicate the sequence cleaved by topoisomerase II in vivo (Borgnetto et al, 1996), the filled box corresponds to the four consecutive dC·dG bp located within the staggered cut. This region spans the P9-induced DNAse I-hypersensitive site and is not bound by D1 or MATH20. Download figure Download PowerPoint Figure 4.D1 Displacement results in a selective loss of HP1. (A) Localization of HP1 (red) in interphase nuclei and mitotic chromosomes of female diploid larval neuroblasts. Samples were counterstained with Lex9F (green) and DAPI (gray). The inset shows a similarly stained interphase nucleus. A region that stains strongly for HP1 alone corresponds to SAT V repeats on chromosome 2, as assessed by P31TR staining (not shown). rDNA repeats are indicated. (B, C) Magnified X chromosomes after staining for HP1 or D1 (red), respectively, and counterstaining with Lex9F (green) and DAPI (gray). (D–E) Localization of HP1 (red) after treatment with 2.5 μM P9. Magnified X chromosomes are shown in (E). Samples were counterstained with Lex9F (green) and DAPI (gray). In (D), arrowheads indicate the HP1-depleted DAPI-bright region that corresponds to SAT III repeats in interphase nuclei after treatment with P9; SAT V repeats on chromosome 2 are also indicated. (F) The localization of HP1 following treatment with P31. The inset shows untreated chromosomes 2 and a nucleus with the SAT V-associated HP1 signal (arrowhead). A general representation of major satellite blocks is schematized in (G), which also shows the approximate location of the white gene on a wm4 X chromosome, within a type I insertion sequence characteristic of some rDNA repeats. ‘C’ denotes the centromere. Scale bars: 5 μm. Download figure Download PowerPoint Figure 3B establishes that P9 dissociates D1/SAT III complexes in vitro. Examination of panel B shows that the D1/SAT III ladder diminishes in a concentration-dependent manner upon addition of P9 (compare lanes 5–7, 8–10 and 11–13 with lanes 2–4). This is accompanied by an increase in the mobility of the free DNA and protein-bound fragments indicative of P9 binding. This displacement reaction was independent of the order in which P9 and D1 were added (data not shown). Taken together, these results demonstrate that the P9 oligopyrrole binds SAT III repeats in vitro and displaces the D1 protein bound to these sequences. DNAse I footprinting experiments demonstrate that the competition between P9 and D1 is the result of nearly identical DNA-binding properties. Figure 3C (lanes 2–5) shows that P9 primarily protects stretches of dA·dT base pairs (W7–W14) of the SAT III repeat from DNAse I digestion. These tracts are also the main interaction sites for D1 (Figure 3D and E). Although both ligands bind similar sequences, some minor differences can be noted, as is manifested by the greater affinity of P9 for longer dA·dT tracts. A similar experiment performed with the artificial MATH20 protein yielded a footprint similar to that of P9 or D1 (lanes 5–7 of panel E). These interaction results are graphically summarized in Figure 3F. In vivo displacement by P9 of D1 from SAT I and III in embryos We tested whether P9 displaces D1 from SAT III in vivo by exposing whole embryos at the cellular blastoderm stage to this compound. Panels D–F of Figure 2 show that P9 induces a dose-dependent decrease of the D1 immunosignal in the chromocenter of nuclei. In untreated embryos, D1 (red) was found to localize predominantly to two foci in each nucleus (panel D) that correspond to SAT I and SAT III as verified by P9F binding (not shown). Exposure of embryos to 10 μM P9 (panel E), however, led to a loss of D1 from some but not all foci, typically resulting in the detection of only one D1-positive region in each nucleus. This displacement of D1 was essentially complete at the highest drug concentration tested (50 μM, panel F). These results extend our in vitro competition experiments and demonstrate that P9 displaces D1 from AT-rich satellite repeats in vivo. D1 displacement from chromosomes of eye disks in vivo The displacement of D1 was studied in more detail on mitotic chromosomes from eye imaginal disks isolated from P9-fed larvae. As shown above for neuroblast chromosomes (Figure 2A), the immunosignal of D1 is also predominantly confined to SAT III and SAT I in eye disk chromosomes isolated from control larvae (no P9, Figure 2G). In contrast, panels H and I show that D1 is displaced if larvae were fed yeast paste containing 100 μM P9. While displacement by P9 of D1 from SAT III was complete (panel H), we observed a stochastic loss of D1 from SAT I in about 20% of the examined mitotic spreads. Panel I shows such an example, where D1 was completely displaced from both SAT I and III repeats. This experiment shows that P9 displaces D1 from chromosomes isolated from eye disks, where the suppression of wm4 PEV is exerted. The above experiments were carried out with large amounts of P9. As our polyamide supply is very limited, we used permeabilized larval brains in the following experiments, where lower concentrations can be used. Squashes of larval brains also have the advantage that they yield mitotic chromosome spreads of excellent quality (Figure 2A–C). As shown in panels J–M of Figure 2, the displacement of D1 in permeabilized brains occurs at a much lower P9 concentration. Notably, we observed that a P9 concentration of 250 nM suffices to displace D1 from SAT III (panel K, red arrowheads), while higher concentrations (1–2.5 μM) are required to evict D1 from SAT I (panels L and M). P9 displaces HP1 from SAT III but not from rDNA repeats The possible implication of HP1 in the suppression of wm4 PEV by P9 was also studied. Figure 4A shows that HP1 (red) is bound to the pericentric regions of mitotic chromosomes, with a predominant association with SAT III and SAT V repeats of chromosomes X and 2, respectively. In contrast, the HP1 immunosignal is undetectable on the SAT I repeats on chromosomes 4 and Y (not shown) and at the tip of the X chromosome. It is important to note that Lex9F was used to counterstain these panels of Figure 4, as it serves to highlight SAT I (not SAT III) repeats that remain green since their HP1 immunosignal remains undetectable (panel A). This conclusion is also supported by an examination of the staining pattern of interphase nuclei. The inset of panel A shows the intense HP1 staining of SAT V and partial staining of the SAT III chromocenter (yellow), while HP1 is excluded from a region (green) that also harbors SAT I repeats. Careful inspection of the HP1 immunosignal over the X chromosome at higher magnification (panel B) shows that its signal extends from the rDNA repeats to the adjacent SAT III array, where staining reproducibly appears in the form of a gradient that diminishes toward the centromere. In contrast to the binding of HP1 to the SAT III and rDNA repeats, the immunosignal of D1 shown for comparison is restricted to the centromere-proximal region of SAT III and only partially overlaps with HP1 (panel C). Panels D and E of Figure 4 show that P9 selectively displaces HP1 from SAT III repeats, but not from the rDNA array. This conclusion is best noted on the enlarged X chromosomes shown in panel E. This micrograph shows the green Lex9F stain of SAT I at the chromosomal tip and the red immunosignal of HP1 over the rDNA repeats, while the SAT III region remains unstained (gray). Hence, following treatment with P9, HP1 is delocalized from the SAT III repeats, but remains associated with the rDNA array. Examination of panel D demonstrates that, in contrast, the protein remains prominently associated with SAT V repeats on chromosome 2. The nuclei shown in panel D also confirm this conclusion. In this case, the loss of HP1 from SAT III is reflected by the appearance of a green–gray–red pattern over the chromocenter, corresponding to the SAT I, SAT III and rDNA repeats, respectively, while the strong HP1 association site on SAT V repeats is unaffected. We tested next the sensitivity of HP1 binding to chromatin to treatment with the P31 polyamide. As shown in the inset of Figure 4F, HP1 is prominently associated with SAT V repeats on chromosome 2. Incubation with P31 led to a selective displacement of HP1 from the pericentric region of chromosome 2, without affecting the association of the protein with SAT III and rDNA repeats on the X chromosome. We conclude from these results that HP1 can be targeted by polyamides in a sequence-specific fashion that reflects the selectivity of P9 and P31 for SAT III and SAT V, respectively. This suggests that the association of HP1 with satellites repeats may occur, at least in part, via DNA sequence recognition (Zhao et al, 2000; Perrini et al, 2004). HP1 delocalization results in the invasion of rDNA repeats by D1 The displacement of HP1 from SAT III repeats might be due to direct competition with P9, predicting that D1 and HP1 may compete for binding to SAT III. Displacement of HP1 from heterochromatin should then result in additional D1 binding to sites normally occupied by HP1. We tested this possibility by feeding a histone deacetylase inhibitor to growing third-instar larvae. Treatments with trichostatin A (TSA) have been shown to result in the delocalization of HP1 from heterochromatin (Taddei et al, 2001). Figure 5A shows the HP1 and D1 localization patterns normally observed in larval neuroblast nuclei. HP1 (red) partially associates with SAT III repeats stained with P9F (green) as well as with SAT V repeats and is largely absent from the nucleoplasm. The localization of D1 is strictly correlated with P9F-positive foci, which correspond to SAT I and SAT III repeats. TSA feeding resulted in a loss of HP1 from heterochromatin and a relocalization of the protein to the nucleoplasm (Figure 5B). Significantly, this displacement was accompanied by an extension of the D1 signal: instead of colocalizing with P9F-positive foci, yielding a characteristic yellow signal (Figure 5A), the D1 antibody also stained an immediately adjacent region, yielding both red and yellow signals. The red-only domain occupied by D1 after TSA treatment corresponds to the rDNA repeats normally associated with HP1 (data not shown and see below). Figure 5.SAT III and rDNA repeats define alternative D1 and HP1 domains. Neuroblasts from control (A) or TSA-fed (B) third-instar larvae were immunostained for HP1 or D1 (red) and counterstained with P9F (green). Individual and merged signals are as indicated in the photographs. TSA feeding induces a delocalization of HP1 to the nucleoplasm and an extension of the D1 signal from SAT III to an adjacent P9F-negative region that corresponds to the rDNA repeats. (C) The results of FISH experiments performed with white and Su(f) probes (red) or a full-length rDNA probe (green). Photographs at the top and bottom show results obtained from wild-type Oregon R or wm4 neuroblasts, respectively. Arrowheads indicate the white signal detected in interphase nuclei. Scale bars: 5 μm. (D) The juxtaposition of the rDNA-linked white gene to SAT III repeats as a result of looping out of rDNA sequences in the nucleolus. Download figure Download PowerPoint SAT III protein composition affects the white gene across the rDNA repeats The delocalization of D1 and HP1 from

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCell cycle changes in Physarum polycephalum histone H1 phosphate: relationship to deoxyribonucleic acid binding and chromosome condensationStuart G. Fischer and Ulrich K. LaemmliCite this: Biochemistry 1980, 19, 10, 2240–2246Publication Date (Print):May 13, 1980Publication History Published online1 May 2002Published inissue 13 May 1980https://doi.org/10.1021/bi00551a038Request reuse permissionsArticle Views31Altmetric-Citations37LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (4 MB) Get e-Alertsclose Get e-Alerts

We have found that two different temperature-sensitive mutations in gene 22, tsA74 and ts22-2, produce high frequencies (up to 85%) of petite phage particles when grown at a permissive or intermediate temperature. Moreover, the ratio of petite to normal particles in a lysate depends upon the temperature at which the phage are grown. These petite phage particles appear to have approximately isometric heads when viewed in the electron microscope, and can be distinguished from normal particles by their sedimentation coefficient and by their buoyant density in CsCl. They are biologically active as detected by their ability to complement a co-infecting amber helper phage. Lysates of both mutants grown at a permissive temperature reveal not only a significant number of petite phage particles in the electron microscope, but also sizeable classes of wider-than-normal particles, particles having abnormally attached tails, and others having more than one tail. Striking protein differences exist between the purified phage particles of tsA74 or ts22-2 and wild-type T4. B1∗, a 61,000 molecular weight head protein, is completely absent from the phage particles of both mutants, and the internal protein IPIII∗ is present in reduced amounts as compared to wild type. The precursor to B1∗ is present in the lysates, but these mutations appear to prevent its incorporation into heads, so it does not become cleaved. The product of gene 22 (P22) is known to be the major protein of the morphogenetic core of the T4 head. Besides the mutations reported here, several mutations which affect head length have been found in gene 23, which codes for the major capsid protein (Doermann et al., 1973b). We suggest a model in which head length is determined by an interaction between the core (P22 and IPIII) and the outer shell (P23).

The morphogenetic core of bacteriophage T4 is a transient structure which exists inside an early head precursor, but which is later proteolytically destroyed at the time of DNA packaging. This structure is of interest because genetic evidence shows that P22 (the major core protein and product of gene 22) is involved in determining the length and shape of the head and in selecting the position of tail attachment. P22 may also be involved in initiation of head assembly and in DNA packaging. We have isolated T4 polyheads with intact cores for study by negative staining in the electron microscope. Polyheads were used because their elongated structure makes them suitable for optical diffraction and computer analysis of electron microscope images. All polyheads (except those from cells defective in gene 22) were found to have highly ordered cylindrical cores consisting of several helical chains wound about a hollow center. Cores of l-canavanine-induced polyheads are about 460 Å in diameter and appear to consist of six helical chains spaced about 115 Å apart axially, and with a pitch angle of about 30°. These polyheads probably have the same core structure as an elongated version of a true head precursor, since giant τ-particles were found to have the same structure. Moreover, canavanine polyheads have the surface structural parameters expected of an elongated prohead. They possess hemispherical caps and the core extends into the end of the particle and has a cap of its own. Since the T4 head surface is thought to have a 5-fold axis of rotational symmetry, our data indicate a symmetry mismatch between core and surface. We suggest that head assembly may be initiated with the formation of a connector between core and surface, which later becomes the point of tail attachment. Polyheads induced by mutations in genes 20, 24 or 40 have cores about 350 Å in diameter consisting of five or six helical chains spaced 131 Å apart axially and wound with a pitch angle of about 40°. Polyheads lacking the internal proteins have a similar, but less stable, core while polyheads induced by an amber mutation in gene 22 apparently have no core at all. Since head length is determined by an interaction between the core (P22) and the surface (P23), we examined polyheads containing proteins altered by mutations which affect head length. In all cases where petite phage particles arise, we observed either weakened core-surface interactions, unstable cores, or an altered intrinsic curvature of the surface subunit P23.

An estimate was made of the amount of DNA packaged into gene 49-defective heads when P49 is activated by a temperature shift. The uptake of DNA into preformed heads following activation of P49 was studied using bromo-deoxyuridine as a label. The rate of inactivation by visible light of the phage matured in the presence of BrdU as well as their buoyant density in CsCl, indicate that over half of the particles package, on the average, at least 25% of the DNA complement following P49 activation. This is a minimum estimate, since the BrdU-labeled DNA has to compete with unlabeled DNA. Analysis on alkaline sucrose gradients of the size of the DNA extracted from phage matured in the presence of BrdU following irradiation reveals that extended irradiation at 313 nm breaks the DNA close to half of its original size. These experiments clearly show that up to half of the DNA can be packaged into the preformed heads made at high temperature following activation of the product of gene 49 (P49), strongly supporting the pathway for phage head maturation described by Laemmli & Favre (1973). The so-called τ-particles, which accumulate in 24-defective cells, can serve as precursors of the mature phage (Bijlenga et al., 1973). We have measured the uptake of BrdU-labeled DNA into τ-particles during their maturation. We find that a very large proportion of DNA made after activation of P24 is apparently incorporated into preformed τ-particles as these particles are converted into mature heads. This indicates that the τ-particles contain very little or no DNA prior to P24 activation and supports the pathway described by Laemmli & Favre (1973).

The insulating properties required to delimit higher-order chromosomal domains have been shown to be shared by a variety of chromatin boundary elements (BEs). Boundary elements have been described in several species, from yeast to human, and we have previously reported the existence of a class of chromatin BEs in Drosophila melanogaster whose insulating activity requires the DNA-binding protein BEAF (boundary element-associated factor). Here we focus on the characterization of a moderately repeated 1.2 kb DNA sequence that encompasses boundary element 28 (BE28). We show that it directionally blocks enhancer/promoter communication in transgenic flies. This sequence contains a BEAF-binding sequence juxtaposed to an AT-rich sequence that harbors a strong nuclease-hypersensitive site. Using a combination of DNA-protein and protein blotting techniques, we found that this region is recognized by the A+T-binding D1 non-histone chromosomal protein of D. melanogaster, and we provide evidence that D1 and BEAF physically interact. In addition, the multicopy BE28 element maps to pericentric regions of the D. melanogaster 2L, 2R and X chromosome arms to which D1 has been shown to localize. In yeast, BEs that mark the periphery of silenced chromosomal domains have recently been shown to block the spreading of heterochromatin assembly. We propose that the BE28 repeat clusters could fulfill a similar function, acting as a local boundary between hetero- and euchromatin in a process involving interactions between the BEAF and D1 proteins.

To map the genomic interaction sites of chromatin proteins, two related methods were developed and experimentally explored in Saccharomyces cerevisiae. The ChIC method (chromatin immunocleavage) consists of tethering a fusion protein (pA-MN) consisting of micrococcal nuclease (MN) and staphylococcal protein A to specifically bound antibodies. The nuclease is kept inactive during the tethering process (no Ca2+). The ChEC method (chromatin endogenous cleavage) consists of expressing fusion proteins in vivo, where MN is C-terminally fused to the proteins of interest. The specifically tethered nucleases are activated with Ca2+ ions to locally introduce double-stranded DNA breaks. We demonstrate that ChIC and ChEC map proteins with a 100–200 bp resolution and excellent specificity. One version of the method is applicable to formaldehyde-fixed nuclei, another to native cells with comparable results. Among various model experiments, these methods were used to address the conformation of yeast telomeres.

Using monoclonal antibodies as probes, we have characterized three antigens with respect to localization in the nucleolus, molecular weight and solubility. Two proteins, of 110,000 and 94,000 apparent molecular weight, were found associated with the ribonucleoprotein fibers. A third protein, with a molecular weight of 40,000, was accumulated at the nucleolar periphery, was present in the nucleoplasm, and may be involved in pre-ribosome maturation and transport.

A procedure is described for immunizing in vitro and stimulating proliferation of specific B-cell lymphocytes. The method is applicable to production of monoclonal antibodies against proteins that are soluble only in denaturing solvents. An induction period is described in which antigen is presented to the B-cell population in the absence of serum. Also, antigen is coupled to mitogenic silica, which allows the effective presentation of both soluble and insoluble antigens. The results indicate hybridomas can be obtained that secrete IgMs directed against highly conserved or weakly immunogenic antigens.

During our search for transcriptional regulators that control the developmentally regulated expression of the enkephalin (ENK) gene, we identified AUF1. ENK, a peptide neurotransmitter, displays precise cell-specific expression in the adult brain. AUF1 (also known as heterogeneous nuclear ribonucleoprotein D) has been known to regulate gene expression through altering the stability of AU-rich mRNAs. We show here that in the developing brain AUF1 proteins are expressed in a spatiotemporally defined manner, and p37 and p40/42 isoforms bind to an AT-rich double-stranded (ds) DNA element of the rat ENK (rENK) gene. This AT-rich dsDNA sequence acts as a cis-regulatory DNA element and is involved in regulating the cell-specific expression of the ENK gene in primary neuronal cultures. The AT-rich dsDNA elements are present at ∼2.5 kb 5′upstream of the rat, human, and mouse ENK genes. AUF1 proteins are shown here to provide direct interaction between these upstream AT-rich DNA sequences and the TATA region of the rENK gene. Double immunohistochemistry demonstrated that in the developing brain AUF1 proteins are expressed by proliferating neural progenitors and by differentiating neurons populating brain regions, which will not express the ENK gene in the adult, suggesting a repressor role for AUF1 proteins during enkephalinergic differentiation. Their subnuclear distribution and interactions with AT-rich DNA suggest that in the developing brain they can be involved in complex nuclear regulatory mechanisms controlling the development- and cell-specific expression of the ENK gene.

The human immunodeficiency virus type 1 (HIV) Rev protein is thought to be involved in the export of unspliced or singly spliced viral mRNAs from the nucleus to the cytoplasm. This function is mediated by a sequence-specific interaction with a cis-acting RNA element, the Rev response element (RRE), present in these intron-containing RNAs. To identify possible host proteins involved in Rev function, we fractionated nuclear cell extracts with a Rev affinity column. A single, tightly associated Rev-binding protein was identified; this protein is the mammalian nucleolar protein B23. The interaction between HIV Rev and B23 is very specific, as it was observed in complex cell extracts. The complex is also very stable toward dissociation by high salt concentrations. Despite the stability of the Rev-B23 protein complex, the addition of RRE, but not control RNA, led to the displacement of B23 and the formation of a specific Rev-RRE complex. The mammalian nucleolar protein B23 or its amphibian counterpart No38 is believed to function as a shuttle receptor for the nuclear import of ribosomal proteins. B23 may also serve as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus to allow further rounds of export of RRE-containing viral RNAs.

9-Aminoacridine (9AA) reversibly inhibits bacteriophage T4 maturation. In the presence of 9AA, cells infected with bacteriophage T4 accumulate an intermediate similar to prohead III on the maturation pathway described by Laenmmli and Favre (J. Mol. Biol., 80, 575–599, 1973). This particle sediments at 550 S if cells are lysed under conditions that help to preserve fragile intermediates. This particle contains about half the normal complement of DBA and is bound to the replicative DNA. The completion of the DNA packaging process occurs following removal of 9AA. This was demonstrated by estimating the amount of newly synthesized DNA packaged into these particles after 9AA removal, using 5-bromo-2′-deoxyuridine as a label. Light inactivation studies showed that 75% of the particles packaged an average of at least 12% of their DNA after 9AA removal. Cleavage of the various head proteins that normally occur during maturation is not affected by 9AA, and the particles which accumulate in cells blocked with 9AA contain the same proteins found in mature heads.

The presentation focuses on the structural rearrangements of the subunits and the processing of the various protein constituents which accompany the maturation events of the head of bacteriophage T 4. The major features of the maturation steps of the head are the following: (a) the viral DNA is pulled into an empty head in a series of events; (b) cleavage of two core proteins, P22 (mol. mass = 31000), to small fragments and the internal protein IP III (mol. mass = 23000) to IP III* (mol. mass = 21000) appears to be intimately linked to the DNA packaging event, whereas the cleavage of the major head protein of the viral coat, P23 (mol. mass = 55000), to P23* (mol. mass = 45000) precedes the DNA packaging event. Recently, we have obtained information about the mechanism by which the viral DNA is pulled into a preformed empty head. Our evidence suggests that the DNA becomes attached to the inside of the empty head and is subsequently collapsed in the interior by the so-called internal peptides. These are highly acidic and derived from a large precursor protein by cleavage.

Conference Abstract| April 01 1981 CHROMOSOME STRUCTURAL STUDIES Ulrich K. Laemmli; Ulrich K. Laemmli 1University of Geneva, 1211 Geneva 4, Switzerland Search for other works by this author on: This Site PubMed Google Scholar Catherine D. Lewis; Catherine D. Lewis 1University of Geneva, 1211 Geneva 4, Switzerland Search for other works by this author on: This Site PubMed Google Scholar Jane S. Lebkowski Jane S. Lebkowski 1University of Geneva, 1211 Geneva 4, Switzerland Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1981) 9 (2): 6P. https://doi.org/10.1042/bst009006pa Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Ulrich K. Laemmli, Catherine D. Lewis, Jane S. Lebkowski; CHROMOSOME STRUCTURAL STUDIES. Biochem Soc Trans 1 April 1981; 9 (2): 6P. doi: https://doi.org/10.1042/bst009006pa Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1981 Biochemical Society1981 Article PDF first page preview Close Modal You do not currently have access to this content.

The Biochemistry Department at the University of Geneva currently has four full professors, a professor emeritus, one assistant professor, two MER (Maître d'enseignement et de Recherche) and a permanent scientific collaborator. The research interests of the members of the Biochemistry Department are described.

ABSTRACT. Recent evidence suggests that metallo-protein interactions are important in maintaining one level of organization of the DNA in histone-depleted chromosomes and nuclei. When treated with the metal chelators, 1,10-phenanthroline, neocuproine, or thiols, but not with EDTA, histone-depleted chromosomes and nuclei unfold their compacted DNA as evidenced by a reduction in their sedimentation coefficient. For nuclei, the s value is reduced by such chelation from a fast form I (18000s) to a slow form II (8500s). With chromosomes, a similar shift from a fast form I (5500s) to a slow form II (1500s) is observed. It is unlikely that this relaxation of the DNA is due to the possible degradative processes of 1,10-phenanthroline known to occur under some conditions.

Summary alternative, complementary procedures that omit the chromatin fractionation-solubilization step. The methTo map the genomic interaction sites of chromatin ods introduced here consist of tethering micrococcal proteins, two related methods were developed and nuclease in an inactive state (no Ca 2 ions) directly experimentally explored in Saccharomyces cerevisiae. (in vivo) or indirectly (via antibodies) to proteins of interThe ChIC method (chromatin immunocleavage) con- est. The specifically bound nuclease is then activated sists of tethering a fusion protein (pA-MN) consisting by Ca 2 ions to locally introduce double-stranded DNA of micrococcal nuclease (MN) and staphylococcal pro- breaks. Mapping of such breaks by molecular techtein A to specifically bound antibodies. The nuclease niques is shown to map proteins with a 100–200 bp is kept inactive during the tethering process (no Ca 2 ). resolution and excellent specificity. One version of the The ChEC method (chromatin endogenous cleavage) method is applicable to formaldehyde-fixed cells, anconsists of expressing fusion proteins in vivo, where other to native cells with comparable results. MN is C-terminally fused to the proteins of interest. The specifically tethered nucleases are activated with Results Ca 2 ions to locally introduce double-stranded DNA