Fang-Lin Sun

Failing to repair DNA double-strand breaks by either nonhomologous end joining (NHEJ) or homologous recombination (HR) poses a threat to genome integrity, and may have roles in the onset of aging and age-related diseases. Recent work indicates an age-related decrease of NHEJ efficiency in mouse models, but whether NHEJ and HR change with age in humans and the underlying mechanisms of such a change remain uncharacterized. Here, using 50 eyelid fibroblast cell lines isolated from healthy donors at the age of 16-75 years, we demonstrate that the efficiency and fidelity of NHEJ, and the efficiency of HR decline with age, leading to increased IR sensitivity in cells isolated from old donors. Mechanistic analysis suggests that decreased expression of XRCC4, Lig4 and Lig3 drives the observed, age-associated decline of NHEJ efficiency and fidelity. Restoration of XRCC4 and Lig4 significantly promotes the fidelity and efficiency of NHEJ in aged fibroblasts. In contrast, essential HR-related factors, such as Rad51, do not change in expression level with age, but Rad51 exhibits a slow kinetics of recruitment to DNA damage sites in aged fibroblasts. Further rescue experiments indicate that restoration of XRCC4 and Lig4 may suppress the onset of stress-induced premature cellular senescence, suggesting that improving NHEJ efficiency and fidelity by targeting the NHEJ pathway holds great potential to delay aging and mitigate aging-related pathologies.

The eukaryotic genome is enormously complex. Experimental observations indicate that in addition to the subdivision of the genome into distinct chromosomes, each chromosome is partitioned into distinct structural and functional domains. This implies the presence of boundaries, DNA elements that mark the border between adjacent domains, allowing them to maintain different functional states. Several different assays have been used to identify and describe boundaries. Elements have been detected that can block enhancer function; these are commonly referred to as "insulators." Other elements have been described within the Bithorax complex (BX-C) of Drosophila melanogaster that delimit adjacent cis-regulatory domains. Recently, boundary elements have been identified that appear to limit the spread of heterochromatin. The identification and characterization of these latter elements, which we will refer to as "barriers," will be the major focus of this minireview. While some elements have both insulator and barrier activity, there are suggestions that these functions may be separable in other cases. Further analysis will be needed to discern whether or not a common mechanism can be established for the different boundary functions. The eukaryotic chromosome is a mosaic of silenced and active domains. The largest blocks of silent chromatin are the heterochromatic masses associated with centromeric regions and telomeres. These regions often remain visibly condensed throughout the cell cycle; have a high content of repetitive sequences; are "gene poor" (although not devoid of genes); and are replicated late in S phase. In addition, there are many instances in which a smaller region of the genome has apparently been silenced by packaging into a heterochromatin-like structure, often in a response to developmental signals. Such "heterochromatin-like domains" impart stable epigenetic silencing (inherited following mitosis) to genes within the domain, independent of the type of promoter. Examples include the silencing imparted by the E and I elements at the HMR and HML loci in yeast (Saccharomyces cerevisiae), and that directed by the polycomb response element (PRE) in Drosophila. Heterochromatin and heterochromatin-like domains appear to share a number of structural characteristics, observed as a general loss of accessibility to nucleases, and a shift to hypoacetylation of the core histones. Genomic alterations provide evidence that the chromatin structure imparting silencing can "spread" to adjacent genes. In Drosophila, a rearrangement with one breakpoint in a heterochromatic domain that places a normally euchromatic gene adjacent to the heterochromatin results in position–effect variegation (PEV), a silencing of that gene in a subset of the cells in which it is normally expressed (reviewed by 6Elgin S.C.R Curr. Opin. Genet. Dev. 1996; 6: 193-202Crossref PubMed Scopus (190) Google Scholar). In yeast, the complex formed by association of the silent information regulator (SIR) proteins with DNA-binding protein RAP1 and core histones can spread from the telomere at a terminal truncation into adjacent regions (reviewed by 9Grunstein M Cell. 1998; 93: 325-328Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). One can infer that discrete boundaries are needed to maintain the mosaic pattern of silent and active domains found under normal conditions; indeed, such "barriers" to the spread of heterochromatin and heterochromatin-like structure have recently been identified (see Table 1) in yeast and in chicken.Table 1Boundary ElementsElementLocusOrganismA: Enhancer BlockingB: Protection of a TransgeneC: Deletion AssayD: Insulate HeterochromatinAssociated ProteinBarriers between heterochromatin and euchromatin:HMR-RHMR locusYeastNDND++SMC,** SMC3**HMR-LHMR locusYeastNDNDND+NDUASrpgTEF2 geneYeastNDNDND+Rap1STARsTelomeresYeastNDNDND+Tbf1p, Reb1pBoundaries within BX-C regulatory region:Fab-7Abdominal-B locusDrosophila+ND+NDNDFab-8Abdominal-B locusDrosophila+NDNDNDNDMcpAbdominal-B locusDrosophilaNDND+NDNDInsulators of enhancer activity:scshsp70 genesDrosophila++ND−Zw5scs′hsp70 genesDrosophila++ND−BEAF-32gypsyTransposable elementDrosophila++ND+SU(HW), Mod(mdg4)eve promotereven-skipped geneDrosophila+NDNDNDGAGAβ-globin HS4β-globin locusChicken++NDNDCTCFRepeat organizerrRNA geneXenopus+NDNDNDxUBF, CTCF*snsEarly histone genesSea urchin+NDNDNDNDHS2-6TCRα/δ-Dad1 locusHuman+NDNDNDNDBEAD1TCRα/δ locusHuman+NDNDNDCTCF*Elements are listed with their associated gene(s) and the organism under study. The tests of boundary function include: (A) blocking enhancer function in a transgenic test construct; (B) providing uniform expression of a bracketed transgene at multiple insertion sites in the genome (not including pericentric heterochromatin); (C) separating two regulatory elements (shown by the impact of deleting the boundary); and (D) blocking the spread of heterochromatin-induced silencing. The table is illustrative, not comprehensive. While the gypsy element is listed as an insulator, it has also been shown to block heterochromatin-induced silencing; however, it has not been observed at a normal heterochromatin/euchromatin boundary. * indicates a protein interaction demonstrated in vitro only. ** indicates a protein interaction demonstrated only by a genetic effect. Data from Bell and Felsenfeld, 1999, and the references cited therein; Donze et al., 1999; Bi and Broach, 1999; Fourel et al., 1999; Mihaly et al., 1998; and additional references [Cuvier, O., Hart, C.M., and Laemmli, U.K. (1998) Mol. Cell. Biol. 18, 7478–7486; Gaszner, M., Vazquez, J., and Schedl, P. (1999) Genes Dev. 13, 2098–2107; Zhong, X.-P., and Krangel, M.S. (1999) J. Immunol. 163, 295–300; Zhou, J., Ashe, H., Burks, C., and Levine, M. (1999) Development 126, 3057–3065]. Open table in a new tab Elements are listed with their associated gene(s) and the organism under study. The tests of boundary function include: (A) blocking enhancer function in a transgenic test construct; (B) providing uniform expression of a bracketed transgene at multiple insertion sites in the genome (not including pericentric heterochromatin); (C) separating two regulatory elements (shown by the impact of deleting the boundary); and (D) blocking the spread of heterochromatin-induced silencing. The table is illustrative, not comprehensive. While the gypsy element is listed as an insulator, it has also been shown to block heterochromatin-induced silencing; however, it has not been observed at a normal heterochromatin/euchromatin boundary. * indicates a protein interaction demonstrated in vitro only. ** indicates a protein interaction demonstrated only by a genetic effect. Data from Bell and Felsenfeld, 1999, and the references cited therein; Donze et al., 1999; Bi and Broach, 1999; Fourel et al., 1999; Mihaly et al., 1998; and additional references [Cuvier, O., Hart, C.M., and Laemmli, U.K. (1998) Mol. Cell. Biol. 18, 7478–7486; Gaszner, M., Vazquez, J., and Schedl, P. (1999) Genes Dev. 13, 2098–2107; Zhong, X.-P., and Krangel, M.S. (1999) J. Immunol. 163, 295–300; Zhou, J., Ashe, H., Burks, C., and Levine, M. (1999) Development 126, 3057–3065]. In addition, the promiscuous behavior of many regulatory elements also requires that the genome be partitioned into discrete regulatory domains. Enhancers are known to function over large distances, and have the potential to affect many different promoters. Some resolution of the problem of enhancer promiscuity has come with the recognition of a class of DNA elements that serve as "insulators" of enhancer function (see Table 1). The defining members of the group, scs and scs′, flank two hsp70 genes of Drosophila. These elements can block communication between an enhancer and a promoter if they are positioned between them. Insulator activity, so defined, has now been associated with a number of sequence elements in Drosophila and higher eukaryotes (8Geyer P.K Curr. Opin. Genet. Develop. 1997; 7: 242-248Crossref PubMed Scopus (190) Google Scholar, 1Bell A.C Felsenfeld G Curr. Opin. Genet. Dev. 1999; 9: 191-198Crossref PubMed Scopus (190) Google Scholar). Another type of boundary element has been identified within the Bithorax complex (BX-C) of Drosophila. The BX-C complex contains a tandem array of regulatory elements that control the expression of the Abd-B gene in successive parasegments of the organism. Genetic and molecular analysis has led to the identification of three elements, Mcp, Fab-7, and Fab-8, which appear to delimit adjacent cis-regulatory elements. Fab-7 lies between the regulatory regions iab-6 (normally active in parasegment 11) and iab-7 (normally inactive in parasegment 11 but active in parasegment 12). Deletion of Fab-7 results in a complex gain- and loss-of-function phenotype in parasegment 11; in some groups of cells both iab-6 and iab-7 are active, while in other groups of cells both are inactive (13Mihaly J Hogga I Gausz J Gyurkovics H Karch F Development. 1997; 124: 1809-1820PubMed Google Scholar). This and other observations imply that Fab-7 separates (and constrains) the active and inactive states of these regulatory elements. Both Fab-7 and Fab-8 have been shown to block enhancer function in direct tests using transgenic constructs; however, one can infer that this is not a part of their activity in their normal context, as all of the regulatory elements in the complex interact with the Abd-B promoter when active (14Mihaly J Hogga I Barges S Galloni M Mishra R.K Hagstrom K Muller M Schedl P Sipos L Gausz J et al.Cell. Mol. Life Sci. 1998; 54: 60-70Crossref PubMed Scopus (110) Google Scholar). The yeast S. cerevisiae maintains silent copies of its mating type genes in two loci, HMR and HML. Silencing requires the binding of proteins including Rap1, Abf1p, Sir1p, and ORC to specific sites E and I (see Figure 1), and is dependent on the SIR proteins 2–4. The boundaries of the silent HMR domain, HMR-L and HMR-R, have been inferred from the pattern of susceptibility to nuclease digestion (12Loo S Rine J Science. 1994; 264: 1768-1771Crossref PubMed Scopus (181) Google Scholar). 4Donze D Adams C.R Rine J Kamakaka R.T Genes Dev. 1999; 13: 698-708Crossref PubMed Scopus (307) Google Scholar have now used a URA3 reporter gene to provide a functional assessment. A test URA3 gene placed a few hundred base pairs to either side of HMR-E shows silencing, while one placed 2840 bp to the left, beyond the inferred boundary, does not. This is not simply the consequence of a limited pool of silencing proteins; the silent domain was expanded 2-fold in size (by inserting a fragment of DNA) with no loss of silencing. However, deletion of HMR-R results in the spread of silencing, repressing expression of a URA3 reporter gene. Both HMR-R and HMR-L are able to block silencing of the MATa1 gene by the HMR-E silencer in a plasmid construct; control "stuffer" fragments had no such effect. In addition, HMR-R can protect a URA3 gene from telomere position effect. Examination of heterozygous strains showed that HMR-R provides protection from silencing for an adjacent URA3 reporter gene only in a cis configuration, not in trans (4Donze D Adams C.R Rine J Kamakaka R.T Genes Dev. 1999; 13: 698-708Crossref PubMed Scopus (307) Google Scholar). Thus, the HMR boundary elements limit the activity of silencers to a single contiguous chromatin domain. The data suggest that these elements are "barriers," preventing the spread of a heterochromatin structure. Recent analyses of native yeast telomeres have also identified elements there that appear to limit the heterochromatin to appropriate domains, again blocking the spread of silencing (7Fourel G Revardel E Koering C.E Gilson E EMBO J. 1999; 18: 2522-2537Crossref PubMed Scopus (190) Google Scholar, 17Pryde F.E Louis E.J EMBO J. 1999; 18: 2538-2550Crossref PubMed Scopus (232) Google Scholar). While epigenetic silencing in general is promoter independent, some loci can escape silencing. Such occurrences are well documented on the inactive X chromosome in mammals and in variegating regions of Drosophila. Yeast is no exception; some genes, including TEF1 and TEF2 (encoding translation elongation factors) are refractory to the silencing normally imposed by residence in the HM domains. 3Bi X Broach J.R Genes Dev. 1999; 13: 1089-1101Crossref PubMed Scopus (114) Google Scholar recently demonstrated that the TEF UASrpg (upstream activation sequence of ribosome protein genes), in addition to its ability to enhance ribosome protein gene transcription, also has all of the attributes of a barrier element, blocking the effects of the HML-E silencer on gene expression, and blocking the physical spread of heterochromatin (assayed by changes in supercoiling). The barrier activity does not require transcription, but is associated with a specific cluster of binding sites for Rap1, a very abundant DNA-binding protein that can impact gene expression in different ways, depending on context. It will be of particular interest to learn what features of this cluster (organization of the Rap1 sites, other associated factors, etc.) determine its activity as an enhancer, and what features are necessary for its activity as a barrier for the silencing element. Can we suggest a mechanism for barrier activity? Heterochromatin formation, and concomitant silencing, is generally associated with an overall loss of DNA accessibility to nucleases; a shift to histone hypoacetylation; the loss of accessible regulatory regions (DNase I hypersensitive [DH] sites); an altered nucleosome array, suggestive of more regular spacing; and a shift in topology to more negative supercoiling (see 6Elgin S.C.R Curr. Opin. Genet. Dev. 1996; 6: 193-202Crossref PubMed Scopus (190) Google Scholar, 9Grunstein M Cell. 1998; 93: 325-328Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 3Bi X Broach J.R Genes Dev. 1999; 13: 1089-1101Crossref PubMed Scopus (114) Google Scholar). All of these properties suggest a "closed-up," relatively uniform nucleosome array with a high percentage of the DNA bound to the octamer core. If this form spreads, once initiated, then anything that significantly disrupts the nucleosome array could serve as a barrier. A cluster of protein-binding sites, such as the Rap1 sites of the UASrpg, might perform this function. A genetic analysis of the pol II–related transcription factors predicted to bind to the HMR-R DNA (which includes a Ty1 LTR and a tRNA gene) failed to find a functional link; but the loss of SMC1 and SMC3 disrupted boundary function significantly (4Donze D Adams C.R Rine J Kamakaka R.T Genes Dev. 1999; 13: 698-708Crossref PubMed Scopus (307) Google Scholar). The SMC proteins are required for chromosome condensation and cohesion, and might play a role in organizing interphase chromatin. This finding points to a barrier mechanism related to chromatin organization, or at the least argues that normal chromosome architecture is necessary to maintain barriers to heterochromatin. A recent study has shown that the chicken β-globin gene insulator, HS4, serves not only as an insulator of enhancer function, but also lies at the boundary between two domains of chromatin structure. In the mature erythrocyte, the β-globin gene cluster lies in an accessible domain; the 33 kb region is 3–5 times more sensitive to digestion by DNase I (compared to a transcriptionally silent gene) and shows elevated histone acetylation. 5′ HS4, a constitutive DH site, is positioned roughly at the boundary of the domain. It behaves as an insulator, blocking enhancer function in tests in either Drosophila or chicken cells. Immediately upstream, a folate receptor gene is separated from the β-globin locus by a 16 kb region of silent chromatin (see Figure 2). This region is packaged in a micrococcal nuclease–resistant chromatin structure; its DNA is highly methylated. An upstream boundary can be inferred abutting the regulatory region (observed as a DH site) of the folate receptor gene, although insulator activity has not yet been tested. Both genes are expressed in the erythropoietic cell lineage, the folate receptor gene during the earlier CFU-E stage, and the β-globin gene with terminal differentiation. The presence of a block of condensed chromatin between the two genes may facilitate their independent regulation (16Prioleau M.-N Nony P Simpson M Felsenfeld G EMBO J. 1999; 18: 4035-4048Crossref PubMed Scopus (142) Google Scholar). Might the HS4 element serve as a barrier to heterochromatin as well as an insulator? Further analysis has shown that the chicken HS4 element can protect a stably integrated gene, not only from local position effects, but also from the gradual extinction of activity commonly observed on propagation of transformed cells (15Pikaart M.J Recillas-Targa F Felsenfeld G Genes Dev. 1998; 12: 2852-2862Crossref PubMed Scopus (333) Google Scholar). Reporter constructs with and without flanking 1.2 kb HS4 elements, based on the chicken βA-globin gene, provided expression of an antigenic IL2R cell surface marker. The presence of the flanking HS4 elements resulted in uniform and stable expression of the transgene; in the absence of HS4 elements, expression of IL2R was usually extinct in over 90% of the cells after 80 days of growth. Sorting the uninsulated lines on the basis of expression, the investigators demonstrated a loss of DNase I hypersensitivity and an increase in DNA methylation in the promoter region of the inactive transgene. Chromatin immunoprecipitation assays showed preferential association of acetylated histones H3 and H4 with the IL2R transgene from an insulated line, but not from an inactive, uninsulated line. Significant reactivation of the uninsulated lines could be achieved using trichostatin A and butyrate (which inhibit histone deacetylases) or 5-azacytidine (which inhibits DNA methylation). Extinction of expression is completely prevented by flanking the transgene with the HS4 elements; the protected transgene retains nuclease sensitivity and histone acetylation characteristics typical of active genes. (There is no clear correlation between the presence of the HS4 insulators and the level of DNA methylation, however.) The results indicate that HS4 elements can create a barrier, insulating euchromatin from a heterochromatin-like domain (15Pikaart M.J Recillas-Targa F Felsenfeld G Genes Dev. 1998; 12: 2852-2862Crossref PubMed Scopus (333) Google Scholar). A 42 bp fragment of the HS4 element that provides insulator activity has been identified. This sequence is a binding site for CTCF, a highly conserved zinc finger protein found in vertebrates. However, while the CTCF-binding sites appear to be necessary and sufficient for the enhancer blocking activity, other sequences appear to be needed to prevent extinction of expression of a reporter gene as discussed above (2Bell A.C West A.G Felsenfeld G Cell. 1999; 98: 387-396Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). These results suggest that insulator activity is distinct from barrier activity, but that the insulator can be part of a larger complex that functions as a barrier to limit the spread of heterochromatic silencing. The functional tests we have used have identified different boundary elements with different functions; however, our testing is incomplete (see Table 1). Is there a fundamental difference between insulators and barriers, or might they operate by a common mechanism? Can all insulators serve as barriers, and vice versa? Barriers serve as the boundary between heterochromatin and euchromatin, while insulators operate within euchromatic domains. The insulator element by itself does not normally activate or inactivate either the promoter or the enhancer; both can interact with other regulatory elements that are on the same side of the insulator (tested with scs and the gypsy insulator). Thus while these elements insulate enhancer function, there is no indication that they mark the boundary between different modes of chromatin packaging. However, the gypsy insulator, made up of multiple copies of the SU(HW)-binding site, can function as a barrier, blocking the silencing activity of the PRE and protecting a transgene from silencing on insertion into heterochromatin (18Roseman R.R Pirotta V Geyer P.K EMBO J. 1993; 12: 435-442Crossref PubMed Scopus (224) Google Scholar, 19Sigrist C.J.A Pirotta V Genetics. 1997; 147: 209-221Crossref PubMed Google Scholar). In contrast, the scs elements do not protect a mini-white transgene from exhibiting a variegating phenotype when resident in the pericentric heterochromatin (11Kellum R Schedl P Cell. 1991; 64: 941-950Abstract Full Text PDF PubMed Scopus (480) Google Scholar). Indeed, previous observations showing the spreading of gene silencing over hundreds of kilobases and multiple genes (as seen in PEV) imply that many insulators capable of establishing domains of enhancer function are not able to serve as barriers to heterochromatin spreading. One would like to know whether the barriers to heterochromatin spreading characterized in yeast can serve as insulators, if separated from silencing elements. A distinction between insulators and the boundary elements of the BX-C complex is implied by a recent analysis in which the Fab-7 boundary element was replaced by either the scs or gypsy insulator. Both elements can substitute for the Fab-7 activity, preventing adventitious interaction between the iab-6 and iab-7 regulatory domains; however, the scs or gypsy insulators also blocked interaction of the distal cis-regulatory domains with the Abd-B promoter, with deleterious effect (14Mihaly J Hogga I Barges S Galloni M Mishra R.K Hagstrom K Muller M Schedl P Sipos L Gausz J et al.Cell. Mol. Life Sci. 1998; 54: 60-70Crossref PubMed Scopus (110) Google Scholar). Thus while Fab-7 can act as an insulator in a transgenic construct, it apparently does not have such an effect in its normal context, implying a distinction between these types of boundaries. How do elements that insulate enhancers work? Might they also act by disrupting chromatin structure? The various models fall into two general categories: those involving a local interaction between the proteins of the insulator element and the proteins of the enhancer (or other regulatory element), and those in which insulator activity is coupled to a structural role in higher-order chromatin/nuclear organization (10Kellum R Elgin S.C.R Curr. Biol. 1998; 8: R521-R524Abstract Full Text Full Text PDF PubMed Google Scholar, 1Bell A.C Felsenfeld G Curr. Opin. Genet. Dev. 1999; 9: 191-198Crossref PubMed Scopus (190) Google Scholar). At present, no single model satisfies all observations. Additional clues are being derived from genetic approaches. A recent analysis of the Drosophila genes Chip and Nipped-B has suggested that their protein products play a role in the interaction of remote enhancers with their target gene promoters; it has been suggested that SU(HW)-binding sites (gypsy insulator) block enhancer function by disrupting these interactions (5Dorsett D Curr. Opin. Genet. Dev. 1999; 9: 505-514Crossref PubMed Scopus (159) Google Scholar). If insulators function by disrupting the cis interactions that bring an enhancer to a promoter, might barriers function by disrupting the cis interactions that bring together elements needed to maintain a silent state? Such a model could allow some insulators to function as barriers, and vice versa, without requiring that all do so, depending on the protein complement at a given boundary element. Given the current gaps in our knowledge, any such model will be highly speculative. No doubt further clues to the mechanism of both insulator and barrier activity will be obtained by identifying and characterizing the proteins that interact with these elements (see Table 1), and analyzing the complexes that they form. Only by establishing the requirements for each activity will we be able to identify the relationships between these different types of boundaries, and understand how the cell maintains its mosaic genome.

The small fourth chromosome of Drosophila melanogaster (3.5% of the genome) presents a puzzle. Cytological analysis suggests that the bulk of the fourth, including the portion that appears banded in the polytene chromosomes, is heterochromatic; the banded region includes blocks of middle repetitious DNA associated with heterochromatin protein 1 (HP1). However, genetic screens indicate 50–75 genes in this region, a density similar to that in other euchromatic portions of the genome. Using a P element containing an hsp70 - white gene and a copy of hsp26 (marked with a fragment of plant DNA designated pt ), we have identified domains that allow for full expression of the white marker (R domains), and others that induce a variegating phenotype (V domains). In the former case, the hsp26 - pt gene shows an accessibility and heat-shock-inducible activity similar to that seen in euchromatin, whereas in the latter case, accessibility and inducible expression are reduced to levels typical of heterochromatin. Mapping by in situ hybridization and by hybridization of flanking DNA sequences to a collection of cosmid and bacterial artificial chromosome clones shows that the R domains (euchromatin-like) and V domains (heterochromatin-like) are interspersed. Examination of the effect of genetic modifiers on the variegating transgenes shows some differences among these domains. The results suggest that heterochromatic and euchromatic domains are interspersed and closely associated within this 1.2-megabase region of the genome.

We have used line HS-2 of Drosophila melanogaster, carrying a silenced transgene in the pericentric heterochromatin, to investigate in detail the chromatin structure imposed by this environment. Digestion of the chromatin with micrococcal nuclease (MNase) shows a nucleosome array with extensive long-range order, indicating regular spacing, and with well-defined MNase cleavage fragments, indicating a smaller MNase target in the linker region. The repeating unit is ca. 10 bp larger than that observed for bulk Drosophila chromatin. The silenced transgene shows both a loss of DNase I-hypersensitive sites and decreased sensitivity to DNase I digestion within an array of nucleosomes lacking such sites; within such an array, sensitivity to digestion by MNase is unchanged. The ordered nucleosome array extends across the regulatory region of the transgene, a shift that could explain the loss of transgene expression in heterochromatin. Highly regular nucleosome arrays are observed over several endogenous heterochromatic sequences, indicating that this is a general feature of heterochromatin. However, genes normally active within heterochromatin (rolled and light) do not show this pattern, suggesting that the altered chromatin structure observed is associated with regions that are silent, rather than being a property of the domain as a whole. The results indicate that long-range nucleosomal ordering is linked with the heterochromatic packaging that imposes gene silencing.

The two main problems in hypersonic vehicles are wave drag and aerodynamic heating. This work proposes a novel method for drag reduction by combining spikes with the plasma synthetic jet actuator (PSJA). Numerical simulations are performed to better understand the drag reduction mechanism with an incoming flow at Mach 6. The results suggest that the flow field is affected primarily by the diffracted wave and synthetic jet. The maximum drag reduction reaches 47.7% with the plasma spike compared with the opposing jet. A better drag reduction effect is achieved when increasing the energy density of the PSJA, while the propagation velocity of the diffracted wave remains constant. A wider and faster jet is obtained with a larger PSJA orifice diameter. However, the control time of the jet shortens. A mode conversion occurs when the orifice diameter is 1 mm. Furthermore, the maximum drag reduction rate increases from 37.6% to 49.0% when the length diameter ratio (L/D) increases from 0.5 to 1.5. The effect of spike length on drag reduction decreases gradually at greater lengths.

The dynamics and function of ribosomal proteins in the cell nucleus remain enigmatic. Here we provide evidence that specific components of Drosophila melanogaster ribosomes copurify with linker histone H1. Using various experimental approaches, we demonstrate that this association of nuclear ribosomal proteins with histone H1 is specific, and that colocalization occurs on condensed chromatin in vivo. Chromatin immunoprecipitation analysis confirmed that specific ribosomal proteins are associated with chromatin in a histone H1-dependent manner. Overexpression of either histone H1 or ribosomal protein L22 in Drosophila cells resulted in global suppression of the same set of genes, while depletion of H1 and L22 caused up-regulation of tested genes, suggesting that H1 and ribosomal proteins are essential for transcriptional gene repression. Overall, this study provides evidence for a previously undefined link between ribosomal proteins and chromatin, and suggests a role for this association in transcriptional regulation in higher eukaryotes.

Heterochromatin protein 1 (HP1) was originally described as a non-histone chromosomal protein and is required for transcriptional gene silencing and the formation of heterochromatin. Although it is localized primarily at pericentric heterochromatin, a scattered distribution over a large number of euchromatic loci is also evident. Here, we provide evidence that Drosophila HP1 is essential for the maintenance of active transcription of euchromatic genes functionally involved in cell-cycle progression, including those required for DNA replication and mitosis. Depletion of HP1 in proliferating embryonic cells caused aberrant progression of the cell cycle at S phase and G2/M phase, linked to aberrant chromosome segregation, cytokinesis, and an increase in apoptosis. The chromosomal distribution of Aurora B, and the level of phosphorylation of histone H3 serine 10 were also altered in the absence of HP1. Using chromatin immunoprecipitation analysis, we further demonstrate that the promoters of a number of cell-cycle regulator genes are bound to HP1, supporting a direct role for HP1 in their active transcription. Overall, our data suggest that HP1 is essential for the maintenance of cell-cycle progression and the transcription of cell-cycle regulatory genes. The results also support the view that HP1 is a positive regulator of transcription in euchromatin.

hMOF is the major acetyltransferase of histone H4 lysine 16 (H4K16) in humans, but its biological function is not well understood. In this study, hMOF was found to be more frequently highly expressed in non-small cell lung cancer (NSCLC) than corresponding normal tissues (P < 0.001). In addition, up-regulation of H4K16 acetylation was also more frequent in NSCLC than normal tissues (P = 0.002). Furthermore, hMOF promotes the cell proliferation, migration and adhesion of NSCLC cell lines. Microarray analysis and chromatin immunoprecipitation (ChIP) assays suggest that hMOF modulates proliferation and metastasis by regulating histone H4K16 acetylation at the promoter regions of downstream target genes. Moreover, hMOF promotes S phase entry via Skp2. These findings suggest that hMOF contributes to NSCLC tumorigenesis.

Cancer cells reprogram their energy metabolic system from the mitochondrial oxidative phosphorylation (OXPHOS) pathway to a glucose-dependent aerobic glycolysis pathway. This metabolic reprogramming phenomenon is known as the Warburg effect, a significant hallmark of cancer. However, the detailed mechanisms underlying this event or triggering this reprogramming remain largely unclear. Here, we found that histone H2B monoubiquitination (H2Bub1) negatively regulates the Warburg effect and tumorigenesis in human lung cancer cells (H1299 and A549 cell lines) likely through controlling the expression of multiple mitochondrial respiratory genes, which are essential for OXPHOS. Moreover, our work also suggested that pyruvate kinase M2 (PKM2), the rate-limiting enzyme of glycolysis, can directly interact with H2B in vivo and in vitro and negatively regulate the level of H2Bub1. The inhibition of cell proliferation and nude mice xenograft of human lung cancer cells induced by PKM2 knockdown can be partially rescued through lowering H2Bub1 levels, which indicates that the oncogenic function of PKM2 is achieved, at least partially, through the control of H2Bub1. Furthermore, PKM2 and H2Bub1 levels are negatively correlated in cancer specimens. Therefore, these findings not only provide a novel mechanism triggering the Warburg effect that is mediated through an epigenetic pathway (H2Bub1) but also reveal a novel metabolic regulator (PKM2) for the epigenetic mark H2Bub1. Thus, the PKM2-H2Bub1 axis may become a promising cancer therapeutic target.

Significance Hepatocellular carcinoma (HCC) is the sixth most common type of cancer, and its mortality rate continues to increase. We generated knock-in reporter mouse models for measuring DNA double-strand break (DSB) repair efficiency and demonstrated that both DSB repair pathways are up-regulated in mouse HCCs. We then found that activation of PARP1 and DNA-PKcs is critical to HCC survival both in vitro and in vivo. Targeting the two proteins to block both DSB repair pathways by combining olaparib and NU7441 synergistically inhibits HCC growth in both orthotopic HCC mouse and human PDX models. Our work not only establishes versatile tools for in vivo analysis of DNA repair, but also implies an effective combination therapy for HCC.

It is well established that the small GTPase Ras promotes tumor initiation by activating at least three different mediators: Raf, PI3K, and Ras-like (Ral) guanine nucleotide exchange factors. However, the exact mechanisms that underlie these different Ras signaling pathways, which are involved in tumor progression, remain to be elucidated. In this study, we report that the Ras-PI3K pathway, but not Raf or the Ral guanine nucleotide exchange factors, specifically targets the acetylation of H3 at lysine 56 (H3K56ac), thereby regulating tumor cell activity. We demonstrate that the Ras-PI3K-induced reduction in H3K56ac is associated with the proliferation and migration of tumor cells by targeting the transcription of tumor-associated genes. The depletion of the histone deacetyltransferases Sirt1 and Sirt2 rescues the Ras-PI3K-induced decrease in H3K56ac, gene transcription, tumor cell proliferation, and tumor cell migration. Furthermore, we demonstrate that the Ras-PI3K-AKT pathway regulates H3K56ac via the MDM2-dependent degradation of CREB-binding protein/p300. Taken together, the results of this study demonstrate that the Ras-PI3K signaling pathway targets specific epigenetic modifications in tumor cells.

Histone H2B lysine 120 monoubiquitination is required for embryonic stem cell differentiation

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients.

Autophagy is an evolutionarily conserved cellular process that primarily participates in lysosome-mediated protein degradation. Although autophagy is a cytoplasmic event, how epigenetic pathways are involved in the regulation of autophagy remains incompletely understood. Here, we found that H2B monoubiquitination (H2Bub1) is down-regulated in cells under starvation conditions and that the decrease in H2Bub1 results in the activation of autophagy. We also identified that the deubiquitinase USP44 is responsible for the starvation-induced decrease in H2Bub1. Furthermore, the changes in H2Bub1 affect the transcription of genes involved in the regulation of autophagy. Therefore, this study reveals a novel epigenetic pathway for the regulation of autophagy through H2Bub1.

Lung cancer is one of the most lethal cancers due to its highly metastatic spreading. The motility of lung cancer cells is regulated by paracrine factors, such as TGF-β, in the tumor microenvironment through the induction of epithelial-to-mesenchymal transition (EMT). The stability of microtubules is reported to be associated with the EMT process and the migration of cancer cells. Here, we observed that RCC1 domain-containing protein 1 (RCCD1) is highly expressed in non-small cell lung cancer (NSCLC) patients with poor prognosis, and RCCD1 is much higher expressed in tumor tissues compared with adjacent normal tissues. Depletion of RCCD1 using siRNAs significantly inhibits the migration of lung cancer cells. Subsequent studies reveal that the loss of RCCD1 results in upregulation of acetylated α-tubulin levels and stabilizes cytoskeletal microtubules. Mechanistically, we observed that RCCD1 modulates the stability of microtubules through interacting with JMJD5. Furthermore, RCCD1 depletion significantly attenuates the TGF-β-induced EMT process, as assessed by altered expression of epithelial and mesenchymal markers (Occludin, Vimentin and Snail), and inhibits TGF-β-induced cell migration. Collectively, these findings support RCCD1 as a novel regulator of TGF-β-induced EMT in NSCLC.

Epigenetics plays critical roles in controlling stem cell self-renewal and differentiation. Histone H1 is one of the most critical chromatin regulators, but its role in adult stem cell regulation remains unclear. Here we report that H1 is intrinsically required in the regulation of germline stem cells (GSCs) in the Drosophila ovary. The loss of H1 from GSCs causes their premature differentiation through activation of the key GSC differentiation factor bam. Interestingly, the acetylated H4 lysine 16 (H4K16ac) is selectively augmented in the H1-depleted GSCs. Furthermore, overexpression of mof reduces H1 association on chromatin. In contrast, the knocking down of mof significantly rescues the GSC loss phenotype. Taken together, these results suggest that H1 functions intrinsically to promote GSC self-renewal by antagonizing MOF function. Since H1 and H4K16 acetylation are highly conserved from fly to human, the findings from this study might be applicable to stem cells in other systems.

Maintaining an appropriate cellular concentration of p53 is critical for cell survival and normal development in various organisms. In this study, we provide evidence that the human E2 ubiquitin-conjugating enzyme RAD6 plays a critical role in regulating p53 protein levels under both normal and stress conditions. Knockdown and overexpression of RAD6 affected p53 turnover and transcription. We showed that RAD6 can form a ternary complex with MDM2 and p53 that contributes to the degradation of p53. Chromatin immunoprecipitation (ChIP) analysis showed that RAD6 also binds to the promoter and coding regions of the p53 gene and modulates the levels of H3K4 and K79 methylation on local chromatin. When the cells were exposed to stress stimuli, the RAD6-MDM2-p53 ternary complex was disrupted; RAD6 was then recruited to the chromatin of the p53 gene, resulting in an increase in histone methylation and p53 transcription. Further studies showed that stress-induced p53 transcriptional activation, cell apoptosis, and disrupted cell cycle progression are all RAD6 dependent. Overall, this work demonstrates that RAD6 regulates p53 levels in a "yin-yang" manner through a combination of two distinct mechanisms in mammalian cells.

Dysregulation of the homeostatic balance of histone H3 di- and tri-methyl lysine 27 (H3K27me2/3) levels caused by the mis-sense mutation of histone H3 (H3K27M) is reported to be associated with various types of cancers. In this study, we found that reduction in H3K27me2/3 caused by H3.1K27M, a mutation of H3 variants found in patients with diffuse intrinsic pontine glioma (DIPG), dramatically attenuated the presence of 53BP1 (also known as TP53BP1) foci and the capability of non-homologous end joining (NHEJ) in human dermal fibroblasts. H3.1K27M mutant cells showed increased rates of genomic insertions/deletions and copy number variations, as well as an increase in p53-dependent apoptosis. We further showed that both hypo-H3K27me2/3 and H3.1K27M interacted with FANCD2, a central player in the choice of DNA repair pathway. H3.1K27M triggered the accumulation of FANCD2 on chromatin, suggesting an interaction between H3.1K27M and FANCD2. Interestingly, knockdown of FANCD2 in H3.1K27M cells recovered the number of 53BP1-positive foci, NHEJ efficiency and apoptosis rate. Although these findings in HDF cells may differ from the endogenous regulation of the H3.1K27M mutant in the specific tumor context of DIPG, our results suggest a new model by which H3K27me2/3 facilitates NHEJ and the maintenance of genome stability.This article has an associated First Person interview with the first author of the paper.

The global features of H3K4 and H3K27 trimethylations (H3K4me3 and H3K27me3) have been well studied in recent years, but most of these studies were performed in mammalian cell lines. In this work, we generated the genome-wide maps of H3K4me3 and H3K27me3 of mouse cerebrum and testis using ChIP-seq and their high-coverage transcriptomes using ribominus RNA-seq with SOLiD technology. We examined the global patterns of H3K4me3 and H3K27me3 in both tissues and found that modifications are closely-associated with tissue-specific expression, function and development. Moreover, we revealed that H3K4me3 and H3K27me3 rarely occur in silent genes, which contradicts the findings in previous studies. Finally, we observed that bivalent domains, with both H3K4me3 and H3K27me3, existed ubiquitously in both tissues and demonstrated an invariable preference for the regulation of developmentally-related genes. However, the bivalent domains tend towards a "winner-takes-all" approach to regulate the expression of associated genes. We also verified the above results in mouse ES cells. As expected, the results in ES cells are consistent with those in cerebrum and testis. In conclusion, we present two very important findings. One is that H3K4me3 and H3K27me3 rarely occur in silent genes. The other is that bivalent domains may adopt a "winner-takes-all" principle to regulate gene expression.

Precise mitotic spindle assembly is a guarantee of proper chromosome segregation during mitosis. Chromosome instability caused by disturbed mitosis is one of the major features of various types of cancer. JMJD5 has been reported to be involved in epigenetic regulation of gene expression in the nucleus, but little is known about its function in mitotic process. Here we report the unexpected localization and function of JMJD5 in mitotic progression. JMJD5 partially accumulates on mitotic spindles during mitosis, and depletion of JMJD5 results in significant mitotic arrest, spindle assembly defects, and sustained activation of the spindle assembly checkpoint (SAC). Inactivating SAC can efficiently reverse the mitotic arrest caused by JMJD5 depletion. Moreover, JMJD5 is found to interact with tubulin proteins and associate with microtubules during mitosis. JMJD5-depleted cells show a significant reduction of α-tubulin acetylation level on mitotic spindles and fail to generate enough interkinetochore tension to satisfy the SAC. Further, JMJD5 depletion also increases the susceptibility of HeLa cells to the antimicrotubule agent. Taken together, these results suggest that JMJD5 plays an important role in regulating mitotic progression, probably by modulating the stability of spindle microtubules.

Core histone modifications play an important role in chromatin remodeling and transcriptional regulation. Histone acetylation is one of the best-studied gene modifications and has been shown to be involved in numerous important biological processes. Herein, we demonstrated that the depletion of histone deacetylase 3 (Hdac3) in Drosophila melanogaster resulted in a reduction in body size. Further genetic studies showed that Hdac3 counteracted the organ overgrowth induced by overexpression of insulin receptor (InR), phosphoinositide 3-kinase (PI3K) or S6 kinase (S6K), and the growth regulation by Hdac3 was mediated through the deacetylation of histone H4 at lysine 16 (H4K16). Consistently, the alterations of H4K16 acetylation (H4K16ac) induced by the overexpression or depletion of males-absent-on-the-first (MOF), a histone acetyltransferase that specifically targets H4K16, resulted in changes in body size. Furthermore, we found that H4K16ac was modulated by PI3K signaling cascades. The activation of the PI3K pathway caused a reduction in H4K16ac, whereas the inactivation of the PI3K pathway resulted in an increase in H4K16ac. The increase in H4K16ac by the depletion of Hdac3 counteracted the PI3K-induced tissue overgrowth and PI3K-mediated alterations in the transcription profile. Overall, our studies indicated that Hdac3 served as an important regulator of the PI3K pathway and revealed a novel link between histone acetylation and growth control.

Chromatin is a highly organized and dynamic structure in eukaryotic cells. The change of chromatin structure is essential in many cellular processes, such as gene transcription, DNA damage repair and others. Anti-silencing function 1 (ASF1) is a histone chaperone that participates in chromatin higher-order organization and is required for appropriate chromatin assembly. In this study, we identified the E2 ubiquitin-conjugating enzyme RAD6 as an evolutionary conserved interacting protein of ASF1 in D. melanogaster and H. sapiens that promotes the turnover of ASF1A by cooperating with a well-known E3 ligase, MDM2, via ubiquitin-proteasome pathway in H. sapiens. Further functional analyses indicated that the interplay between RAD6 and ASF1A associates with tumorigenesis. Together, these data suggest that the RAD6-MDM2 ubiquitin ligase machinery is critical for the degradation of chromatin-related proteins.

The turnover of tumor suppressor p53 is critical for its role in various cellular events. However, the pathway that regulates the turnover of the Drosophila melanogaster DMP53 is largely unknown. Here, we provide evidence for the first time that the E2 ligase, Drosophila homolog of Rad6 (dRad6/Dhr6), plays an important role in the regulation of DMP53 turnover. Depletion of dRad6 results in DMP53 accumulation, whereas overexpression of dRad6 causes enhanced DMP53 degradation. We show that dRad6 specifically interacts with DMP53 at the transcriptional activation domain and regulates DMP53 ubiquitination. Loss of dRad6 function in transgenic flies leads to lethalities and altered morphogenesis. The dRad6-induced defects in cell proliferation and apoptosis are found to be DMP53-dependent. The loss of dRad6 induces an accumulation of DMP53 that enhances the activation of apoptotic genes and leads to apoptosis in the presence of stress stimuli. In contrast to that, the E3 ligase is the primary factor that regulates p53 turnover in mammals, and this work demonstrates that the E2 ligase dRad6 is critical for the control of DMP53 degradation in Drosophila.

Histone H3 lysine 4 trimethylation (H3K4me3) is well known to occur in the promoter region of genes for transcription activation. However, when investigating the H3K4me3 profiles in the mouse cerebrum and testis, we discovered that H3K4me3 also has a significant enrichment at the 3' end of actively transcribed (sense) genes, named as 3'-H3K4me3. 3'-H3K4me3 is associated with ~15% of protein-coding genes in both tissues. In addition, we examined the transcriptional initiation signals including RNA polymerase II (RNAPII) binding sites and 5'-CAGE-tag that marks transcriptional start sites. Interestingly, we found that 3'-H3K4me3 is associated with the initiation of antisense transcription. Furthermore, 3'-H3K4me3 modification levels correlate positively with the antisense expression levels of the associated sense genes, implying that 3'-H3K4me3 is involved in the activation of antisense transcription. Taken together, our findings suggest that H3K4me3 may be involved in the regulation of antisense transcription that initiates from the 3' end of sense genes. In addition, a positive correlation was also observed between the expression of antisense and the associated sense genes with 3'-H3K4me3 modification. More importantly, we observed the 3'-H3K4me3 enrichment among genes in human, fruitfly and Arabidopsis, and found that the sequences of 3'-H3K4me3-marked regions are highly conserved and essentially indistinguishable from known promoters in vertebrate. Therefore, we speculate that these 3'-H3K4me3-marked regions may serve as potential promoters for antisense transcription and 3'-H3K4me3 appear to be a universal epigenetic feature in eukaryotes. Our results provide a novel insight into the epigenetic roles of H3K4me3 and the regulatory mechanism of antisense transcription.

Microtubules play essential roles in mitosis, cell migration, and intracellular trafficking. Drugs that target microtubules have demonstrated great clinical success in cancer treatment due to their capacity to impair microtubule dynamics in both mitotic and interphase stages. In a previous report, we demonstrated that JMJD5 associated with mitotic spindle and was required for proper mitosis. However, it remains elusive whether JMJD5 could regulate the stability of cytoskeletal microtubules and whether it affects the efficacy of microtubule-targeting agents. In this study, we find that JMJD5 localizes not only to the nucleus, a fraction of it also localizes to the cytoplasm. JMJD5 depletion decreases the acetylation and detyrosination of α-tubulin, both of which are markers of microtubule stability. In addition, microtubules in JMJD5-depleted cells are more sensitive to nocodazole-induced depolymerization, whereas JMJD5 overexpression increases α-tubulin detyrosination and enhances the resistance of microtubules to nocodazole. Mechanistic studies revealed that JMJD5 regulates MAP1B protein levels and that MAP1B overexpression rescued the microtubule destabilization induced by JMJD5 depletion. Furthermore, JMJD5 depletion significantly promoted apoptosis in cancer cells treated with the microtubule-targeting anti-cancer drugs vinblastine or colchicine. Together, these findings suggest that JMJD5 is required to regulate the stability of cytoskeletal microtubules and that JMJD5 depletion increases the susceptibility of cancer cells to microtubule-destabilizing agents.

Enhancing photosynthetic efficiency is a major goal to improve crop yields under agricultural field conditions and is associated with chloroplast biosynthesis and development. In this study, we demonstrate that GOLDEN2-LIKE 1a (BnGLK1a) plays an important role in the regulating chloroplast development and photosynthetic efficiency. Overexpressing BnGLK1a resulted in significant increases in chlorophyll content, the number of thylakoid membrane layers and photosynthetic efficiency in B. napus, while knocking down BnGLK1a transcript levels through RNA interference (RNAi) had the opposite effects. A yeast two-hybrid screen revealed that BnGLK1a interacts with the abscisic acid receptor PYRABACTIN RESISTANCE 1-LIKE 1-2 (BnPYL1-2) and CONSTITUTIVE PHOTOMORPHOGENIC 9 SIGNALOSOME 5A subunit (BnCSN5A), which play essential roles in regulating chloroplast development and photosynthesis. In agreement with this, BnGLK1a-RNAi lines of B. napus display hypersensitivity to ABA response. Importantly, overexpression of BnGLK1a resulted in a 10% increase in thousand-seed weight, whereas seeds from BnGLK1a-RNAi lines were 16% lighter than wild type. We propose that BnGLK1a could be a potential target in breeding for improving rapeseed productivity. Our results not only provide the insight into the mechanisms of BnGLK1a function, but also offer a potential approach to improving the productivity of Brassica species.

To further understand the relationship between nucleosome-space occupancy (NO) and global transcriptional activity in mammals, we acquired a set of genome-wide nucleosome distribution and transcriptome data from the mouse cerebrum and testis based on ChIP (H3)-seq and RNA-seq, respectively. We identified a nearly consistent NO patterns among three mouse tissues--cerebrum, testis, and ESCs--and found, through clustering analysis for transcriptional activation, that the NO variations among chromosomes are closely associated with distinct expression levels between house-keeping (HK) genes and tissue-specific (TS) genes. Both TS and HK genes form clusters albeit the obvious majority. This feature implies that NO patterns, i.e. nucleosome binding and clustering, are coupled with gene clustering that may be functionally and evolutionarily conserved in regulating gene expression among different cell types.

RAD6, an E2 ubiquitin-conjugating enzyme, is a key node for determining different DNA damage repair pathways, controlling both the error-prone and the error-free DNA damage repair pathways through differential regulation of the ubiquitination of the proliferating cell nuclear antigen (PCNA) protein. However, whether other pathways are involved in the RAD6-mediated regulation of DNA damage repair is still unclear. To deeply understand the molecular mechanisms of RAD6 in DNA damage repair, we performed a proteomic analysis and identified the changes of the protein-protein interaction (PPI) networks of RAD6 before and after X-ray irradiation. Furthermore, our study indicated that a proteasome-related event is likely involved in the DNA damage repair process. Moreover, we found that RAD6 promotes proteasome activity and nuclear translocation by enhancing the degradation of PSMF1 and the lamin B receptor (LBR). Therefore, we provide a novel pathway that is employed by RAD6 in response to DNA damage.

To investigate the effects of morroniside on H2O2-induced apoptosis in nerve cells.Human neuroblastoma cell line SH-SY5Y cells were pre-incubaed with morroniside (1, 10, and 100 micromol x L(-1)) for 24 h prior to exposure to H2O2 (500 micromol x L(-1)) for 18 h. The activity of reactive SOD was measured by a biochemical assay. The expression of caspase-3, caspase-9, Bcl-2 and Bax was determined by Wastern blotting method.Pretreatment of the cells with morroniside (10 and 100 micromol x L(-1)) increasd SOD activity by 14% (P<0.01) and 11% (P<0.05) in comparison with cells exposed only to H2O2. Morroniside (1, 10, 100 micromol x L(-1)) lowered caspase-3 level by 31% (P<0.01), 103% (P<0.001) and 95% (P<0.001), decreased caspase-9 content by 71% (P<0.001), 132% (P<0.001) and 37% (P<0.05), and increasd Bcl-1 level by 88% (P<0.01), 121% (P<0.001) and 60% (P<0.01) respectively but no significant change occurred in Bax level in comparison with cells exposed only to H2O2.Morroniside has neuroprotection effect against H2O2-induced oxidation injury in nerve cell.

This paper examines the impact on the Chinese grain market of the global food problem caused by the Russian-Ukrainian conflict.It is conducted in two main aspects.First, the Russia-Ukraine conflict was found to have caused a global food problem.In turn, the impact of Chinese food was examined.Hypothesis testing was used to explain the impact on the Chinese grain market.It was found that after the Russia-Ukraine conflict, grain futures prices in China increased significantly compared to the pre-conflict period.Then, the regression model is applied to examine changes in the grain stocks of leading Chinese companies in the context of the grain futures that have been studied.In turn, the impact of global food issues on the stock prices of grain companies in the Chinese stock market is explained.It is then found that the AAR and CAAR data are obtained by averaging the AR and CAR data for the eight companies.Therefore, it can be concluded that the Russian-Ukrainian conflict started to have an impact on the entire Chinese grain market ten days after it occurred and that it also had a different effect on the leading Chinese grain companies.

The classic definition of epigenetics is “the study of changes in gene function that are heritable but do not entail a change in DNA sequence.” The epigenetic phenomenon was first observed in Drosophila melanogaster in the 1930s. Since the 1970s, a series of epigenetic markers were found.

Epigenetics has emerged since the late 1980s. In the twenty-first century, with advances in technological means, this new discipline is developing in an unprecedentedly rapid speed. Increasingly more biophysicists, developmental biologists, chemists, bioinformaticians, and geneticists are participating in the exploration of epigenetics.

The Major Research Plan of “Epigenetic Mechanisms of Cell Programming and Reprogramming” (hereinafter referred to as “this MRP”) was launched by the National Natural Science Foundation of China (NSFC) during the 11th Five-Year Plan period, which was started in October 2008 and concluded at the end of 2016.

Abstract Heterochromatin marked by trimethylated histone 3 at lysine 9 (H3K9me3) plays fundamental roles in reprogramming to direct cell fate determination in higher eukaryotes. However, the upstream factors that guide the establishment and spreading of H3K9me3-heterochromatin, leading to human developmental malformations, remain elusive. In this study, we found that Cdk13, a member of RNA polymerase II (RNAPII) kinase, suppresses congenital heart syndrome by preventing global facultative H3K9me3-heterochromatin spreading. Additionally, Cdk13 directs the phosphorylation of a large set of heterochromatin proteins at specific sites, which are required for the interaction between HP1 and histone H3K9 methyltransferases. Furthermore, we identified a compound, an inhibitor of heterochromatin regulators, that can alleviate syndromic heart defects in Cdk 13-mutant mice through the inhibition of H3K9me3-heterochromatin spreading. In summary, this study reveals a novel role and mechanism of Cdk13-triggered facultative H3K9me3-heterochromatin spreading in human genetic disease and paves the way for the treatment of congenital heart syndrome.

This article has been withdrawn by the authors.

SUMMARY Environmental stress-induced epigenetic changes are inherited by germlines and profoundly affect the behavior and physiology of subsequent generations. However, the mechanisms by which acquired transgenerational epigenetic imprints are established in the parental germline remain poorly understood. In this study, we used Caenorhabditis elegans as a model system and demonstrated that a key node of metabolism-Pod-2, an acetyl-CoA carboxylase, together with vitellogenins (Vits), cholesterol-binding/transport proteins, guide establishment of the acquired transgenerational epigenetic modifications H3K27me3 in the parental germline. The loss of function of a Vit; its transporter or receptor; or its binding partner, pod-2, resulted in the loss of acquired epigenetic memory in the germline. Our findings indicate that lipid metabolism is a critical mediator for establishing transgenerational epigenetic imprints in the germline.

Abstract The insulin-like growth factors IGFl and IGF2 are mitogenic polypeptides which are structurally and functionally homologous to the hormone insulin. Both single chain peptides play important roles in pathways regulating growth and development (1, 2). Their action is mediated by specific interactions with cell surface receptors and is further modified by at least six different IGF-binding proteins. Two IGF receptors have been identified. The type-1 receptor has a higher affinity for IGFl than for IGF2 and is related to the insulin receptor. The type-2 receptor, which is also a receptor for mannose-6-phosphate, binds IGF2 with high affinity, but is structurally distinct from the type-1 and insulin receptors. IGFl and IGF2 mediate their effects on growth and development through the type-1 receptor (3).

Core histone modifications play an important role in chromatin remodeling and transcriptional regulation. Histone acetylation is one of the best-studied gene modifications and has been shown to be involved in numerous important biological processes. Herein, we demonstrated that the depletion of histone deacetylase 3 (Hdac3) in Drosophila melanogaster resulted in a reduction in body size. Further genetic studies showed that Hdac3 counteracted the organ overgrowth induced by overexpression of insulin receptor (InR), phosphoinositide 3-kinase (PI3K) or S6 kinase (S6K), and the growth regulation by Hdac3 was mediated through the deacetylation of histone H4 at lysine 16 (H4K16). Consistently, the alterations of H4K16 acetylation (H4K16ac) induced by the overexpression or depletion of males-absent-on-the-first (MOF), a histone acetyltransferase that specifically targets H4K16, resulted in changes in body size. Furthermore, we found that H4K16ac was modulated by PI3K signaling cascades. The activation of the PI3K pathway caused a reduction in H4K16ac, whereas the inactivation of the PI3K pathway resulted in an increase in H4K16ac. The increase in H4K16ac by the depletion of Hdac3 counteracted the PI3K-induced tissue overgrowth and PI3K-mediated alterations in the transcription profile. Overall, our studies indicated that Hdac3 served as an important regulator of the PI3K pathway and revealed a novel link between histone acetylation and growth control.