Changes In Chromatin Architecture Research Articles

ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27Kip1 was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCFSkp2 ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCFSkp2 pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.

Epigenetic regulation of the ribosomal cistron seasonally modulates enrichment of H2A.Z and H2A.Zub in response to different environmental inputs in carp (Cyprinus carpio)

BackgroundThe specific deposition of histone variants into chromatin is an important epigenetic mechanism that contributes to gene regulation through chromatin architectural changes. The histone variant H2A.Z is essential in higher eukaryotes, and its incorporation within chromatin is a relevant process for gene expression and genome stability. However, the dual positive and negative roles of H2A.Z in gene regulation still remain unclear. We previously reported that acclimatization in common carp fish (Cyprinus carpio) involves cyclical seasonal gene reprogramming as an adaptation response to its natural environment, when rRNA synthesis and processing are profoundly affected. Epigenetic mechanisms primarily contribute to the transcriptional modulation of ribosomal genes concomitant with the acclimatization process, thus significantly regulating this process. The aim of this study was to describe the presence of several H2A.Z subtypes in carp, and assess the role of H2A.Z on the ribosomal cistron in summer- and winter-acclimatized carp.ResultsThis paper reports for the first time about the transcriptional expression of four different H2A.Z subtypes belonging to the same organism. Remarkably, a novel H2A.Z.7 was found, which corresponds to a tissue-specific histone subtype that contains seven amino acid residues longer than the canonical H2A.Z. Moreover, H2A.Z enrichment through the ribosomal cistron was significantly higher during summer, when rRNA transcription and processing are highly active, than it was in winter. Similar patterns of H2A.Z enrichment are found in two seasonally active promoters for genes transcribed by RNA polymerase II, the L41 and Δ9-desaturase genes. Interestingly, ubiquitylated-H2A.Z (H2A.Zub) was strongly enriched on regulatory regions of the ribosomal cistron in summer-acclimatized carp. Additionally, H2A.Z was present in both heterochromatin and euchromatin states on ribosomal cistron and RNA polymerase II promoters.ConclusionsOur study revealed seasonally-dependent H2A.Z enrichment for active ribosomal cistron and RNA polymerase II promoters during the carp environmental adaptation. Moreover, seasonal H2A.Zub enrichment appears as a specific mechanism contributing to the regulation of chromatin architecture under natural conditions. The existence of several H2A.Z subtypes in carp suggests that the epigenetic regulation in this species constitutes a complex and finely tuned mechanism developed to cope with seasonal environmental changes that occur in its habitat.

The expanding role ofPARPs in the establishment and maintenance of heterochromatin

Poly(ADP-ribose) polymerases (PARPs) are enzymes that transfer poly(ADP-ribose) (PAR) groups to target proteins, and thereby affect various nuclear and cytoplasmic processes. The activity of PARP family members, such as PARP1 and PARP2, is tied to cellular signalling pathways, and, through poly(ADP-ribosyl)ation, they ultimately promote changes in chromatin architecture, gene expression, and the location and activity of proteins that mediate signalling responses. A growing body of evidence suggest that PARPs, particularly PARP1 and PARP2, also operate at heterochromatic regions such as the inactive X chromosome, telomeres, pericentric heterochromatin and silent ribosomal RNA (rRNA) genes. Both proteins localize to heterochromatic sites and often associate with or poly(ADP-ribosyl)ate histones and heterochromatin-binding proteins, thereby modulating their activities. In this review, we describe current knowledge concerning the role of PARPs in establishment and inheritance of heterochromatic structures, and highlight how their contribution affects biological outcomes.

ADP-ribose polymer depletion leads to nuclear Ctcf re-localization and chromatin rearrangement

Ctcf (CCCTC-binding factor) directly induces Parp [poly(ADP-ribose) polymerase] 1 activity and its PARylation [poly(ADPribosyl)ation] in the absence of DNA damage. Ctcf, in turn, is a substrate for this post-synthetic modification and as such it is covalently and non-covalently modified by PARs (ADP-ribose polymers). Moreover, PARylation is able to protect certain DNA regions bound by Ctcf from DNA methylation. We recently reported that de novo methylation of Ctcf target sequences due to overexpression of Parg [poly(ADP-ribose)glycohydrolase] induces loss of Ctcf binding. Considering this, we investigate to what extent PARP activity is able to affect nuclear distribution of Ctcf in the present study. Notably, Ctcf lost its diffuse nuclear localization following PAR (ADP-ribose polymer) depletion and accumulated at the periphery of the nucleus where it was linked with nuclear pore complex proteins remaining external to the perinuclear Lamin B1 ring. We demonstrated that PAR depletion-dependent perinuclear localization of Ctcf was due to its blockage from entering the nucleus. Besides Ctcf nuclear delocalization, the outcome of PAR depletion led to changes in chromatin architecture. Immunofluorescence analyses indicated DNA redistribution, a generalized genomic hypermethylation and an increase of inactive compared with active chromatin marks in Parg-overexpressing or Ctcf-silenced cells. Together these results underline the importance of the cross-talk between Parp1 and Ctcf in the maintenance of nuclear organization.

Elucidating the temporal dynamics of chromatin-associated protein release upon DNA digestion by quantitative proteomic approach

Chromatin is a highly dynamic well organized nucleoprotein complex of DNA and proteins that controls DNA-dependent processes such as transcription, replication, repair and many others. Chromatin structure is regulated by various chromatin associated proteins, post-translational modifications of histones and DNA methylation, but a complete picture of structural changes in chromatin architecture is unclear due to the lack of comprehensive data of chromatin-associated proteins and their bindings to different chromatin regions. This study temporally released chromatin-associated proteins by DNase I and MNase treatment and profiled them by exponentially modified protein abundance index (emPAI) based label-free quantitative proteomics. We identified 694 high confidence proteins, with 160 known chromatin-associated proteins. Identified proteins were functionally classified into histones, non-histones involved in architectural maintenance, proteins involved in DNA replication and repair, transcription machinery, transcription regulation, other chromatin proteins, cell cycle proteins and several novel proteins. Numerous proteins presumed to be chromatin associated were identified and their chromatin interactions were explored. The comprehensive differential chromatin bound proteome might expand our knowledge of the proteins that were associated with different chromatin regions, which could be very useful in elucidating chromatin biology.

Abstract 4098: COX-2 expression in human pancreatic cancer cells is associated with an epithelial phenotype

Abstract Epithelial to mesenchymal transition is a developmental pathway that has been shown to have a direct role in tumor progression and drug resistance. Increasing evidence suggests that EMT (and MET) leads to global changes in chromatin architecture that modifies expression of numerous genes. Interestingly, reports in the literature indicate a possible association between cyclooxygenase-2 (COX-2) expression and EMT. COX-2 is upregulated in response to inflammation, and is known to be overexpressed in multiple malignancies, including pancreatic cancer. As a critical mediator of prostaglandin production, COX-2 has proangiogenic properties associated with increased tumor growth and invasion, and is also thought to play a role in the carcinogenic process. In this study, we report that COX-2 expression is associated with an epithelial phenotype among a panel of human pancreatic cancer cells. Using chromatin immunoprecipitation (ChIP), we demonstrate increased acetylated H3 at the COX-2 promoter region in the epithelial cell lines versus mesenchymal cell lines. Treatment with a Type-I selective HDAC inhibitor SNDX-275 increased levels of acetylation at the COX-2 promoter region in mesenchymal cell lines and subsequently promoted COX-2 gene expression. Overall, these data point to an epigenetic suppression of COX-2 expression in mesenchymal pancreatic cancer cells that can be reversed by Type-I HDAC inhibitors. Our results suggest that COX2 inhibitors will preferentially target “epithelial” pancreatic cancers and that combinations of Cox2 and HDAC inhibitors may display increased anti-tumor activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4098. doi:1538-7445.AM2012-4098

Purkinje Cell Degeneration in pcd Mice Reveals Large Scale Chromatin Reorganization and Gene Silencing Linked to Defective DNA Repair

DNA repair protects neurons against spontaneous or disease-associated DNA damage. Dysfunctions of this mechanism underlie a growing list of neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes the loss of nna1 expression and is associated with the postnatal degeneration of PCs. This PC degeneration dramatically affects nuclear architecture and provides an excellent model to elucidate the nuclear mechanisms involved in a whole array of neurodegenerative disorders. We used immunocytochemistry for histone variants and components of the DNA damage response, an in situ transcription assay, and in situ hybridization for telomeres to analyze changes in chromatin architecture and function. We demonstrate that the phosphorylation of H2AX, a DNA damage signal, and the trimethylation of the histone H4K20, a repressive mark, in extensive domains of genome are epigenetic hallmarks of chromatin in degenerating PCs. These histone modifications are associated with a large scale reorganization of chromatin, telomere clustering, and heterochromatin-induced gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail to be concentrated in the damaged chromatin compartments, even though the expression levels of their coding genes were slightly up-regulated. Although the mechanism by which Nna1 loss of function leads to PC neurodegeneration is undefined, the progressive accumulation of DNA damage in chromosome territories irreversibly compromises global gene transcription and seems to trigger PC degeneration and death.

Abstract 488: Reprogramming cancer cells into terminal differentiation via induced pluripotency

Abstract Induced pluripotent stem cells (iPSCs) are defined as somatic cells that have been “reprogrammed” (using either the four transcriptional factors Oct4, Sox2, Klf4 and c-Myc or Oct4, Sox2, Nanog, and Lin28) to exhibit embryonic stem cell-like pluripotent differentiation properties. The generation of iPSCs was a great achievement for the field of cell transplantation and tissue engineering. More recently multiple cancer cell lines (e.g., leukemic cells, melanoma cells & gastric cancer cells) have been successfully reprogrammed to the iPSC-state, at least as defined by (1) expression of genes specific to undifferentiated embryonic stem cells; and (2) pluripotency as defined by embryoid body formation in vitro and early markers of commitment to various differentiation lineages. However, the question that remains unanswered is: can the cancer derived iPSCs complete maturation along a given lineage, be it the lineage from which it was derived (i.e., originally prematurely blocked) or an alternative lineage? If the former were demonstrated it would in essence answer the question of whether global changes in chromatin architecture or ‘epigenetics’ (which are believed to underlie the mechanism of nuclear iPSC reprogramming) can supersede the specific genetic changes (i.e., mutations) in DNA that resulted in the lineage specific differentiation block and subsequent tumorigenesis. To attempt to answer these questions we generated iPSC-cancer cells from sarcoma cell lines representing undifferentiated sarcomas, osteosarcoma cells, liposarcoma cells, and sarcomas of unknown lineage. While the original sarcoma cell lines could not be differentiated in vitro into mature connective tissue cells, their iPSC-counterparts readily differentiated into multiple mature connective tissue lineages. Furthermore, assessment of proliferation indicated that acquisition of the mature phenotype was concurrent with cessation of proliferation; and thus abrogation of tumorigenicity. We are currently (1) performing gene expression, miRNA, and global DNA promoter methylation profiling in order to assess the mechanism by which iPSC-based reprogramming of cancer cells over-rides the inherent genetic mutations in these cells and restores the capacity for terminal differentiation; and (2) extending our work to carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 488. doi:10.1158/1538-7445.AM2011-488

Role of HMGB Proteins in Chromatin Dynamics and Telomere Maintenance in Arabidopsis thaliana

Chromosome stability is conditioned by functional chromatin structure of chromosome ends - telomeres. Organisation and regulation of telomere maintenance represent a complex process whose details still remain enigmatic, especially in plants. Several telomere-binding or telomere-associated proteins and distinct epigenetic marks have been shown to influence telomere length and telomerase activity. HMGB proteins play important role in dynamic changes of chromatin structure and are involved in regulation of cellular processes of key importance, such as replication, transcription, recombination and DNA-repair. HMGB proteins in plants are more diversified than in other eukaryotes. Here, we summarise the roles of plant HMGB proteins in regulation of chromatin structure and dynamics and report on the newly identified role of AtHMGB1 protein in the regulation of plant telomere length. Astonishingly, contrary to mice mHMGB1 homologue, AtHMGB1 does not affect telomerase activity and AtHMGB1 loss or overexpression does not cause any obvious changes in chromatin architecture.

Induction of Senescence by Doxorubicin Is Associated with Upregulated Mir-375 and Induction of Autophagy In CML Cell Line K562

AbstractAbstract 1843Senescence is a specialized form of growth arrest that it is generally irreversible and can be induced by telomere attrition, oxidative stress, oncogene expression and DNA damage signaling. Senescent cells display a typical upregulated senescence-associated (SA)-b-galactosidase activity and novel changes in chromatin architecture, the formation of SA heterochromatic foci (SAHF). Changes in gene expression, such as upregulated p16, p53, and p21 expression and silencing of E2F target genes, have been characterized to promote the establishment of senescence. Besides, by the actions of HP1g, HMGA, and DNMT proteins to produce a repressive chromatin environment, the transcription of proliferation-associated genes can be suppressed. Therefore, senescence has been suggested to functions as a natural brake to tumor development. In this study, we first sought to establish an in vitro senescence model using doxorubicin (DOX) and paclitaxel to treat CML cell line K562. We found that 50 nM DOX induced senescence, but did not induce apoptosis. In contrast, 10 nM and 100 nM paclitaxel induced apoptosis but did not induce senescence, and lower doses of paclitaxel (1 nM and 5 nM) had no effect on the cells. p53 and p16-pRb are the two major senescence pathways. Since p53 and p16 are homozygously deleted in the K562 cells, the DOX-induced senescence in K562 cells is supposed to be established by a pathway independent of p53 and p16-pRb pathways. Indeed, the expression of the typical SA-premalignant cell markers (CDC6, Ki67, p19, p38, PU1, DNMT1, HMGA1, HP1g) did not change in the DOX-induced senescent K562 cells although the typical SA-b-galactosidase staining and SAHF were apparent. MicroRNA profiling revealed that miR-375 was upregulated in DOX-induced senescent K562 cells. Treatment with miR-375 inhibitor could rescue the proliferation ability suppressed by DOX (p < 0.05). The identification of miR-375 targets should help us to elucidate the substitution pathway that is responsible for the DOX-induced senescence in the absence of both p16 and p53 genes. With the observation that DOX treatment induced cells entering senescence but eventually lead to cell death, we also investigated if the alternative mode of cell death, autophagy, was involved. By examining the expression patterns of the 26 human autophagy-related (ATG) genes, a 12-fold increase of ATG9B at day 4 and a 20-fold increase of ATG18 at day 2 after DOX treatment were noted. Both ATG9 and ATG18 are crucial for the formation of autophagosome. Hence, in addition to the upregulation of miR-375, our results also demonstrated that senescence induced by DOX in K562 cells is associated with the initiation of autophagy. Updated results on the identification of miR-375 targets and the regulation of senescence and autophagy pathway will be presented at the meeting. Disclosures:No relevant conflicts of interest to declare.

The role of nucleosome positioning in the evolution of gene regulation.

Author SummaryDivergence in gene regulation plays a major role in organismal evolution. Evidence suggests that changes in the packaging of eukaryotic genomes into chromatin can underlie the evolution of divergent gene expression patterns. Here, we explore the role of chromatin structure in regulatory evolution by whole-genome measurements of nucleosome positions and mRNA levels in 12 yeast species spanning ∼250 million years of evolution. We find several distinct ways in which changes in chromatin structure are associated with changes in gene expression. These include changes in promoter accessibility, changes in promoter chromatin architecture, and changes in the accessibility of specific transcription factor binding sites. In many cases, changes in chromatin architecture are coupled to physiological diversity, including the evolution of a respiration- or fermentation-based lifestyle, mating behavior, salt tolerance, and broad aspects of genomic structure. Together, our data will provide a rich resource for future investigations into the interplay between chromatin structure, gene regulation, and evolution.

Epigenetic control of signaling networks involved in interleukin-18-induced cardiac hypertrophy and its attenuation by pan-histone deacetylase inhibitors

Background Daily intra-peritoneal injection of IL-18 for one week in Balb/c mice led to the development of severe cardiac hypertrophy that was accompanied by induction of fetal gene expression. Both anatomical and molecular features of cardiac hypertrophy were ameliorated by pan-histone deacetylase inhibitors (HDACIs), m-carboxycinnamic acid bis-hydroxamide (CBHA) and trichostatin A (TSA). Materials and methods Treatment of mice with IL-18, with or without CBHA or TSA, caused dynamic remodeling of cardiac chromatin as judged by altered posttranslational modifications of the constituent histones. Administration of either TSA or CBHA in mice, even in the presence of IL-18, induced hyper-acetylation of histone H3 at lysine 9 demonstrating the unique action of HDAC inhibitors. Epigenetic changes in chromatin architecture were accompanied by reprogramming of cardiac hypertrophy-specific gene expression. Comparative profiles of cardiac gene expression of vehicle-treated mice with animals that received IL-18±CBHA or TSA revealed that 184 genes were differentially expressed genes (DEGs) and a subset of 38 genes was oppositely regulated by IL-18 and TSA. In contrast, the hearts of mice treated with IL-18 and/or CBHA elicited 147 DEGs; IL-18 robustly up-regulated 58 genes that were blocked by CBHA or IL-18+CBHA. Results and conclusion Ingenuity Pathway Analysis of DEGs revealed that both HDACIs profoundly impacted a number of intracellular signaling pathways. Global gene expression analyses of H9c2 cardiac myocytes incubated with CBHA or TSA for 6 and 24 hr were also carried out. We observed that the signatures of DEGs evoked by TSA and CBHA after 6 and at 24h treatment were unique. Nevertheless, it appeared that both HDACIs altered the course of PI3KPTEN-Akt/PKB, Ca ++ /calmodulin and p38-ERK-MAPKJNK signaling pathways to regulate gene expression via activation of Ap1, HNF4A, Myc and NFkB. Informed by the putative signaling networks deduced from the DEGs seen in the heart, we investigated the expression of phosphatase and tensin homolog (PTEN), a key regulator of the PI3K-AKT/PKB-NFkB pathway. Both TSA and CBHA induced robust expression of PTEN mRNA and protein in the heart. Based on these data we conclude that both TSA and CBHA attenuated anatomical and functional manifestations of IL-18-induced cardiac hypertrophy by epigenetic induction of PTEN to selectively block the PI3K-AKT/PKB-NFkB signaling.

Abstract 3935: H3K56 is deacetylated in response to UV-irradiation and recovery of acetylation is regulated by ATR and ATM checkpoint kinases

Abstract DNA damage results in cell cycle arrest by checkpoint activation until successful completion of DNA repair. Cell cycle arrest and DNA repair processes involve changes in chromatin architecture, which help access of proteins involved in these processes. After DNA repair, the chromatin structure and cell cycle progression are restored, but the mechanism is largely unknown. Recent studies implicated a role of histone chaperones, CAF1 and ASF1 in DNA damage repair through histone modification as well as histone assembly and disassembly. CAF1 depletion can lead to a G1 and S phase checkpoint response, in an ATR-dependent manner. In yeast, ASF1 chaperone associates with Rtt109 HAT complex which is required for H3K56 acetylation and is important for checkpoint recovery. Recently, Das et. al. showed that mammalian ASF1 interacts with p300/CBP histone acetylase and acetylates H3K56, which is deposited at the damage site in a CAF1-dependent manner. In contrast, Tjeertes et. al. showed that H3K56Ac is present in undamaged cells and is reduced in response to DNA damage. Due to the contradictory nature of H3K56 acetylation from these studies, we wanted to establish a solid foundation related to the nature of this important UV damage response event in human cells. For this, cells were irradiated and the status of H3K56 acetylation was determined immediately and up to 48 hr after UV-irradiation. Our data unambiguously show that in comparison to the steady levels of control actin, H3K56 acetylation decreased gradually and was completely eliminated at 24 hr following 20 J/m2 UV dose. However, H3K56 acetylation exhibited full recovery after 48 hr at 2.5, 5 and 10 J/m2 UV doses. Thus, UV-irradiation causes H3K56 deacetylation, which is promptly restored possibly due to the successful repair and checkpoint recovery. We observed a similar effect on H3K56 deacetylation in response to UV damage using NHF cells. Since, H3K56 acetylation has been implicated in checkpoint recovery after completion of DNA repair, we tested whether H3K56 acetylation/deacetylation is regulated by ATR and ATM checkpoint kinases. Interestingly, in Seckel cells (lower ATR level) H3K56 acetylation is dramatically reduced even in the absence of UV-irradiation suggesting that H3K56 acetylation is regulated by ATR. In contrast, in A-T cells (ATM-deficient), H3K56 acetylation did not change in the absence of UV-irradiation. In A-T cells, H3K56 is deacetylated in response to UV-irradiation as in NHF cells, but the acetylation did not recover after 48 hr post-repair. These data clearly indicate a role of ATR/ATM in recovery of H3K56 acetylation. We propose that H3K56 acetylation presents a histone mark needed for ATR/ATM-mediated checkpoint recovery. (This work was supported by NIH grants CA93413, ES2388 and ES12991) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3935.

Drosophila Heat Shock System as a General Model to Investigate Transcriptional Regulation

Whereas the regulation of a gene is uniquely tailored to respond to specific biological needs, general transcriptional mechanisms are used by diversely regulated genes within and across species. The primary mode of regulation is achieved by modulating specific steps in the transcription cycle of RNA polymerase II (Pol II). Pol II "pausing" has recently been identified as a prevalent rate-limiting and regulated step in the transcription cycle. Many sequence-specific transcription factors (TFs) modulate the duration of the pause by directly or indirectly recruiting positive transcription elongation factor b (P-TEFb) kinase, which promotes escape of Pol II from the pause into productive elongation. These specialized TFs find their target-binding sites by discriminating between DNA sequence elements based on the chromatin context in which these elements reside and can result in productive changes in gene expression or nonfunctional "promiscuous" binding. The binding of a TF can precipitate drastic changes in chromatin architecture that can be both dependent and independent of active Pol II transcription. Here, we highlight heat-shock-mediated gene transcription as a model system in which to study common mechanistic features of gene regulation.

Activation of ATM depends on chromatin interactions occurring before induction of DNA damage

Efficient and correct responses to double stranded breaks (DSB) in chromosomal DNA are critical for maintaining genomic stability and preventing chromosomal alterations leading to cancer1. The generation of DSB is associated with structural changes in chromatin and the activation of the protein kinase ataxia-telangiectasia mutated (ATM), a key regulator of the signaling network of the cellular response to DSB 2,3. The interrelationship between DSB-induced changes in chromatin architecture and the activation of ATM is unclear 3. Here we show that the nucleosome-binding protein HMGN1 modulates the interaction of ATM with chromatin both prior to and after DSB formation thereby optimizing its activation. Loss of HMGN1, or ablation of its ability to bind to chromatin, reduces the levels of IR-induced ATM autophosphorylation and the activation of several ATM targets. IR treatments lead to a global increase in the acetylation of Lys14 of histone H3 (H3K14) in an HMGN1 dependent manner and treatment of cells with a histone deacetylase inhibitor bypasses the HMGN1 requirement for efficient ATM activation. Thus, by regulating the levels of histone modifications, HMGN1 affects ATM activation. Our studies identify a new mediator of ATM activation and demonstrate a direct link between the steady-state intranuclear organization of ATM and the kinetics of its activation following DNA damage.

The coactivators CBP/p300 and the histone chaperone NAP1 promote transcription-independent nucleosome eviction at the HTLV-1 promoter

The human T cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma. The multifunctional virally encoded oncoprotein Tax is responsible for malignant transformation and potent activation of HTLV-1 transcription. Tax, in complex with phosphorylated cAMP response element binding protein (pCREB), strongly recruits the cellular coactivators CREB binding protein (CBP)/p300 to the viral promoter concomitant with transcriptional activation. Although the mechanism of activator/coactivator-mediated transcriptional activation is poorly understood, the recruitment of CBP/p300 by regulatory factors appears to function, in part, by promoting changes in chromatin architecture that are permissive to transcriptional activation. Here, we show that CBP/p300 recruitment promotes histone acetylation and eviction of the histone octamer from the chromatin-assembled HTLV-1 promoter template in vitro. Nucleosome disassembly is strictly acetyl-CoA dependent and is not linked to ATP utilization. We find that the histone chaperone, nucleosome assembly protein 1 (NAP1), cooperates with CBP/p300 in eviction of the acetylated histones from the chromatin template. These findings reveal a unique mechanism in which the DNA-bound Tax/pCREB complex recruits CBP/p300, and together with NAP1, the coactivators cooperate to dramatically reduce nucleosome occupancy at the viral promoter in an acetylation-dependent and transcription-independent fashion.

DNA Methylation in Health, Disease, and Cancer

The spatial arrangement and three-dimensional structure of DNA in the nucleus is controlled through the interdigitation of DNA binding proteins such as histones and their modifiers, the Polycomb-Trithorax proteins, and the DNA methyltransferase enzymes. DNA methylation forms the foundation of chromatin and is crucial to epigenetic gene regulation in mammals. Disease pathogenesis mediated through infectious agents, inflammation, aging, or genetic damage often involves changes in gene expression. In particular, cellular transformation coincides with multiple changes in chromatin architecture, many of which appear to affect genome integrity and gene expression. Infectious agents, such as viruses directly affect genome structure and induce methylation of particular sequences to suppress host immune responses. Hyperproliferative tissues such as those in the gastrointestinal tract and colon have been shown to gradually acquire aberrant promoter hypermethylation. Here we review recent findings on altered DNA methylation in human disease, with particular focus on cancer and the increasingly large number of genes subject to tumor-specific promoter hypermethylation and the possible role of aberrant methylation in tumor development.

Rtt109 Is Required for Proper H3K56 Acetylation

Histone acetylation has been shown to be required for the proper regulation of many cellular processes including transcription, DNA repair, and chromatin assembly. Acetylation of histone H3 on lysine 56 (H3K56) occurs both during the premeiotic and mitotic S phase and persists throughout DNA damage repair. To learn more about the molecular mechanism of H3K56 acetylation and factors required for this process, we surveyed the genome of the yeast Saccharomyces cerevisiae to identify genes necessary for this process. A comparative global proteomic screen identified several factors required for global H3K56 acetylation, which included histone chaperone Asf1 and a protein of an unknown function Rtt109 but not Spt10. Our results indicate that the loss of Rtt109 results in the loss of H3K56 acetylation, both on bulk histone and on chromatin, similar to that of asf1Delta or the K56Q mutation. RTT109 deletion exhibits sensitivity to DNA damaging agents similar to that of asf1Delta and H3K56Q mutants. Furthermore, Rtt109 and H3K56 acetylation appear to correlate with actively transcribed genes and associate with the elongating form of polymerase II in yeast. This histone modification is also associated with some of the transcriptionally active puff sites in Drosophila. Our results indicate a new role for the Rtt109 protein in the proper regulation of H3K56 acetylation.

Packing for the germy: the role of histone H4 Ser1 phosphorylation in chromatin compaction and germ cell development: Figure 1.

Genetic and epigenetic information is passed to the next generation through germ cells. In this issue of Genes and Development, Krishnamoorthy et al. (2006) have elegantly demonstrated that a conserved histone modification, phosphorylation of Ser1 on histone 4 (H4 S1ph) is involved in chromatin compaction during sporulation in yeast, and that it is an evolutionarily conserved mark found during Drosophila melanogaster and mouse germ cell development. Post-translational modifications of core histones (PTMs) and incorporation of histone variants/replacement proteins during gametogenesis provide an exceptional example of the relationship between chromatin structure and function. In these instances, histones fulfill their more traditional role as structural regulators and DNA packaging proteins, while providing modificationor variant-specific regulation. Significant changes in chromatin architecture are mediated in part by evolutionarily conserved modifications of histone within the nucleosomes, including ubiquitination, methylation, acetylation, and phosphorylation (Workman and Kingston 1998; Shilatifard 2006). These histone modifications serve to alter chromatin structure and thus regulate transcription factor accessibility to chromatin and affect transcriptional readout. During metazoan gametogenesis, cells incorporate somatic and gamete-specific histone variants, as well as generalized histone modifications into their chromatin prior to replacement by DNA packaging proteins (Lewis et al. 2003). Spermatogenesis in Drosophila requires the differentiation of round spermatids into an elongated, needle-like structure, with a 200-fold drop in nuclear volume. This is accomplished in part by modification of histones, as well as a highly orchestrated succession of histone replacements by sperm-specific histone variants, then by basic transition proteins, and finally protamines (Sassone-Corsi 2002; Govin et al. 2004; Kimmins and Sassone-Corsi 2005). The appearance of these new histone subtypes and specific histone modification patterns and their correlation with genome compaction, sperm function, and increased fertility, comprise a germ cellspecific histone modification pattern that alters chromatin structure during meiosis. Saccharomyces cerevisae must protect its genome from damage while simultaneously replicating, recombining, and properly segregating haploid genomes to individual spores. In response to nutrient deprivation, diploid S. cerevisae cells undergo meiosis to produce four stable, metabolically inert haploid spores. Of ∼6000 protein-encoding genes in yeast, >1000 show significant changes in expression during the four stages of sporulation—early, middle, mid-late, and late (Chu et al. 1998). These stages result from a sequential cascade of transcription factors and a specialized mitogen-activated protein (MAP) kinase signaling pathway. During meiosis there is strong correlation between a gene’s expression pattern and its involvement in a particular biological process. Early genes are typically involved in pairing of homologous chromosomes and recombination, middle genes are required for nuclear division and spore formation, and mid-late genes include those necessary for the outer layer of the spore wall (Chu et al. 1998). The metaphase peak of post-translational phosphorylation of S10 of histone H3 from late G2 to telophase is a hallmark of mitosis and meiosis in yeast and metazoan species, and this particular modification correlates with chromatin condensation (Hsu et al. 2000; Nowak and Corces 2004). However, mutations altering S10 of H3 do not alter mitosis, meiosis, or cell growth, suggesting a possible redundancy of its function (Hsu et al. 2000; Barber et al. 2004). In this issue of Genes and Development, Krishnamoorthy et al. (2006) re-examined the function of the previously described phosphorylation of S1 of histone H4 in chromatin compaction during sporulation. They found H4Ser1ph to be required for proper chromatin compaction during sporulation, and also for gametogenesis. It is significant that this histone modification is evolutionarily conserved, from Drosophila to mammals. Corresponding author. E-MAIL shilatia@slu.edu; FAX (314) 977-5737. Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1477706.

Epigenetic Regulation during B Cell Differentiation Controls CIITA Promoter Accessibility

B cell to plasma cell maturation is marked by the loss of MHC class II expression. This loss is due to the silencing of the MHC class II transcriptional coactivator CIITA. In this study, experiments to identify the molecular mechanism responsible for CIITA silencing were conducted. CIITA is expressed from four promoters in humans, of which promoter III (pIII) controls the majority of B cell-mediated expression. Chromatin immunoprecipitation assays were used to establish the histone code for pIII and determine the differences between B cells and plasma cells. Specific histone modifications associated with accessible promoters and transcriptionally active genes were observed at pIII in B cells but not in plasma cells. A reciprocal exchange of histone H3 lysine 9 acetylation to methylation was also observed between B cells and plasma cells. The lack of histone acetylation correlated with an absence of transcription factor binding to pIII, particularly that of Sp1, PU.1, CREB, and E47. Intriguingly, changes in chromatin architecture of the 13-kb region encompassing all CIITA promoters showed a remarkable deficit in histone H3 and H4 acetylation in plasma cells, suggesting that the mechanism of silencing is global. When primary B cells were differentiated ex vivo, most of the histone marks associated with pIII activation and expression were lost within 24 h. The results demonstrate that CIITA silencing occurs by controlling chromatin accessibility through a multistep mechanism that includes the loss of histone acetylation and transcription factor binding, and the acquisition of repressive histone methylation marks.