Advertisement

Identification of a Functional Network of Human Epigenetic Silencing Factors*

  • Andrey Poleshko
    Footnotes
    Affiliations
    Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
    Search for articles by this author
  • Margret B. Einarson
    Affiliations
    Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
    Search for articles by this author
  • Natalia Shalginskikh
    Affiliations
    Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
    Search for articles by this author
  • Rugang Zhang
    Affiliations
    Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
    Search for articles by this author
  • Peter D. Adams
    Footnotes
    Affiliations
    Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
    Search for articles by this author
  • Anna Marie Skalka
    Affiliations
    Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
    Search for articles by this author
  • Richard A. Katz
    Correspondence
    Supported by the Pennsylvania Department of Health and by NIH Grants NS053666 and DK082498. To whom correspondence should be addressed: Institute for Cancer Research, Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111. Tel.: 215-728-3668; Fax: 215-728-2778;
    Affiliations
    Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants CA71515 and CA06927. This work was also supported by an appropriation from the Commonwealth of Pennsylvania.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S6.
    1 Recipient of an American Association for Cancer Research Centennial Predoctoral Fellowship in Cancer Research.
    2 Present address: The Beatson Institute for Cancer Research, Garscube Estate, Switchback Rd., Bearsden, Glasgow G61 1BD, United Kingdom.
Open AccessPublished:October 30, 2009DOI:https://doi.org/10.1074/jbc.M109.064667
      Epigenetic silencing is mediated by families of factors that place, remove, read, and transmit repressive histone and DNA methylation marks on chromatin. How the roles for these functionally diverse factors are specified and integrated is the subject of intense study. To address these questions, HeLa cells harboring epigenetically silent green fluorescent protein reporter genes were interrogated with a small interference RNA library targeting 200 predicted epigenetic regulators, including potential activators, silencers, chromatin remodelers, and ancillary factors. Using this approach, individual, or combinatorial requirements for specific epigenetic silencing factors could be detected by measuring green fluorescent protein reactivation after small interference RNA-based factor knockdown. In our analyses, we identified a specific subset of 15 epigenetic factors that are candidates for participation in a functional epigenetic silencing network in human cells. These factors include histone deacetylase 1, de novo DNA methyltransferase 3A, components of the polycomb PRC1 complex (RING1 and HPH2), and the histone lysine methyltransferases KMT1E and KMT5C. Roles were also detected for two TRIM protein family members, the cohesin component Rad21, and the histone chaperone CHAF1A (CAF-1 p150). Remarkably, combinatorial knockdown of factors was not required for reactivation, indicating little functional redundancy. Consistent with this interpretation, knockdown of either KMT1E or CHAF1A resulted in a loss of multiple histone-repressive marks and concomitant gain of activation marks on the promoter during reactivation. These results reveal how functionally diverse factors may cooperate to maintain gene silencing during normal development or in disease. Furthermore, the findings suggest an avenue for discovery of new targets for epigenetic therapies.

      Introduction

      Epigenetic processes control the binary on-off states of specific gene sets, thereby creating heritable transcription patterns that drive development and maintain cellular identity. It is now well established that chromatin-based mechanisms underlie such epigenetic control, largely mediated by intricate chemical marks that are placed or removed by chromatin-modifying enzymes (
      • Bernstein B.E.
      • Meissner A.
      • Lander E.S.
      ,
      • Grewal S.I.
      • Jia S.
      ,
      • Klose R.J.
      • Bird A.P.
      ,
      • Kouzarides T.
      ,
      • Martin C.
      • Zhang Y.
      ,
      • Strahl B.D.
      • Allis C.D.
      ,
      • Taverna S.D.
      • Li H.
      • Ruthenburg A.J.
      • Allis C.D.
      • Patel D.J.
      ,
      • Shi Y.
      • Whetstine J.R.
      ).
      The prominent epigenetic regulatory marks on eukaryotic chromatin are histone modifications and DNA cytosine methylation (5meCpG), which are placed by enzyme complexes containing members of the histone modifying and DNA methyltransferase (DNMT)
      The abbreviations used are: DNMT
      DNA methyltransferase
      DNMTi
      DNMT inhibitor
      GFP
      green fluorescent protein
      siRNA
      small interfering RNA
      FACS
      fluorescence-activated cell sorting
      LTR
      long terminal repeat
      CMV
      cytomegalovirus
      HDAC
      histone deacetylase
      HDACi
      HDAC inhibitor
      MBD
      methyl-CpG-binding protein
      KAT
      lysine acetyltransferase
      KMT
      lysine methyltransferase
      KDM
      lysine demethylase
      KMT
      lysine methyltransferase
      PcG
      polycomb group
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      ChIP
      chromatin immunoprecipitation
      TIF1
      transcriptional intermediary factor 1
      TSA
      trichostatin A
      5-azaC
      5-azacytidine
      TRIM
      tripartite motif
      CAF-1
      chromatin assembly factor-1
      PRC1
      polycomb repressor complex
      EF1α
      elongation factor 1α
      H3K9
      histone H3 lysine 9
      pol
      polymerase
      TSS
      transcriptional start site.
      families, respectively. The histone tails that protrude from nucleosomes are thus decorated by a variety of position-specific “histone code” marks, including acetyl, methyl, and ubiquitin lysine modifications. In contrast, DNA-based epigenetic regulation is limited to DNA methylation. Histone marks may be either activating or repressive, whereas the 5meCpG DNA mark is strictly repressive. The presence or absence of these chromatin marks provide cues for recruitment of downstream protein effectors that positively or negatively affect transcription or may also directly influence chromatin structure and function.
      The heritable transcriptional off-state is denoted “epigenetic silencing.” The corresponding silent regions are generally characterized by hypoacetylated histones, histone H3 lysine 9 (H3K9) di- or trimethylation (H3K9me2/3), and DNA hypermethylation. These marks guide the formation of heterochromatic-like features over gene promoters or broader areas (
      • Grewal S.I.
      • Jia S.
      ). Histone deacetylases (HDACs) are generally viewed as repressive epigenetic regulators that maintain the hypoacetylated histone state, thereby antagonizing the transcription-promoting activities of lysine acetyltransferases (KATs). The repressive H3K9 methyl histone mark is placed by lysine methyltranferases (KMTs) and is read by the effector heterochromatin protein 1 (
      • Grewal S.I.
      • Jia S.
      ). Repressive methyl marks can potentially be antagonized by lysine demethylase (KDM) activities (
      • Shi Y.
      • Whetstine J.R.
      ). The 5meCpG DNA marks, placed by DNMT enzymes, are read by methyl-CpG-binding domain proteins (MBDs) to promote silencing (
      • Klose R.J.
      • Bird A.P.
      ). Both HDAC- and DNMT-based epigenetic silencing can sometimes be reversed by chemical inhibitors (
      • Bolden J.E.
      • Peart M.J.
      • Johnstone R.W.
      ,
      • Esteller M.
      ,
      • Feinberg A.P.
      • Tycko B.
      ,
      • Hake S.B.
      • Xiao A.
      • Allis C.D.
      ,
      • Jones P.A.
      • Baylin S.B.
      ,
      • Marks P.A.
      • Breslow R.
      ,
      • Yoo C.B.
      • Jones P.A.
      ). HDAC inhibitors (HDACi) act by favoring KAT-mediated activating acetylation marks, whereas DNMT inhibitors (DNMTi) cause a passive loss of the repressive DNA methylation marks during cell division.
      Deregulation of epigenetic silencing can lead to inappropriate shut-off of specific genes, a process that underlies a variety of human diseases, including cancer (
      • Esteller M.
      ,
      • Feinberg A.P.
      • Tycko B.
      ,
      • Hake S.B.
      • Xiao A.
      • Allis C.D.
      ,
      • Jones P.A.
      • Baylin S.B.
      ,
      • Yoo C.B.
      • Jones P.A.
      ,
      • Feinberg A.P.
      ). In addition, silencing of viral genomes by epigenetic mechanisms can contribute to pathogenesis by promoting a latent viral state (
      • Lieberman P.M.
      ). In both cases, reversal of epigenetic silencing by inhibitors (e.g. HDACi) may provide therapeutic benefits (
      • Bolden J.E.
      • Peart M.J.
      • Johnstone R.W.
      ,
      • Esteller M.
      ,
      • Feinberg A.P.
      • Tycko B.
      ,
      • Hake S.B.
      • Xiao A.
      • Allis C.D.
      ,
      • Jones P.A.
      • Baylin S.B.
      ,
      • Marks P.A.
      • Breslow R.
      ,
      • Yoo C.B.
      • Jones P.A.
      ). There is substantial interest in identifying functional networks of epigenetic silencing factors, because such knowledge may provide additional therapeutic targets. However, the processes surrounding enzymatic placement and removal, as well as decoding, of epigenetic marks are highly complex. For example, a variety of combinatorial, temporal, dynamic, and context-dependent histone modifications have been described (
      • Grewal S.I.
      • Jia S.
      ,
      • Klose R.J.
      • Bird A.P.
      ,
      • Kouzarides T.
      ,
      • Martin C.
      • Zhang Y.
      ,
      • Taverna S.D.
      • Li H.
      • Ruthenburg A.J.
      • Allis C.D.
      • Patel D.J.
      ,
      • Berger S.L.
      ,
      • Ruthenburg A.J.
      • Allis C.D.
      • Wysocka J.
      ,
      • Ruthenburg A.J.
      • Li H.
      • Patel D.J.
      • Allis C.D.
      ,
      • Li B.
      • Carey M.
      • Workman J.L.
      ). In view of these vast complexities, we have implemented a functional assay to identify silencing factor repertoires. This strategy has uncovered an epigenetic network in human cells and provides a general method for the identification of factors that may serve as targets for epigenetic therapies.

      DISCUSSION

      The mechanisms that underlie epigenetic control include DNA methylation and histone modification. Recent work has revealed that the placement and decoding of these modifications is a more intricate process than once believed, characterized by intra- and inter-histone modification cross-talk (
      • Cedar H.
      • Bergman Y.
      ,
      • Fischle W.
      ), histone-DNA methylation cross-talk (
      • Cedar H.
      • Bergman Y.
      ,
      • Fischle W.
      ), and dynamic histone modifications during transcription (
      • Berger S.L.
      ,
      • Li B.
      • Carey M.
      • Workman J.L.
      ). A detailed understanding of these processes is important, because deregulation of epigenetic modifications can cause disease. In particular, inappropriate epigenetic silencing may lead to various disease states, and the reversal of silencing appears to be an avenue for therapeutic intervention (
      • Bolden J.E.
      • Peart M.J.
      • Johnstone R.W.
      ,
      • Esteller M.
      ,
      • Feinberg A.P.
      • Tycko B.
      ,
      • Hake S.B.
      • Xiao A.
      • Allis C.D.
      ,
      • Jones P.A.
      • Baylin S.B.
      ,
      • Marks P.A.
      • Breslow R.
      ,
      • Yoo C.B.
      • Jones P.A.
      ). Epigenetic drugs, such as HDACi and DNMTi, relieve epigenetic silencing by inhibiting (or depleting) enzymes that modify chromatin; however, current epigenetic therapy targets are limited to these two enzyme families. Our previous study provided a proof-of-concept for the use of a siRNA-based knockdown strategy to identify functional roles for epigenetic silencing factors (
      • Poleshko A.
      • Palagin I.
      • Zhang R.
      • Boimel P.
      • Castagna C.
      • Adams P.D.
      • Skalka A.M.
      • Katz R.A.
      ). In this report we implemented a functional screen to determine the identity, and potential breadth, of epigenetic silencing factor networks in a human cell line.
      Our approach employed a comprehensive siRNA library targeting human epigenetic regulators. The goal was to identify which, among the set of diverse factors, were critical to maintain silencing in the human HeLa cell line. A gene-by-gene siRNA interrogation was used, based on the validated principle that knockdown of essential silencing factors would result in reactivation of reporter genes. In this system, the GFP-silent reporter serves as a beacon to monitor the effects of global depletion of epigenetic regulators and enables a one-step, quantifiable assay. The results were validated at several levels, including analyses of up to four to seven independent siRNAs per target gene (supplemental Figs. S1 and S2). The readout was not binary, because the GFP signal produced during reactivation showed characteristic target-specific trends. For example, individual KMT1E and CHAF1A siRNAs consistently produced more robust GFP signals, as compared with DNMT3A siRNAs. These target-specific differences may reflect each factor's particular function, abundance, or half-life after mRNA knockdown.
      We considered whether experimental parameters might contribute to the factor set that we have identified. First, the silent GFP reporter genes in the cell population used in our study are widely dispersed (
      • Cedar H.
      • Bergman Y.
      ,
      • Fischle W.
      ), presumably neutralizing position-specific factor roles. Indeed, analyses of cell populations harboring dispersed reporter genes, or individual cell clones harboring reporter genes at a limited number of sites (Fig. 4), indicate that the factor set is highly independent of the chromosomal position of the reporter gene. One interpretation of these results is that the set represents a functional repertoire of factors that predominates in HeLa cells. Our analyses included reporter cells that utilize different promoters to drive the silent GFP gene, and the factor hits were largely promoter-independent (Table 1); as such, the factor set does not appear to represent a promoter-specific transcriptional corepressor complex. However, some differences were noted, the most significant of which appears to be a reduced role for KMT1E for silencing of the EF1α promoter-driven GFP gene. Another feature of the reporter system that might contribute to factor roles was the selection of GFP-silent cells by transient treatment with an HDACi (TSA) or a DNMTi (5-azaC). These selection strategies provided a strong technical advantage, as a means to enrich for a cell population in which all cells harbored silent reporter genes. However, selection for either HDAC- or DNMT-mediated silencing revealed a similar factor set by siRNA screening. We conclude, overall, that the factor set that we have identified is promoter and position independent, and is not biased by the reporter cell selection procedure. In fact, the similar factor sets identified using DNMTi and HDACi selection supports the idea of significant cross-talk between the DNA methylation and histone modification machineries (
      • Cedar H.
      • Bergman Y.
      ,
      • Fischle W.
      ).
      Our experimental strategy proved to be robust, because siRNA analyses of the 200 candidates yielded a validated set of 15 hits (Table 1 and Fig. 8). The hits included members of diverse protein families. To obtain an overview of the known relationships among the hits, we used STRING (available on-line), a data base of protein interactions. This analysis detected a core network of factor interactions that include CHAF1A, MBD1, MBD3, KMT1E, HDAC1, and DNMT3A, as well as others. In supplemental Fig. S6 we provide a depiction of these interactions. A general model for roles for these and other factors is shown in Fig. 8. The potential functional roles of factors, and their interactions, are discussed below.
      Figure thumbnail gr8
      FIGURE 8Summary of selected hits, displayed as a physical model, based on obtained and published results. The model summarizes roles for histone modifiers and displays highly stylized and generic interactions in some cases.
      Two factors identified in the screen, KMT1E (SETDB1) and KMT5C (SUV420H2), are enzymes that are predicted to place repressive histone methyl modifications, H3K9me3 and H4K20me3, respectively. H3K9 methylation recruits heterochromatin protein 1 to mediate silencing, while the H4K20 methylation may block an activating modification, acetylation of histone H4 (
      • Latham J.A.
      • Dent S.Y.
      ). These marks were detected on the GFP-silent promoter by ChIP analysis (Fig. 6, A and B). We interpret these results to indicate that the activities of these enzymes are required to maintain these repressive marks on the GFP-silent promoter and, accordingly, the silent state of the GFP gene.
      We also detected requirements for PRC1 proteins, RING1 and a partner HPH2, consistent with their predicted function in placement of the repressive monoubiquitin mark on histone H2A (H2Aub, Fig. 8) (
      • Wang H.
      • Wang L.
      • Erdjument-Bromage H.
      • Vidal M.
      • Tempst P.
      • Jones R.S.
      • Zhang Y.
      ). Taken together with the findings described above, a constellation of repressive marks on histones H3, H4, and H2A appears to be required to maintain silencing (Fig. 8). Roles were also detected for two histone demethylase enzymes KDM2A and KDM4A that may promote silencing by removal of H3K36me2/3 modifications that are associated with the bodies of transcriptionally active genes (
      • Allis C.D.
      • Berger S.L.
      • Cote J.
      • Dent S.
      • Jenuwien T.
      • Kouzarides T.
      • Pillus L.
      • Reinberg D.
      • Shi Y.
      • Shiekhattar R.
      • Shilatifard A.
      • Workman J.
      • Zhang Y.
      ,
      • Frescas D.
      • Guardavaccaro D.
      • Kuchay S.M.
      • Kato H.
      • Poleshko A.
      • Basrur V.
      • Elenitoba-Johnson K.S.
      • Katz R.A.
      • Pagano M.
      ).
      Numerous examples of bidirectional cross-talk between the DNA methylation and histone modification machineries have been described (
      • Cedar H.
      • Bergman Y.
      ,
      • Fischle W.
      ), and such interactions are believed to reflect a self-reinforcing silencing mechanism (
      • Feinberg A.P.
      • Tycko B.
      ). Here, we derived GFP-silent reporter cells that were selected by response to an HDACi. However, siRNA analyses revealed roles for KMT1E, DNMT3A, and repressive MBD proteins (MBD1 and MBD3) among other factors (Fig. 8). siRNA analyses of GFP-silent reporter cells that were derived by reciprocal selection with the DNMT inhibitor 5-azaC also revealed roles for DNMT3A and KMT1E, as well as several other factors common to the HDACi selection strategy (Fig. 5). These results provide strong support for cross-talk between the DNA methylation and histone modification machineries in maintaining silencing in this system.
      The siRNA library targeted nine histone chaperones and chromatin assembly factors, including CHAF1A. Knockdown of CHAF1A, but not the two other CAF-1 subunits (p60 and p48), nor other chaperones, resulted in reporter gene reactivation. Knockdown of CHAF1A also provoked (i) a coordinated loss of the repressive H3K9me3 and H4K20me3 histone marks, (ii) an increase in histone acetyl activating marks, and (iii) an increase RNA pol II occupancy (Fig. 6B). Furthermore, reactivation by CHAF1A siRNA was inhibited in S-phase-arrested cells (Fig. 6C). In contrast, such cell cycle arrest slightly enhanced GFP reactivation by siRNAs targeting several other factors. Taken together, our results favor a model whereby CHAF1A plays a role in maintenance of epigenetic silencing through S-phase. This role is seemingly not limited to the maintenance of the H3K9me3 mark, as we also observed loss of the H4K20me3 mark. Interestingly, mouse embryonic cells that are deficient or null for CHAF1A show a reduction of both H3K9me3 and H4K20me3 marks in pericentric heterochromatin (
      • Quivy J.P.
      • Gérard A.
      • Cook A.J.
      • Roche D.
      • Almouzni G.
      ). Here we found that CHAF1A plays a major role in maintenance of these marks in non-embryonic human cells. Importantly, our results demonstrate a correlation between CHAF1A function, histone marks, and maintenance of gene silencing through S-phase. The discrimination of such a role illustrates the ability of this system to detect highly diverse, but coordinated, functions of silencing factors. Indeed, three factors that we identified, MBD1, CHAF1A, and KMT1E, have been implicated in collaborating during S-phase to transmit the H3K9me mark to replicated chromatin, as guided by DNA methylation (Fig. 8) (
      • Sarraf S.A.
      • Stancheva I.
      ).
      Based on our current findings, as well as previous studies (
      • Poleshko A.
      • Palagin I.
      • Zhang R.
      • Boimel P.
      • Castagna C.
      • Adams P.D.
      • Skalka A.M.
      • Katz R.A.
      ,
      • Narezkina A.
      • Taganov K.D.
      • Litwin S.
      • Stoyanova R.
      • Hayashi J.
      • Seeger C.
      • Skalka A.M.
      • Katz R.A.
      ), we believe that the silent GFP transgenes described here may provide a nucleation site for heterochromatin formation, which in turn leads to reporter gene silencing. In particular, the identification of coordinated H3K9 and H4K20 methylation marks, and their apparent maintenance by CHAF1A, supports this notion. We have also detected widespread DNA methylation within and around the GFP transgene (data not shown), again consistent with heterochromatin formation. The cues that initiate these processes are currently unknown. However, our experimental system has proven to be powerful for identification of factors that are required to sustain the silent state.
      Of the remaining factors, TRIM24 (TIF1α) and TRIM33 (TIF1γ) are interacting members of the TIF1 family of transcriptional regulators that can function as repressors (
      • Peng H.
      • Feldman I.
      • Rauscher 3rd, F.J.
      ), and RAD21 is a member of the cohesin family that has been implicated in transcriptional control (
      • Peters J.M.
      • Tedeschi A.
      • Schmitz J.
      ). Two factors, ZMYND8 (RACK7), a putative transcriptional regulator, and PBRM1 (BAF180), a component of the human SWI/SNF chromatin-remodeling complex PBAF, have not yet been directly implicated in epigenetic silencing.
      Our findings have revealed that each identified factor can be singularly targeted for depletion to promote reactivation of a silent gene. These results indicate a surprising lack of redundancy within, or between factor families. The apparent high level of interdependence among the functioning set may be explained, in part, by the fact that the presence of each factor is required to maintain one or more physical complexes. In this regard, it has been demonstrated that chromatin-modifying enzymes, in addition to their catalytic activities, can play a scaffolding role. For example, it has been shown that the HMT G9a (KMT1C) can contribute to DNA methylation in an activity-independent manner (
      • Dong K.B.
      • Maksakova I.A.
      • Mohn F.
      • Leung D.
      • Appanah R.
      • Lee S.
      • Yang H.W.
      • Lam L.L.
      • Mager D.L.
      • Schübeler D.
      • Tachibana M.
      • Shinkai Y.
      • Lorincz M.C.
      ).
      In summary, we have identified a set, or network, of specific factors that maintains epigenetic silencing in human cells. The composition of the set may reflect the particular functional repertoire of epigenetic silencing factors that are available in these cells. The experimental approach described herein may be extended to other human cell types, offering the possibility of identifying additional cell or disease type-specific factors. Because knockdown of any member of the set relieves silencing, such factors may serve as targets for the development of novel epigenetic therapies.

      Acknowledgments

      We benefited from the use of the following Fox Chase Cancer Center (FCCC) Facilities: Flow Cytometry and Cell Sorting, Translational Research, and Biostatistics and Bioinformatics. We thank Dr. Yan Zhou from the FCCC Biostatistics and Bioinformatics Facility for assistance. We are also extremely grateful to Dr. Andrew Kossenkov and Olga Tchuvatkina for providing critical advice related to statistical analyses. We also thank Drs. Tim Yen, Alfonso Bellacosa, Jeff Peterson, and Severin Gudima for comments on the manuscript. Lastly, we are grateful to Marie Estes for assistance in preparing the manuscript and Karen Trush for help with artwork.

      REFERENCES

        • Bernstein B.E.
        • Meissner A.
        • Lander E.S.
        Cell. 2007; 128: 669-681
        • Grewal S.I.
        • Jia S.
        Nat. Rev. Genet. 2007; 8: 35-46
        • Klose R.J.
        • Bird A.P.
        Trends Biochem. Sci. 2006; 31: 89-97
        • Kouzarides T.
        Cell. 2007; 128: 693-705
        • Martin C.
        • Zhang Y.
        Curr. Opin. Cell Biol. 2007; 19: 266-272
        • Strahl B.D.
        • Allis C.D.
        Nature. 2000; 403: 41-45
        • Taverna S.D.
        • Li H.
        • Ruthenburg A.J.
        • Allis C.D.
        • Patel D.J.
        Nat. Struct. Mol. Biol. 2007; 14: 1025-1040
        • Shi Y.
        • Whetstine J.R.
        Mol. Cell. 2007; 25: 1-14
        • Bolden J.E.
        • Peart M.J.
        • Johnstone R.W.
        Nat. Rev. Drug Discov. 2006; 5: 769-784
        • Esteller M.
        Br. J. Cancer. 2006; 94: 179-183
        • Feinberg A.P.
        • Tycko B.
        Nat. Rev. Cancer. 2004; 4: 143-153
        • Hake S.B.
        • Xiao A.
        • Allis C.D.
        Br. J. Cancer. 2007; 96: R31-R39
        • Jones P.A.
        • Baylin S.B.
        Cell. 2007; 128: 683-692
        • Marks P.A.
        • Breslow R.
        Nat. Biotechnol. 2007; 25: 84-90
        • Yoo C.B.
        • Jones P.A.
        Nat. Rev. Drug Discov. 2006; 5: 37-50
        • Feinberg A.P.
        Nature. 2007; 447: 433-440
        • Lieberman P.M.
        Trends Microbiol. 2006; 14: 132-140
        • Berger S.L.
        Nature. 2007; 447: 407-412
        • Ruthenburg A.J.
        • Allis C.D.
        • Wysocka J.
        Mol. Cell. 2007; 25: 15-30
        • Ruthenburg A.J.
        • Li H.
        • Patel D.J.
        • Allis C.D.
        Nat. Rev. Mol. Cell Biol. 2007; 8: 983-994
        • Li B.
        • Carey M.
        • Workman J.L.
        Cell. 2007; 128: 707-719
        • Katz R.A.
        • Jack-Scott E.
        • Narezkina A.
        • Palagin I.
        • Boimel P.
        • Kulkosky J.
        • Nicolas E.
        • Greger J.G.
        • Skalka A.M.
        J. Virol. 2007; 81: 2592-2604
        • Poleshko A.
        • Palagin I.
        • Zhang R.
        • Boimel P.
        • Castagna C.
        • Adams P.D.
        • Skalka A.M.
        • Katz R.A.
        J. Virol. 2008; 82: 2313-2323
        • Zhang J.H.
        • Chung T.D.
        • Oldenburg K.R.
        J. Biomol. Screen. 1999; 4: 67-73
        • Allis C.D.
        • Berger S.L.
        • Cote J.
        • Dent S.
        • Jenuwien T.
        • Kouzarides T.
        • Pillus L.
        • Reinberg D.
        • Shi Y.
        • Shiekhattar R.
        • Shilatifard A.
        • Workman J.
        • Zhang Y.
        Cell. 2007; 131: 633-636
        • Schultz D.C.
        • Ayyanathan K.
        • Negorev D.
        • Maul G.G.
        • Rauscher 3rd, F.J.
        Genes Dev. 2002; 16: 919-932
        • Frescas D.
        • Guardavaccaro D.
        • Kuchay S.M.
        • Kato H.
        • Poleshko A.
        • Basrur V.
        • Elenitoba-Johnson K.S.
        • Katz R.A.
        • Pagano M.
        Cell Cycle. 2008; 7: 3539-3547
        • Latham J.A.
        • Dent S.Y.
        Nat. Struct. Mol. Biol. 2007; 14: 1017-1024
        • Wang H.
        • Wang L.
        • Erdjument-Bromage H.
        • Vidal M.
        • Tempst P.
        • Jones R.S.
        • Zhang Y.
        Nature. 2004; 431: 873-878
        • Groothuis T.A.
        • Dantuma N.P.
        • Neefjes J.
        • Salomons F.A.
        Cell Div. 2006; 1: 21
        • Li H.
        • Rauch T.
        • Chen Z.X.
        • Szabó P.E.
        • Riggs A.D.
        • Pfeifer G.P.
        J. Biol. Chem. 2006; 281: 19489-19500
        • Cedar H.
        • Bergman Y.
        Nat. Rev. Genet. 2009; 10: 295-304
        • Fischle W.
        Genes Dev. 2008; 22: 3375-3382
        • Quivy J.P.
        • Gérard A.
        • Cook A.J.
        • Roche D.
        • Almouzni G.
        Nat. Struct. Mol. Biol. 2008; 15: 972-979
        • Sarraf S.A.
        • Stancheva I.
        Mol. Cell. 2004; 15: 595-605
        • Narezkina A.
        • Taganov K.D.
        • Litwin S.
        • Stoyanova R.
        • Hayashi J.
        • Seeger C.
        • Skalka A.M.
        • Katz R.A.
        J. Virol. 2004; 78: 11656-11663
        • Peng H.
        • Feldman I.
        • Rauscher 3rd, F.J.
        J. Mol. Biol. 2002; 320: 629-644
        • Peters J.M.
        • Tedeschi A.
        • Schmitz J.
        Genes Dev. 2008; 22: 3089-4114
        • Dong K.B.
        • Maksakova I.A.
        • Mohn F.
        • Leung D.
        • Appanah R.
        • Lee S.
        • Yang H.W.
        • Lam L.L.
        • Mager D.L.
        • Schübeler D.
        • Tachibana M.
        • Shinkai Y.
        • Lorincz M.C.
        EMBO J. 2008; 27: 2691-2701