Cross-talk between Histone Modifications in Response to Histone Deacetylase Inhibitors
Histones are subject to a wide variety of post-translational modifications that play a central role in gene activation and silencing. We have used histone modification-specific antibodies to demonstrate that two histone modifications involved in gene activation, histone H3 acetylation and H3 lysine 4 methylation, are functionally linked. This interaction, in which the extent of histone H3 acetylation determines both the abundance and the “degree” of H3K4 methylation, plays a major role in the epigenetic response to histone deacetylase inhibitors. A combination of in vivo knockdown experiments and in vitro methyltransferase assays shows that the abundance of H3K4 methylation is regulated by the activities of two opposing enzyme activities, the methyltransferase MLL4, which is stimulated by acetylated substrates, and a novel and as yet unidentified H3K4me3 demethylase. Histones are subject to a wide variety of post-translational modifications that play a central role in gene activation and silencing. We have used histone modification-specific antibodies to demonstrate that two histone modifications involved in gene activation, histone H3 acetylation and H3 lysine 4 methylation, are functionally linked. This interaction, in which the extent of histone H3 acetylation determines both the abundance and the “degree” of H3K4 methylation, plays a major role in the epigenetic response to histone deacetylase inhibitors. A combination of in vivo knockdown experiments and in vitro methyltransferase assays shows that the abundance of H3K4 methylation is regulated by the activities of two opposing enzyme activities, the methyltransferase MLL4, which is stimulated by acetylated substrates, and a novel and as yet unidentified H3K4me3 demethylase. A growing body of evidence suggests that many different types of post-translational histone modifications play key roles in regulating gene expression and that some modifications at least are functionally inter-related (1.Nightingale K.P. O’Neill L.P. Turner B.M. Curr. Opin. Genet. Dev. 2006; 16: 125-136Crossref PubMed Scopus (221) Google Scholar). The linked deposition of distinct modifications can occur both on the same histone tail, e.g. H3S10 phosphorylation and H3K9 acetylation (2.Thomson S. Clayton A.L. Mahadevan L.C. Mol. Cell. 2001; 8: 1231-1241Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) or on different tails, e.g. H2A ubiquitination and H3 methylation (3.Sun Z.W. Allis C.D. Nature. 2002; 418: 104-108Crossref PubMed Scopus (819) Google Scholar), histone acetylation, and methylation (4.Annunziato A.T. Eason M.B. Perry C.A. Biochemistry. 1995; 34: 2916-2924Crossref PubMed Scopus (66) Google Scholar, 5.Hendzel M.J. Davie J.R. Biochem. J. 1991; 273: 753-758Crossref PubMed Scopus (34) Google Scholar). Multiprotein complexes have been identified that are capable of depositing, or removing, different modifications in a coordinated manner (e.g. histone demethylase and deacetylase activity in coREST) (6.Lee M.G. Wynder C. Cooch N. Shiekhattar R. Nature. 2005; 437: 432-435Crossref PubMed Scopus (591) Google Scholar). Similarly, binding proteins are sensitive to combinations of modifications; for example, HP1 binding to the H3 tail requires histone H3K9 methylation but is blocked by H3S10 phosphorylation and H3K14 acetylation (Ref. 7.Mateescu B. England P. Halgand F. Yaniv M. Muchardt C. EMBO Rep. 2004; 5: 490-496Crossref PubMed Scopus (120) Google Scholar, although see Ref. 8.Fischle W. Tseng B.S. Dormann H.L. Ueberheide B.M. Garcia B.A. Shabanowitz J. Hunt D.F. Funabiki H. Allis C.D. Nature. 2005; 438: 1116-1122Crossref PubMed Scopus (724) Google Scholar). This suggests that epigenetic marks are not deposited or recognized in isolation but comprise a complex and inter-related collection of modifications at adjacent residues. The correlation between different histone modifications is particularly clear for histone H3 acetylation and the methylation of histone H3 lysine 4 (H3K4me). Mass spectrometric analysis of the cellular pool of histones indicates that this methyl mark is associated with histone H3 molecules containing high levels of acetylation (9.Zhang K. Siino J.S. Jones P.R. Yau P.M. Bradbury E.M. Proteomics. 2004; 4: 3765-3775Crossref PubMed Scopus (63) Google Scholar). This is consistent with the observed co-localization of these marks, which show related distribution patterns both at a chromosome-wide level during X inactivation (10.O’Neill L.P. Randall T.E. Lavender J. Spotswood H.T. Lee J.T. Turner B.M. Hum. Mol. Genet. 2003; 12: 1783-1790Crossref PubMed Scopus (26) Google Scholar) and over the coding regions of individual genes (11.Schubeler D. MacAlpine D.M. Scalzo D. Wirbelauer C. Kooperberg C. van Leeuwen F. Gottschling D.E. O’Neill L.P. Turner B.M. Delrow J. Bell S.P. Groudine M. Genes Dev. 2004; 18: 1263-1271Crossref PubMed Scopus (631) Google Scholar, 12.Pokholok D.K. Harbison C.T. Levine S. Cole M. Hannett N.M. Lee T.I. Bell G.W. Walker K. Rolfe P.A. Herbolsheimer E. Zeitlinger J. Lewitter F. Gifford D.K. Young R.A. Cell. 2005; 122: 517-527Abstract Full Text Full Text PDF PubMed Scopus (1083) Google Scholar). These correlations may arise due to physical links between histone-modifying enzymes such that they are co-recruited to the same loci. Both MLL1, a histone methyltransferase (HMT) 2The abbreviations used are: HMT, histone methyltransferase; HDAC, histone deacetylase; HDI, histone deacetylase inhibitor; PBS, phosphate-buffered saline; siRNA, small interfering RNA; TAU, Triton-acid-urea.2The abbreviations used are: HMT, histone methyltransferase; HDAC, histone deacetylase; HDI, histone deacetylase inhibitor; PBS, phosphate-buffered saline; siRNA, small interfering RNA; TAU, Triton-acid-urea. that can generate H3K4me marks (13.Milne T.A. Briggs S.D. Brock H.W. Martin M.E. Gibbs D. Allis C.D. Hess J.L. Mol. Cell. 2002; 10: 1107-1117Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar), and Chd1, the chromatin remodeler that is subsequently recruited by this methyl mark, associate with histone acetyltransferase activities (14.Pray-Grant M.G. Daniel J.A. Schieltz D. Yates III, J.R. Grant P.A. Nature. 2005; 433: 434-438Crossref PubMed Scopus (400) Google Scholar, 15.Sims R.J. Chen C.F. Santos-Rosa H. Kouzarides T. Patel S.S. Reinberg D. J. Biol. Chem. 2005; 280: 41789-41792Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), whereas the LSD1 complex that removes some of these methyl marks contains the histone deacetylases HDAC1 and HDAC2 (16.Schneider J. Wood A. Lee J.S. Schuster R. Dueker J. Maguire C. Swanson S.K. Florens L. Washburn M.P. Shilatifard A. Mol. Cell. 2005; 19: 849-856Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). However, the interaction could also arise due to the mechanism of action of these enzymes. For example, the SET domain of MLL1 has a preference for acetylated substrates (13.Milne T.A. Briggs S.D. Brock H.W. Martin M.E. Gibbs D. Allis C.D. Hess J.L. Mol. Cell. 2002; 10: 1107-1117Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar). A further layer of complexity is generated by the heterogeneity of methyl-lysine marks on histones; the lysine ε-amino group can be mono-, di-, or tri-methylated (designated H3K4me1/2/3, respectively). Recent data indicate that both the precise lysine residue modified and degree of methylation have differential effects on transcriptional regulation (17.Santos-Rosa H. Schneider R. Bannister A.J. Sherriff J. Bernstein B.E. Emre N.C. Schreiber S.L. Mellor J. Kouzarides T. Nature. 2002; 419: 407-411Crossref PubMed Scopus (1558) Google Scholar, 18.Morillon A. Karabetsou N. Nair A. Mellor J. Mol. Cell. 2005; 18: 723-734Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), suggesting that a combination of both parameters defines the epigenetic mark and its functional role. In the work described here, we use antibodies specific to the H3K4me epitope in its mono-, di-, and tri-methylated form to demonstrate that the link between histone acetylation and the degree of histone H3K4 methylation plays a major role in the epigenetic response to histone deacetylase inhibitors. A combination of in vivo knockdown approaches and in vitro biochemical analysis demonstrates that this arises due to the activities of two opposing enzyme activities, MLL4, a methyltransferase, and a novel, and as yet unidentified, H3K4me3 demethylase activity. Tissue Culture and Reagents−HeLa and HL60 cells were grown in RPMI medium supplemented with 8% fetal bovine serum (Invitrogen), l-glutamine (Invitrogen), and penicillin (Invitrogen). Histone hyperacetylation was induced by exposure of cells to sodium butyrate (10 mm final concentration), sodium valproate (5 mm), or Trichostatin A (165 nm) for 6 Histone and were cells by cells were by with containing mm sodium and in the was and the was with to the histones with on Histones were on to and with the Histone was by the use of the tail of histone H3 binding was by antibodies and by of antibodies specific for mono-, and H3K4 and for and the of by has been described (11.Schubeler D. MacAlpine D.M. Scalzo D. Wirbelauer C. Kooperberg C. van Leeuwen F. Gottschling D.E. O’Neill L.P. Turner B.M. Delrow J. Bell S.P. Groudine M. Genes Dev. 2004; 18: 1263-1271Crossref PubMed Scopus (631) Google Scholar). and were containing MLL1 and and These were identified by and a (Invitrogen). cells were at with of to the and for were on and subsequently or with mm sodium butyrate for 6 to histone or cells were with The extent of was by with and to the were by at and to was in the and a The extent of but for in and in Histone was as a the expression B. C. J.R. Walker P.A. N. S. Nature. 2003; PubMed Scopus Google Scholar) in by with mm at for The was over and at a containing mm mm mm and The SET domain MLL1 was by the (13.Milne T.A. Briggs S.D. Brock H.W. Martin M.E. Gibbs D. Allis C.D. Hess J.L. Mol. Cell. 2002; 10: 1107-1117Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar) and between the and of the expression The SET was in E. by with mm at The enzyme was over a by a SET was with containing mm and mm and at the of and to a final of mm and to the of the histone H3 tail of a of in were during at with the were with of bovine serum in for at the were with and with of methylation methyltransferase, mm in mm at mm mm mm or the mm mm mm mm The were for the at and with and the modified were with or in bovine serum for at The were with mm and with for at by with and with with of for of were and the at on a For the enzyme were as methylation and for the used in the methylation assays to of histone H3 by a and lysine Histone Both Histone and H3 on a variety of and cells have that and in the levels of histone acetylation, consistent with the high of associated with this histone J. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We have used a of on HL60 cells to is a link between levels of histone acetylation and the methylation of specific growing HL60 with 6 mm sodium valproate A and or mm data not as a and in histone The of the response with the in to in A and the abundance of these histone in cells and the of at different lysine D. K. P. C. J. Biochem. PubMed Scopus Google Scholar). also a in methylation at the degree of on the specific methyl H3K4me3 and levels whereas levels show a small to In of the levels of mono-, di-, or at H3K9 or modifications associated with transcriptional not have induced the same specific in H3K4 methylation in types HL60 cells not and cells with C.A. R. C. Turner B.M. 2005; 19: PubMed Scopus Google Scholar). Histone H3 on the of of cells with show a correlation between the extent of histone acetylation in a and the abundance of histone and at We the acetylation and H3K4 methylation induced by occur on the same H3 molecules by histones on that the distinct histone H3 cells a of and whereas cells acetylated histone to Histones on were and with antibodies specific to distinct H3K4 methyl and A central this is that in cells the of H3K4 methylation or in is associated with acetylated histones histone the that in these cells This suggests that at least group is on H3 for methylation to A is that in cells the different of methylation are associated with different levels of H3 is on and on di-, and H3 and H3K4me3 on and These are consistent with the distribution of marks by (9.Zhang K. Siino J.S. Jones P.R. Yau P.M. Bradbury E.M. Proteomics. 2004; 4: 3765-3775Crossref PubMed Scopus (63) Google Scholar) and not that H3 acetylation is for H3K4 methylation to occur but that the degree of methylation induced by is by the acetylation level of distinct H3 This has a of which are in the H3 H3 as related in cells and by a in the Recent indicate that is associated with a distinct chromatin complex H. D. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), is in regions of and is associated with H3K4me3 E. P.R. K. S. S. A. 2004; PubMed Scopus Google Scholar). was to the induced by were associated with in the abundance or levels of the distinct H3 of histones and HL60 cells on indicates whereas and the H3 the of in suggesting that histone acetylation or the functional that occur in the its deposition This is a a of the H3 in as the of the chromatin associated with and histones and cells were and with antibodies and H3K4 to the distribution of these marks on the distinct histone H3 This shows that in cells the of both and H3K4me3 are between and with methylation associated with This is as not the of these in the histone pool is and indicates that is in these methyl The H3 methyl marks are also with the observed in to and In the deposited is with This shows that the marks induced by are to the of Histone H3K4 to methylation has been of as a and was to be by of with histone S. Genet. 2005; PubMed Scopus Google that indicate that histone H3K4 methylation is linked to the extent of histone acetylation, we to the of this mark histone acetylation levels generate these cells were to to high levels of histone acetylation and the was subsequently and the in histone modifications were by of the induced a and in histone acetylation consistent with the of this mark in chromatin histone was by the of H3K4 methylation, with the extent of of specific methyl marks is also with the of H3K4me3 and at these methyl marks are subject to of analysis of the histones indicates H3 and in on the same histone molecules However, a of the H3K4me3 is associated with and histone H3 at This is as indicates that the of and are on the same histone of histones with not generate these H3 The that a of marks are observed on histone these is also as this is not in cells This suggests that the of on individual histone consistent with the observed in the histone pool the of H3 data indicate that a in H3K4 methylation and that this is linked to the acetylation of individual H3 However, the of this is particularly the complexity of the observed H3K4 is a for and the mechanism of they generate mono-, di-, or tri-methylated is However, the that the activity of the SET domain of MLL1, a H3K4 methyltransferase, is stimulated by acetylated substrates (13.Milne T.A. Briggs S.D. Brock H.W. Martin M.E. Gibbs D. Allis C.D. Hess J.L. Mol. Cell. 2002; 10: 1107-1117Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar) we this activity or that of related enzymes was the of the observed in response to experiments regions of a of that cells levels of individual of the of and a distinct H3K4 that can generate marks B. C. J.R. Walker P.A. N. S. Nature. 2003; PubMed Scopus Google Scholar) not We were to the abundance as specific antibodies are We used a knockdown to the histone for the in H3K4 for and were were grown for to and subsequently with sodium The induced a and in histone acetylation in both and the cells as by not was a in H3K4 methylation in the butyrate of and MLL1, and not this of A and suggesting that these not to the in H3 In of the in A and These have been with consistent In the SET to Histone that is the major activity for the of to acetylated histone H3 a of the mechanism by which the distinct are generated in The HMT, MLL1, is to generate tri-methylated marks T.A. A.J. A. J. Allis C.D. Hess J.L. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), and the activity of its SET domain is to be stimulated by acetylated substrates (13.Milne T.A. Briggs S.D. Brock H.W. Martin M.E. Gibbs D. Allis C.D. Hess J.L. Mol. Cell. 2002; 10: 1107-1117Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar). the SET of MLL1 and MLL4, which are the for the methyltransferase activity of the are related we were to the SET domain of MLL4, we in vitro methylation assays to SET domain could the distribution of H3 methyl observed in experiments the activity of the SET domain MLL1 and the activity of the on and H3 The levels of specific methyl generated were with Both enzymes a of over the A and but the of SET domain activity and the final level of methyl marks and are on acetylated substrates the data show that the of the individual methyl marks are with levels of deposition and in the of the and marks suggesting that methylation In activity is not stimulated on the acetylated that not SET are by the acetylation of The that SET domain activity is stimulated by the of the to the effects of a of modifications on the H3 assays were with a of modified H3 substrates, and and These data are consistent with that H3 acetylation SET domain with in methyl deposition on and H3 phosphorylation also activity to a However, the is that of the in a to in the deposition of methyl A final activity on the same of modified not show in the methylation of different substrates These data also the of the antibodies binding is not by the of adjacent or residues. In both histone H3 acetylation and H3S10 phosphorylation the deposition of distinct methyl mark to different suggesting that the methylation to is the sensitive to A between H3 and H3K4 functional of histone H3 acetylation and H3K4 methylation levels of both H3 acetylation and H3K4me3 are at the transcriptional of D.K. Harbison C.T. Levine S. Cole M. Hannett N.M. Lee T.I. Bell G.W. Walker K. Rolfe P.A. Herbolsheimer E. Zeitlinger J. Lewitter F. Gifford D.K. Young R.A. Cell. 2005; 122: 517-527Abstract Full Text Full Text PDF PubMed Scopus (1083) Google Scholar), and both marks functional by chromatin enzymes with and proteins (14.Pray-Grant M.G. Daniel J.A. Schieltz D. Yates III, J.R. Grant P.A. Nature. 2005; 433: 434-438Crossref PubMed Scopus (400) Google Scholar, 15.Sims R.J. Chen C.F. Santos-Rosa H. Kouzarides T. Patel S.S. Reinberg D. J. Biol. Chem. 2005; 280: 41789-41792Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). 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Jones P.R. Yau P.M. Bradbury E.M. Proteomics. 2004; 4: 3765-3775Crossref PubMed Scopus (63) Google Scholar). related that the level of histone H3 acetylation is for or determines the degree of H3K4 data indicate that in histone acetylation in histone This is clear both the of and and with H3K4me marks or of and Similarly, analysis of the in H3 on demonstrates that histone H3 in histones with patterns of H3K4 marks associated with histones with levels of These are not in or but is by the that are histone H3 methyl of the is that they deposition of that this histone is at H3K4 in chromatin This is in with of histone associated with the of C.A. Mahadevan L.C. 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Proteomics. 2004; 4: 3765-3775Crossref PubMed Scopus (63) Google Scholar), are consistent with H3K4me3 demethylase this suggests that the regulation of histone methylation is to that of histone acetylation, by opposing of enzymes that are to be recruited to by knockdown we demonstrate that the methyltransferase MLL4, but not the related enzymes MLL1, or is for the interaction between acetylation and H3K4 methylation that we in The for is that MLL1 and are but may functional in the complexes they form or abundance or and enzymes are in complexes with activities, and this may be the of many of epigenetic This to be the for lysine MLL1 is a of a complex containing both the histone acetyltransferase activities T.A. A.J. A. J. Allis C.D. Hess J.L. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) and P. J. M. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar) and a that the complex to H3K4me J. T. T.A. A.L. Allis C.D. 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Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). we also a in H3 acetylation suggesting that HDAC1 and in the of the complex to the cellular levels of histone However, the of these suggests that the complex plays a small in regulating the pool of histone acetylation and H3K4 These that both the deposition or of H3K4 methyl marks by enzyme complexes is with the deposition or of marks at adjacent The that a of both and histone H3 is at H3K4 and (9.Zhang K. Siino J.S. Jones P.R. Yau P.M. Bradbury E.M. Proteomics. 2004; 4: 3765-3775Crossref PubMed Scopus (63) Google Scholar) is consistent with this and suggests that between these marks is the at containing acetylated This of H3 to two that the methyltransferase is to associate with histone on histone H3 in a manner to This the linked deposition of both modifications at over a wide of both H3 and of H3K4 methylation the demethylase be to associate with activity that is sensitive to deacetylase However, the mechanism of methyl by by a enzyme or the coordinated activity of demethylase complexes LSD1 and the The data that the abundance of methyl in chromatin a between two opposing enzyme activities, histone methylation and the that the acetylation level of a H3 determines both the degree mono-, di-, or and extent of methylation deposited at H3 lysine 4 could be by two Histone H3 acetylation can the the of activity or by the action of a H3K4me3 demethylase. in vitro methylation assays mechanism in that we show that the activity of the SET domain is stimulated by These data are consistent with that methylation K. M. Mol. Cell. 2005; 19: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the that the to is the sensitive to is novel, as is the that H3K9 acetylation, with H3S10 activity. We can that these and generate a that The that the of and in vivo with different and with is consistent with mechanism that that histone acetylation may the of adjacent although is and the of may the of observed is to that of and marks at the same suggesting that this is a We S. J. and J. Hess for the and with