Spatial Distribution of Di- and Tri-methyl Lysine 36 of Histone H3 at Active Genes

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Résumé

Methylation of lysine 4 of histone H3 (K4/H3) is linked to transcriptional activity, whereas methylation of K9/H3 is tightly associated with gene inactivity. These are well characterized sites of methylation within histones, but there are numerous other, less characterized, sites of modification. In Saccharomyces cerevisiae, methylation of K36/H3 has been linked to active genes, but little is known about this methylation in higher eukaryotes. Here we analyzed for the first time the levels and spatial distribution of di- and tri-methyl (di- and tri-Me) K36/H3 in metazoan genes. We analyzed chicken genes that are developmentally regulated, constitutively active, or inactive. We found that active genes contain high levels of these modifications compared with inactive genes. Furthermore, in actively transcribed regions the levels of di- and tri-Me K36/H3 peak toward the 3′ end of the gene. This is in striking contrast to the distributions of di- and tri-Me K4/H3, which peak early in actively transcribed regions. Thus, di/tri-Me K4/H3 and di/tri-Me K36/H3 are both useful markers of active genes, but their genic distribution indicates differing roles. Our data suggest that the unique spatial distribution of di- and tri-Me K36/H3 plays a role in transcriptional termination and/or early RNA processing. Methylation of lysine 4 of histone H3 (K4/H3) is linked to transcriptional activity, whereas methylation of K9/H3 is tightly associated with gene inactivity. These are well characterized sites of methylation within histones, but there are numerous other, less characterized, sites of modification. In Saccharomyces cerevisiae, methylation of K36/H3 has been linked to active genes, but little is known about this methylation in higher eukaryotes. Here we analyzed for the first time the levels and spatial distribution of di- and tri-methyl (di- and tri-Me) K36/H3 in metazoan genes. We analyzed chicken genes that are developmentally regulated, constitutively active, or inactive. We found that active genes contain high levels of these modifications compared with inactive genes. Furthermore, in actively transcribed regions the levels of di- and tri-Me K36/H3 peak toward the 3′ end of the gene. This is in striking contrast to the distributions of di- and tri-Me K4/H3, which peak early in actively transcribed regions. Thus, di/tri-Me K4/H3 and di/tri-Me K36/H3 are both useful markers of active genes, but their genic distribution indicates differing roles. Our data suggest that the unique spatial distribution of di- and tri-Me K36/H3 plays a role in transcriptional termination and/or early RNA processing. Histone N-terminal tails are subject to a variety of post-translational covalent modifications. These include acetylation, phosphorylation, and methylation, all of which have the potential to alter chromatin architecture and thereby to affect all aspects of DNA processing (1Fischle W. Wang Y. Allis C.D. Curr. Opin. Cell Biol. 2003; 2: 172-183Crossref Scopus (989) Google Scholar, 2Strahl B.D. Allis C.D. Nature. 2002; 403: 41-45Crossref Scopus (6679) Google Scholar, 3Turner B.M. Cell. 1993; 75: 5-8Abstract Full Text PDF PubMed Scopus (310) Google Scholar). Over the last few years it has become increasingly clear that deregulation of the enzymes performing these modifications can be linked to various human pathologies including cancer (4Hake S.B. Xiao A. Allis C.D. Br. J. Cancer. 2004; 90: 761-769Crossref PubMed Scopus (312) Google Scholar, 5Schneider R. Bannister A.J. Kouzarides T. Trends Biochem. Sci. 2002; 27: 396-402Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). For instance, mutations in histone acetyltransferases have been linked to cancer, and histone deacetylase inhibitors are now in the clinician's armory as anti-cancer chemotherapeutic agents (4Hake S.B. Xiao A. Allis C.D. Br. J. Cancer. 2004; 90: 761-769Crossref PubMed Scopus (312) Google Scholar). Histone kinases, such as the aurora kinases, have also been linked to oncogenesis (4Hake S.B. Xiao A. Allis C.D. Br. J. Cancer. 2004; 90: 761-769Crossref PubMed Scopus (312) Google Scholar). More recently evidence has begun to emerge implicating histone methyltransferases in developmental disorders as well as in cancer (6Rayasam G.V. Wendling O. Angrand P.O. Mark M. Niederreither K. Song L. Lerouge T. Hager G.L. Chambon P. Losson R. EMBO J. 2003; 16: 3153-3163Crossref Scopus (282) Google Scholar). In vivo histone lysines may be mono-, di-, or tri-methylated, but little is known concerning the functional interplay between these different levels of methylation. In metazoa the levels of the di- and tri-methyl forms of lysine 4 of histone H3 (K4/H3) have been shown to peak in the 5′ transcribed region of active genes, implicating both in the early phases of transcriptional elongation (7Schneider R. Bannister A.J. Myers F.A. Thorne A.W. Crane-Robinson C. Kouzarides T. Nat. Cell. Biol. 2004; 6: 73-77Crossref PubMed Scopus (617) Google Scholar). Consistent with this the yeast Set1p enzyme that is responsible for methylating K4/H3 is found associated with the initiation-elongation phosphorylated form of RNA pol II, 1The abbreviations used are: pol II, polymerase II; ChIP, chromatin immunoprecipitation. RNAPII-5P (8Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar). Another H3 modification, di-methyl K36/H3, also has been found in the transcribed region of active genes in yeast (9Xiao T. Hall H. Kizer K.O. Shibata Y. Hall M.C. Borchers C.H. Strahl B.D. Genes Dev. 2003; 17: 654-663Crossref PubMed Scopus (326) Google Scholar, 10Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (519) Google Scholar). In this case, the Set2p enzyme that methylates K36/H3 associates with the elongation-termination form of RNA pol II, RNAPII-2P (9Xiao T. Hall H. Kizer K.O. Shibata Y. Hall M.C. Borchers C.H. Strahl B.D. Genes Dev. 2003; 17: 654-663Crossref PubMed Scopus (326) Google Scholar, 10Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (519) Google Scholar). Recently, the mammalian NSD1 protein has been shown to possess K36/H3 methyltransferase activity (6Rayasam G.V. Wendling O. Angrand P.O. Mark M. Niederreither K. Song L. Lerouge T. Hager G.L. Chambon P. Losson R. EMBO J. 2003; 16: 3153-3163Crossref Scopus (282) Google Scholar). Haploinsufficiency of the NSD1 gene leads to Sotos syndrome, which is a neurological disorder characterized by overgrowth in childhood, advanced bone age, craniofacial abnormalities, mental retardation, and possibly a susceptibility to cancer (11Visser R. Matsumoto N. Curr. Opin. Pediatr. 2003; 15: 598-606Crossref PubMed Scopus (43) Google Scholar, 12De Boer L. Duyvenvoorde H.A. Willemstein-Van Hove E.C. Hoogerbrugge C.M. Van Doorn J. Maassen J.A. Karperien M. Wit J.M. Eur. J. Endocrinol. 2004; 151: 333-341Crossref PubMed Scopus (18) Google Scholar). In addition, translocation of the NSD1 gene leads to the development of certain leukemias (13Al-Mulla N. Belgaumi A.F. Teebi A. J. Pediatr. Hematol. Oncol. 2004; 26: 204-208Crossref PubMed Scopus (19) Google Scholar). Taken together, these facts suggest that deregulation of K36/H3 methylation and the resulting effect on transcription may be crucial events in cellular transformation. Mapping studies have analyzed the spatial distribution of di-methyl K36/H3 in active yeast genes and have found that this modification is generally enriched in transcribed regions, but no definitive trend within transcribed regions has been observed (9Xiao T. Hall H. Kizer K.O. Shibata Y. Hall M.C. Borchers C.H. Strahl B.D. Genes Dev. 2003; 17: 654-663Crossref PubMed Scopus (326) Google Scholar, 10Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (519) Google Scholar). Furthermore, recent experiments performed in mammalian cells have mapped di-Me K36/H3 to active genes but not inactive genes (14Zhou M. Deng L. Lacoste V. Park H.U. Pumfery A. Kashanchi F. Brady J.N. Kumar A. J. Virol. 2004; 78: 13522-13533Crossref PubMed Scopus (63) Google Scholar). However, to date nothing is known concerning the role of tri-Me K36/H3. Here, we describe the characterization of anti tri-Me K36/H3 antisera and use the antibody to map tri-Me K36/H3 to active metazoan genes. Analysis of the spatial distribution of di- and tri-Me K36/H3 within active genes indicates that the levels of these modifications increase toward the 3′ end of actively transcribing genes. Antibodies—Di-methyl K36/H3-specific antibodies were purchased from Upstate Biotechnology. Tri-methyl K36/H3-specific antibodies are now available from Abcam Ltd. Nucleosome Preparation and Chromatin Immunoprecipitation— Chicken mononucleosomes were prepared and chromatin immunoprecipitations (ChIPs) performed essentially as described previously (20Hebbes T.R. Clayton A.L. Thorne A.W. Crane-Robinson C. EMBO J. 1994; 13: 1823-1830Crossref PubMed Scopus (484) Google Scholar, 21Myers F.A. Evans D.R. Clayton A.L. Thorne A.W. Crane-Robinson C. J. Biol. Chem. 2001; 276: 20197-20205Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Typically, 150 μg of sucrose gradient-purified input mononucleosomes (as DNA) were dialyzed into the immunoprecipitation buffer (150 mm NaCl, 0.02% SDS, 20 mm Tris·HCl, pH 8.0, 2 mm EDTA, 0.9% Triton X-100) and immunoprecipitated at 4 °C with 10 μg of affinity-purified antibodies in the presence of protein A/G beads (Amersham Biosciences). The beads were then washed five times with immunoprecipitation buffer and twice with wash buffer (350 mm NaCl, 0.02% SDS, 20 mm Tris·HCl, pH 8.0, 2 mm EDTA, 0.9% Triton X-100). The histones and DNA from the input, unbound (supernatant), and bound fractions were recovered as described (20Hebbes T.R. Clayton A.L. Thorne A.W. Crane-Robinson C. EMBO J. 1994; 13: 1823-1830Crossref PubMed Scopus (484) Google Scholar). Quantitative Real-time PCR—Using an ABI 7000 real-time PCR system and the TaqMan PCR Master Mix protocol (Applied Biosystems), differences in the DNA content of the bound and input fractions were determined. Bound to input (B/I) ratios were determined for each amplicon by taking a fixed aliquot of the DNA extracted from the input and bound nucleosomal samples. These were amplified together with a defined amount of genomic DNA on the same plate, using the latter to construct the standard curve. B/I values for any one amplicon are thus in the correct quantitative ratio to B/I values for other amplicons measured for the same ChIP, because this procedure compensates for differences in the PCR efficiencies of different probe/primer combinations and for nucleosome representation. However, B/I values derived using different ChIPs cannot be quantitatively compared because of variations in the efficiency of individual immunoprecipitations. All data within each graphical representation are derived from a single representative ChIP experiment; repeat ChIPs were found to give the same relative spatial distributions of B/I values across all amplicons. PCR reactions were carried out in triplicate using fixed amounts of template DNA from each fraction: 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The threshold (CT) was set such that the fluorescence signal was above the base-line noise but as low as possible in the exponential amplification phase. For each amplicon a standard curve was determined by plotting the number of cycles at which the fluorescence crossed the threshold (crossing values) against increasing amounts of genomic DNA template. Based on this standard curve, the DNA sequence content of the input and bound fractions was determined. All primers and TaqMan probes were designed using ABI Primer Express software. The PCR products were substantially shorter than 200 bp, the length of DNA in a nucleosome. Primer and TaqMan sequences are available on request. To better evaluate the role of methylation of K36/H3 in transcription in metazoans, we asked whether the tri-methylation of K36/H3 is more tightly associated with gene activity or inactivity and determined the spatial distribution of this modification within several genes. To this end we performed ChIPs with anti tri-Me K36/H3 antibodies and mapped the presence of this modification in the promoter and transcribed region of developmentally regulated, constitutively active and inactive chicken genes. Input chromatin was in the form of mononucleosomes purified from the nuclei of 15-day chicken embryo erythrocytes; the DNA sequence content of the antibody-bound nucleosomes was analyzed by real-time PCR with short amplicons. The resulting mapping is thus at the mononucleosome level. The experimental methods used were exactly as described for our previous determination of the distribution of di- and tri-Me K4/H3 (7Schneider R. Bannister A.J. Myers F.A. Thorne A.W. Crane-Robinson C. Kouzarides T. Nat. Cell. Biol. 2004; 6: 73-77Crossref PubMed Scopus (617) Google Scholar). First, the specificity of the antibody against tri-Me K36/H3 was verified. This was done by using the antibodies in Western blots of various histone preparations (Fig. 1). We prepared total cellular extracts from three strains of Schizosaccharomyces pombe: wild type; a set1 knock-out lacking the Set1p K4/H3 methyltransferase; and a set2 knock-out, which lacks the Set2p K36/H3 methyltransferase. The antibody specifically recognizes a band corresponding to histone H3 in the wild type strain. Importantly, this recognition is maintained in the set1 knock-out strain but is lost in the set2 knock-out strain (Fig. 1A). This confirms the specificity of the antibodies for Me K36/H3. Furthermore, recognition of the H3 epitope is specifically blocked by an H3 peptide containing tri-Me Lys-36 but not by peptides bearing mono-Me or di-Me Lys-36, nor is it affected by peptides containing tri-Me lysine at positions other than Lys-36 (Fig. 1B and data not shown). We conclude that the antibody is specific for the tri-methylated form of K36/H3. To map the distribution of tri-Me K36/H3 by native ChIP, the promoters and transcribed regions of the four genes in the chicken β-globin locus were the first target to be analyzed (15Felsenfeld G. Gene. 1993; 135: 119-124Crossref PubMed Scopus (34) Google Scholar). These genes were chosen because they are a highly characterized model gene system that includes both active and inactive genes. In 15-day chicken embryo erythrocytes the adult and hatching genes (βA, βH) are active, whereas the embryonic rho and epsilon genes (βρ, βϵ) are inactive (Fig. 2a). Although there is little detectable tri-Me K36/H3 either in the 16 kb of heterochromatin that lies upstream of the locus (Fig. 2b, bar 1) or in the promoters or transcribed regions of the inactive βρ and βϵ genes (Fig. 2b, bars 2, 3, 8, and 9), at the promoters (bars 4 and 6) and in particular within the transcribed regions of the active βA and βH genes (bars 5 and 7) the levels are substantial. These data imply that tri-Me K36/H3 is enriched mostly in the transcribed region of active genes. This hypothesis was tested by analyzing the upstream (5′) transcribed regions of a number of constitutively active and tissue-specific genes and comparing enrichments of tri-Me K36/H3 to those found in similar regions of inactive genes. Fig. 2c clearly shows that tri-Me K36/H3 is enriched at active genes, whereas at several tissue-specific genes not expressed in erythrocytes, very low but not zero levels of tri-Me K36/H3 are present in their transcribed regions. It is striking that there appears to be significantly more tri-Me K36/H3 in the transcribed region of the βA gene when compared with the level at the other active globin gene, βH (Fig. 2b). This might have two explanations: (i) more tri-Me K36/H3 is found associated with the βA gene because it is the most active gene in the β-globin locus in 15-day embryos; or (ii) because the amplicon analyzed in the βA gene is near the 3′ end of the transcribed region but that analyzed for the βH gene is more toward the 5′ end of its transcribed region, the distribution of tri-Me K36/H3 might increase toward the 3′-ends of transcribed regions. If the latter assumption is correct and it is a general phenomenon, then the levels of tri-Me K36/H3 at other active genes should increase toward the 3′ ends of their transcribed regions. Analysis of a further four active genes, vimentin, DNAPKcs, L30, and MHCII demonstrated that this was indeed the case (Fig. 3). Each of the four genes displayed significantly increased levels of tri-Me K36/H3 toward the 3′ ends of their transcribed regions. Previously we have shown that di- and tri-Me K4/H3 are also markers of active transcription but that they peak in the early (5′) transcribed regions of active genes (7Schneider R. Bannister A.J. Myers F.A. Thorne A.W. Crane-Robinson C. Kouzarides T. Nat. Cell. Biol. 2004; 6: 73-77Crossref PubMed Scopus (617) Google Scholar). From the data presented here, tri-Me K36/H3 may also be regarded as a marker of active transcription, but its distribution appears to be different from that of methylated K4/H3. To determine more precisely the spatial distribution of tri-Me K36/H3 at active genes, two active genes were analyzed in more detail: carbonic anhydrase (CA, tissue-specific, 17 kb) and GAPDH (housekeeping gene, 4 kb) (see Fig. 4). For comparison, the distribution of tri-Me K4/H3 is displayed together with that of tri-Me K36/H3. Across both the CA and GAPDH genes there is a clear enrichment of tri-Me K36/H3 toward the 3′ ends of their transcribed regions, in sharp contrast to tri-Me K4/H3, which is confined to the most 5′ transcribed regions, as reported previously (7Schneider R. Bannister A.J. Myers F.A. Thorne A.W. Crane-Robinson C. Kouzarides T. Nat. Cell. Biol. 2004; 6: 73-77Crossref PubMed Scopus (617) Google Scholar). We also determined the spatial distribution of di-Me K36/H3 across the CA and GAPDH genes and found a strikingly similar distribution to that of tri-Me K36/H3 (Fig. 4). In Saccharomyces cerevisiae di-Me K36/H3 has been implicated in both the activation and repression of genes, although the mechanism(s) involved are far from clear (9Xiao T. Hall H. Kizer K.O. Shibata Y. Hall M.C. Borchers C.H. Strahl B.D. Genes Dev. 2003; 17: 654-663Crossref PubMed Scopus (326) Google Scholar, 10Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (519) Google Scholar, 16Strahl B.D. Grant P.A. Briggs S.D. Sun Z. Bone J.R. Caldwell J.A. Mollah S. Cook R.G. Shabanowitz J. Hunt D.F. Allis C.D. Mol. Cell. Biol. 2002; 22: 1298-1306Crossref PubMed Scopus (433) Google Scholar, 17Landry J. Sutton A. Hesman T. Min J. Rui-Ming X. Johnston M. Sternglanz R. Mol. Cell. Biol. 2003; 23: 5972-5978Crossref PubMed Scopus (51) Google Scholar). The yeast Set2p enzyme methylates K36/H3 and associates with RNA polymerase II (RNA pol II), which is phosphorylated at Ser-2 within its C-terminal domain repeats (9Xiao T. Hall H. Kizer K.O. Shibata Y. Hall M.C. Borchers C.H. Strahl B.D. Genes Dev. 2003; 17: 654-663Crossref PubMed Scopus (326) Google Scholar, 10Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (519) Google Scholar). Ser-2 phosphorylated RNA pol II (RNAPII-2P) is the transcriptionally active elongating form that transcribes the DNA into RNA. The observation that this form of RNA pol II Set2p and K36/H3, in the of transcriptional This with the observation that methylated K36/H3 is present within active but di- and tri-Me K36/H3 levels increase in a toward the ends of transcribed The increase be to methyltransferase as the elongating RNA pol II further or because the activity of the histone methyltransferase toward the 3′ end of genes. The above data clearly that in di- and tri-Me K36/H3 is associated with the of active transcription, but Me We previously reported that di- and tri-Me K4/H3 levels peak in the early transcribed region of the active genes analyzed (7Schneider R. Bannister A.J. Myers F.A. Thorne A.W. Crane-Robinson C. Kouzarides T. Nat. Cell. Biol. 2004; 6: 73-77Crossref PubMed Scopus (617) Google Scholar). This to suggest that K4/H3 has an role in the early of transcriptional In in the present we that di- and tri-Me K36/H3 peak toward the 3′ end of actively transcribing regions, that K36/H3 has a different role than that of di- and tri-Me K4/H3. Furthermore, we no differences between the spatial distributions of di- and tri-Me K36/H3, that these modifications may have very similar or Me K36/H3 modification levels a at the end of the GAPDH gene but nucleosomes the end of the four times CA gene, the peak might at an fixed from the of However, the gene is kb and the 3′ amplicon (Fig. is in the last of the peak of tri-Me K36/H3 is clearly to the end of the transcribed region, its and might be involved in transcription termination or have a role in transcriptionally linked early processing. Consistent with the the transcribing RNA polymerase which Set2p has been shown to be involved in the early of RNA processing G. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). Furthermore, the yeast protein a a recently shown to be in involved in which is a that in vivo in with transcription Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). the the mapping studies reported suggest that methylated K4/H3 and methylated K36/H3 have at active genes.

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La notice

Revue

Journal of Biological Chemistry

Thématique

Genomics and Chromatin Dynamics

Domaine

Biochemistry, Genetics and Molecular Biology

Établissements canadiens

—

Organismes subventionnaires

Directorate for Biological SciencesEuropean CommissionCancer Research UK

Mots-clés

Histone H3MethylationGeneBiologyHistoneLysineGeneticsDNA methylationEpigeneticsRegulation of gene expressionMolecular biologyGene expressionAmino acid

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