Arginine Methylation of the Histone H3 Tail Impedes Effector Binding

Histone tail post-translational modification results in changes in cellular processes, either by generating or blocking docking sites for histone code readers or by altering the higher order chromatin structure. H3K4me3 is known to mark the promoter regions of active transcription. Proteins bind H3K4 in a methyl-dependent manner and aid in the recruitment of histone-remodeling enzymes and transcriptional cofactors. The H3K4me3 binders harbor methyl-specific chromatin binding domains, including plant homeodomain, Chromo, and tudor domains. Structural analysis of the plant homeodomains present in effector proteins, as well as the WD40 repeats of WDR5, reveals critical contacts between residues in these domains and H3R2. The intimate contact between H3R2 and these domain types leads to the hypothesis that methylation of this arginine residue antagonizes the binding of effector proteins to the N-terminal tail of H3. Here we show that H3 tail binding effector proteins are indeed sensitive to H3R2 methylation and that PRMT6, not CARM1/PRMT4, is the primary methyltransferase acting on this site. We have tested the expression of a select group of H3K4 effector-regulated genes in PRMT6 knockdown cells and found that their levels are altered. Thus, PRMT6 methylates H3R2 and is a negative regulator of N-terminal H3 tail binding. Histone tail post-translational modification results in changes in cellular processes, either by generating or blocking docking sites for histone code readers or by altering the higher order chromatin structure. H3K4me3 is known to mark the promoter regions of active transcription. Proteins bind H3K4 in a methyl-dependent manner and aid in the recruitment of histone-remodeling enzymes and transcriptional cofactors. The H3K4me3 binders harbor methyl-specific chromatin binding domains, including plant homeodomain, Chromo, and tudor domains. Structural analysis of the plant homeodomains present in effector proteins, as well as the WD40 repeats of WDR5, reveals critical contacts between residues in these domains and H3R2. The intimate contact between H3R2 and these domain types leads to the hypothesis that methylation of this arginine residue antagonizes the binding of effector proteins to the N-terminal tail of H3. Here we show that H3 tail binding effector proteins are indeed sensitive to H3R2 methylation and that PRMT6, not CARM1/PRMT4, is the primary methyltransferase acting on this site. We have tested the expression of a select group of H3K4 effector-regulated genes in PRMT6 knockdown cells and found that their levels are altered. Thus, PRMT6 methylates H3R2 and is a negative regulator of N-terminal H3 tail binding. The tight packing of DNA into chromatin creates a need for mechanisms to relax chromatin and expose DNA for transcription, replication, and DNA repair (1Ito T. J. Biochem. (Tokyo). 2007; 141: 609-614Crossref PubMed Scopus (103) Google Scholar). One of the mechanisms used by the cell to access DNA is the post-translational modification of histone tails. Specifically, methylation of histone tails generates a docking site for effector proteins, which aid in the recruitment of other enzymes necessary for the function at hand. In general, methylation of histone residues lysines 4 and 36 on H3 are correlated with active gene regions, whereas methylation of lysines 9 and 27 on H3 is correlated with repressed gene regions, although exceptions exist (2Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2205) Google Scholar). The domain types that bind histone tails include the Chromodomain, tudor domains, MBT domains, WD40 repeats, and PHD 5The abbreviations used are: PHDplant homeodomainPRMTprotein arginine N-methyltransferaseCARM1coactivator-associated arginine methyltransferaseGSTglutathione S-transferaseshRNAshort hairpin RNAChIPchromatin immunoprecipitationEembryonic day. fingers (3Kim J. Daniel J. Espejo A. Lake A. Krishna M. Xia L. Zhang Y. Bedford M.T. EMBO Rep. 2006; 7: 397-403Crossref PubMed Scopus (396) Google Scholar, 4Shi X. Kachirskaia I. Walter K.L. Kuo J.H. Lake A. Davrazou F. Chan S.M. Martin D.G. Fingerman I.M. Briggs S.D. Howe L. Utz P.J. Kutateladze T.G. Lugovskoy A.A. Bedford M.T. Gozani O. J. Biol. Chem. 2007; 282: 2450-2455Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 5Sims R.J. II I Reinberg D. Genes Dev. 2006; 20: 2779-2786Crossref PubMed Scopus (208) Google Scholar). plant homeodomain protein arginine N-methyltransferase coactivator-associated arginine methyltransferase glutathione S-transferase short hairpin RNA chromatin immunoprecipitation embryonic day. Recently, two groups reported that select PHD fingers have the propensity to bind trimethyl lysine 4 on H3 (6Shi X. Hong T. Walter K.L. Ewalt M. Michishita E. Hung T. Carney D. Pena P. Lan F. Kaadige M.R. Lacoste N. Cayrou C. Davrazou F. Saha A. Cairns B.R. Ayer D.E. Kutateladze T.G. Shi Y. Cote J. Chua K.F. Gozani O. Nature. 2006; 442: 96-99Crossref PubMed Scopus (2) Google Scholar, 7Wysocka J. Swigut T. Xiao H. Milne T.A. Kwon S.Y. Landry J. Kauer M. Tackett A.J. Chait B.T. Badenhorst P. Wu C. Allis C.D. Nature. 2006; 442: 86-90Crossref PubMed Scopus (911) Google Scholar). The structures further showed important aromatic residues in the PHD that cage the methylated lysine but also revealed critical contacts made between the arginine at the second position of the H3 tail and the PHD (8Pena P.V. Davrazou F. Shi X. Walter K.L. Verkhusha V.V. Gozani O. Zhao R. Kutateladze T.G. Nature. 2006; 442: 100-103Crossref PubMed Scopus (568) Google Scholar, 9Ruthenburg A.J. Wang W. Graybosch D.M. Li H. Allis C.D. Patel D.J. Verdine G.L. Nat. Struct. Mol. Biol. 2006; 13: 704-712Crossref PubMed Scopus (199) Google Scholar). During this same time, the WD40 domain of WDR5 was reported to complex with the H3 tail (10Wysocka J. Swigut T. Milne T.A. Dou Y. Zhang X. Burlingame A.L. Roeder R.G. Brivanlou A.H. Allis C.D. Cell. 2005; 121: 859-872Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). The structure of the WD40 repeats of WDR5 revealed arginine 2 of H3, and not lysine 4, buried within the donut hole of the large domain (11Couture J.F. Collazo E. Trievel R.C. Nat. Struct. Mol. Biol. 2006; 13: 698-703Crossref PubMed Scopus (196) Google Scholar, 12Li H. Ilin S. Wang W. Duncan E.M. Wysocka J. Allis C.D. Patel D.J. Nature. 2006; 442: 91-95Crossref PubMed Scopus (189) Google Scholar). Specifically, four amino acids in WDR5 critically interact with arginine 2 (11Couture J.F. Collazo E. Trievel R.C. Nat. Struct. Mol. Biol. 2006; 13: 698-703Crossref PubMed Scopus (196) Google Scholar). In addition, the tudor domains of JMJD2A also bind in an H3K4me3-dependent manner, and again, the H3R2 residue forms critical interactions with an Asp residue of one of the tudor domains (13Huang Y. Fang J. Bedford M.T. Zhang Y. Xu R.M. Science. 2006; 312: 748-751Crossref PubMed Scopus (375) Google Scholar). The analysis of the structures of these three different domain types bound to the N-terminal tail of histone H3 led us to ask whether methylation of this arginine, reportedly by CARM1 (14Schurter B.T. Koh S.S. Chen D. Bunick G.J. Harp J.M. Hanson B.L. Henschen-Edman A. Mackay D.R. Stallcup M.R. Aswad D.W. Biochemistry. 2001; 40: 5747-5756Crossref PubMed Scopus (284) Google Scholar), could block binding of these domain types to the histone. If true, methylation of arginine 2 would be a critical epigenetic mark that will antagonize lysine 4 methylation by blocking effector protein binding. Currently, little is known about H3R2 methylation, including the in vivo enzyme acting on this site, whether it can exist in combination with H3K4 methylation, and how broad an effect H3R2 methylation has on H3 effector binding. To answer these questions, we screened a collection of PRMTs to identify the H3R2-methylating enzyme. Here we show that PRMT6, an enzyme with restricted nuclear localization (15Frankel A. Yadav N. Lee J. Branscombe T.L. Clarke S. Bedford M.T. J. Biol. Chem. 2002; 277: 3537-3543Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar), and not CARM1, methylates H3R2 in vitro, and H3R2 methylation is still present in CARM1-null embryos. We also screened a panel of histone binding domains for sensitivity to H3R2 methylation. We found that a large number of H3 binding domains are sensitive to H3R2 methylation and add additional domains to the list of methyl-specific chromatin binders. In vitro methylation experiments show that R2me2 and K4me3 can exist on the same histone molecule. In addition, finally, the transcriptional activity of select genes, known to be affected by the transcriptional regulatory complexes containing the H3R2me2a-sensitive effectors, is altered when PRMT6 levels are reduced or increased. In Vitro Methylation Reactions–The GST-PRMT1, GST-CARM1, and GST-PRMT6 were expressed and purified as described previously (18Cheng D. Cote J. Shaaban S. Bedford M.T. Mol. Cell. 2007; 25: 71-83Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). In vitro methylation reactions were performed in a final volume of 30 μl of phosphate-buffered saline (pH = 7.4). The reaction contained 0.5–1.0 μg of substrate and 1 μg of recombinant GST-PRMT. All methylation reactions were carried out in the presence of 0.42 μm [3H]S-adenosyl-l-[methyl-3H]methionine (79 Ci/mmol from a 7.5 μm stock solution; PerkinElmer Life Sciences). The reaction was incubated at 30 °C for 1 h and then subjected to fluorography by separation on SDS-PAGE, transferred to a polyvinylidene fluoride membrane, treated with En3Hance™ (PerkinElmer Life Sciences), and exposed to film for 1–3 days at –80 °C. Acid Extraction of Histones–HEK293 and U2OS cells were grown to 80% confluency. Cells were suspended in reticulocyte standard buffer (10 mm Tris-HCl, pH 7.4; 10 mm NaCl; 3 mm MgCl2) and then centrifuged. The pellet was resuspended in reticulocyte standard buffer plus 0.5% Nonidet P-40, placed on ice for 10 min, and then centrifuged again (2500 × g). Nuclei were resuspended in 5 mm MgCl2, an equal volume of 0.8 m HCl was added, and histones were extracted for 20 min on ice. Histones (in supernatant) were precipitated with 50% (w/v) trichloroacetic acid and centrifuged at 8,000 × g. The pellet was washed twice with cold acetone and then resuspended in deionized water and 2 μl of 1.0 m Tris-HCl, pH 8.8. Histological Analysis and Antibodies–E18.5 embryos with their abdomens perforated were fixed in formalin and embedded in paraffin wax. Embryos were sectioned at 3 μm and subjected to immunohistochemical localization ofαCARM1 (Upstate Biotechnology), αH3R2me2a (Abcam), and αH3R17me2a (Upstate Biotechnology). Staining was performed using the En-Vision system (DAKO), and the counterstain was hematoxylin. The H3R2me1 antibody is from Abcam, and the PRMT6 antibody is from Bethyl Laboratories, Inc. Peptide Pulldowns–Biotinylated histone tail peptides (15 μg) were immobilized on 8 μl of streptavidin beads (Pierce) in 500 μl of pulldown buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm dithiothreitol, and 0.1% Nonidet P-40 (v/v), 1 μm ZnSO4) for 2 h at room temperature. Immobilized peptide bead complexes were washed three times with pulldown buffer. 1 μg of GST fusion protein and 400 μl of pulldown buffer were added to beads and rocked overnight at 4 °C. The beads were then washed five times with pulldown buffer, boiled in protein loading buffer, fractionated by 10% SDS-polyacrylamide gel electrophoresis, and subjected to Western blot analysis using an anti-GST antibody. Stable shRNA Line Generation–Plasmids designed to express shRNAs targeting nucleotides 995–1014 of human PRMT6 (GenBank™ accession number AY043278) were constructed by using pSUPER RNA interference system from Oligoengine (Seattle, WA) according to the manufacturer's protocol. The insert target sequences are 5′-gatccccGCAAGACACGGACGTTTCAttcaagagaTGAAACGTCCGTGTCTTGCtttttggaaa-3′ (forward) and agcttttccaaaaaGCAAGACACGGACGTTTCAtctcttgaaTGAAACGTCCGTGTCTTGCggg-5′ (reverse). U2OS cells were then transfected with pSUPERretro vector encoding the PRMT6-shRNA using Lipofectamine 2000 (Invitrogen). A polyclonal population was selected in 2 μg/ml puromycin, and ring cloning was performed. Expression of PRMT6 in each clone was analyzed by Western blots in triplicate. RNA Isolation and Quantitative real-time-PCR–Cells at 80% confluency were trypsinized and collected. The pellet was washed in phosphate-buffered saline, and 10% was used to make a whole cell extract for Western analysis on PRMT6 protein levels. The remaining cells were spun down and then RNA-isolated following the Qiagen RNeasy mini prep manufacturer's protocol. An on-column DNA digestion was performed in each RNA sample preparation. cDNA was prepared from total RNA using the Applied Biosystems high capacity cDNA archive kit following the manufacturer's protocol. Real-time prevalidated gene primer sets were purchased from the Applied Biosystems “Assays-on-Demand” and analyzed with the Applied Biosystems 7900HT real-time PCR instrument using the TaqMan universal master mix. 18 S RNA was used as the internal control. ChIP Analysis–U2OS cells at 80% confluency were transfected with 20 μg of 3×FLAG-ING2 vector (6Shi X. Hong T. Walter K.L. Ewalt M. Michishita E. Hung T. Carney D. Pena P. Lan F. Kaadige M.R. Lacoste N. Cayrou C. Davrazou F. Saha A. Cairns B.R. Ayer D.E. Kutateladze T.G. Shi Y. Cote J. Chua K.F. Gozani O. Nature. 2006; 442: 96-99Crossref PubMed Scopus (2) Google Scholar). 24 h after transfection, ChIP analysis was performed following the Upstate Biotechnology ChIP assay kit protocol (catalog number 17-295). For the immunoprecipitation, 2 μgofthe αFLAG antibody (Sigma, catalog number F-3165) was used for each condition and incubated with the cross-linked complexes overnight at 4°C. PCR of the cyclin D1 promoter on the input and isolated DNA was performed using Advantage 2 polymerase (Clontech 639201). The cyclin D1 promoter primer sequences were as follows: forward, 5′-GATTTTCTTTCAAACAACGTGGTTAC-3′, and reverse, 5′-TCTTGGTGACCATTTGGAGACA-3′. PRMT6 Is the H3R2 Arginine Methyltransferase–CARM1 is reported to methylate H3R2, based on peptide mapping of in vitro methylated H3 (14Schurter B.T. Koh S.S. Chen D. Bunick G.J. Harp J.M. Hanson B.L. Henschen-Edman A. Mackay D.R. Stallcup M.R. Aswad D.W. Biochemistry. 2001; 40: 5747-5756Crossref PubMed Scopus (284) Google Scholar). We decided to further study this using recombinant PRMTs and CARM1 knock-out mice. Based on the previous reports, we hypothesized that CARM1-null embryos would lose immune reactivity with an H3R2me2a-specific antibody. Surprisingly, immunostaining of wild-type and CARM1-null E18.5 embryos reveals a loss of H3R17 methylation, but not of H2R2 methylation, upon CARM1 loss (Fig. 1A). We then performed in vitro methylation experiments on calf thymus core histones using a set of recombinant PRMTs. Both CARM1 and PRMT6 robustly methylated H3 (Fig. 1B). PRMT1 methylates histone H4 and H2A. Next, calf thymus H3 was again methylated in vitro with this same set of PRMTs. The methylated H3 was then used for fluorography and Western analysis, using antibodies specific for histones methylated at different sites. The specificity of the antibodies was tested and only recognizes H3 when modified at the indicated site (data not shown). Surprisingly, although CARM1 dramatically increased the amount of R17 methylation, it did not increase the levels of arginine 2 methylation. PRMT6, and to a lesser degree PRMT1, catalyzed the methylation of H3R2 in vitro (Fig. 1C). Therefore we conclude that PRMT6 is the primary enzyme responsible for H3R2 methylation. Knockdown of PRMT6 Decreases H3R2me2a, whereas Overexpression Increases H3R2me2a–To further validate the data obtained from the in vitro methylation experiments, we overexpressed PRMT6 in HEK293 cells (and HeLa cells, data not shown), which clearly led to an increase in H3R2me2a on bulk histones (Fig. 2A). If overexpression of PRMT6 results in increased H3R2me2a levels, one would expect that by knocking down endogenous PRMT6 levels, we would see a decrease in H3R2me2a methylation levels on bulk histones. Indeed, this is what was observed when we generated a stable PRMT6 knockdown cell line in U2OS cells (Fig. 2B). These data further support the finding that PRMT6 is the H3R2 methyltransferase. H3 Binding Domains Are Sensitive to H3R2 Methylation–The published structures of the ING2 PHD finger, the WDR5 WD40 domain, and the JMJD2A tudor domains all show important contacts between domain residues and arginine 2 of H3. Thus, we were interested in looking at the effects of H3R2 methylation on the binding of these domain types. To address this, we synthesized biotinylated peptide and tested the ability of recombinant PHD, tudor, and WD40 domains to bind unmodified (H3K4me0), H3K4me, and the H3R2me2aK4me3 “dual” modified peptide using a peptide pulldown approach. The selected domains tested included the tudors of JMJD2A, the WD40 domains of WDR5, and several PHD domains that have been reported to bind H3K4me3 (ING2 and BPTF) and others tested here for the first time (PHF2, DATF1, and RAG2). Clearly, the majority of domains tested in this assay are sensitive to H3R2 methylation, as by reduced binding to the peptide as with the H3K4me3 peptide (Fig. not all domains are that proteins are not by this Kuo A.J. S. S. D. M. Carney D. P. Walter K.L. Utz P.J. Shi Y. Kutateladze T.G. W. Gozani O. Nature. 2007; PubMed Scopus Google Scholar, S. Kuo A.J. Carney D. Gozani O. W. S. A. 2007; PubMed Scopus Google Scholar). WDR5 the unmodified H3 N-terminal tail peptide of H3K4 methylation. again, we see that H3R2 methylation this binding. PRMT6 H3 of 4 Methylation to this it is whether the combination of H3K4me3 and on the same histone in To address this we performed an in vitro methylation using histone to amino acids of H3 containing of methylation were subjected to in vitro methylation by of the peptides revealed that PRMT6 has the ability to methylate all the H3 peptides of the lysine 4 methylation (Fig. although methylation of the H3K4me3 substrate is the other peptides Thus, PRMT6 can down the R2me2 whereas H3K4 is methylated in The endogenous levels of the mark and the of the mark are analysis of these two that not and are in E. F. L. L. C. L. Nat. Biol. 2006; PubMed Scopus Google Scholar). The ability of PRMT6 to in vitro methylate a peptide that is methylated at H3K4 that this modification can exist in vivo and the that it is not after the mark is lysine the loss of the H3K4me3 We have not tested the ability of proteins to methylate an H3R2me2a A large number of enzymes have been reported to methylate the H3K4 site Y. Mol. Cell. 2007; 25: Full Text Full Text PDF PubMed Scopus Google Scholar). PRMT6 and D1 H3R2 methylation binding of WDR5 and ING2 chromatin binding domains to H3 N-terminal tail we hypothesized that the recruitment of these complexes to gene be when PRMT6 is or when PRMT6 is We RNA from transfected U2OS cells, U2OS cells that were transfected with a PRMT6 expression vector and U2OS cells for real-time PCR analysis on and cyclin D1 The expression of these genes is by the and protein complexes (6Shi X. Hong T. Walter K.L. Ewalt M. Michishita E. Hung T. Carney D. Pena P. Lan F. Kaadige M.R. Lacoste N. Cayrou C. Davrazou F. Saha A. Cairns B.R. Ayer D.E. Kutateladze T.G. Shi Y. Cote J. Chua K.F. Gozani O. Nature. 2006; 442: 96-99Crossref PubMed Scopus (2) Google Scholar, Y. Milne T.A. A.J. Lee S. Lee Verdine G.L. Allis C.D. Roeder R.G. Nat. Struct. Mol. Biol. 2006; 13: PubMed Scopus Google Scholar). Specifically, the complex transcription, whereas the complex cyclin D1 after DNA In with overexpression of PRMT6 whereas PRMT6 knockdown increased expression (Fig. In addition, of cyclin D1 after by the was in the PRMT6 knockdown cells and in the cells (Fig. The of cyclin D1 after in the cells is to the increased binding of the ING2 H3R2 methylation antagonizes ING2 and PRMT6 knockdown bulk H3R2 methylation levels. Indeed, ChIP analysis of the increased of ING2 at the cyclin D1 when PRMT6 is down (Fig. and Here we a number of proteins sensitive to arginine 2 (Fig. We also showed that the H3R2 can be methylated by PRMT6 when lysine 4 is previously One can cellular PRMT6 to active genes to a H3R2 methylation by PRMT6 would proteins and their transcriptional this H3K4 be to this mark to the this E. F. L. L. C. L. Nat. Biol. 2006; PubMed Scopus Google show a between R2me2 and K4me3 on regions bound by the that the mark is present at H3K4 recruitment is one in a modified on H3. study the of a histone code and will the to on the effects histone have on each other and effector protein binding. described is the between methylation and binding to is by Allis C.D. H. W. 2006; PubMed Scopus Google Scholar). We performed a for genes affected by PRMT6 activity and found that and cyclin D1 is to PRMT6 levels, methylation of We for and for and we also Gozani for the vector and critical core are by of

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.