the spectrum (Figure 1a). Furthermore, other CAD spectra conserved throughout eukaryotes. Finally, we

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1 996 Brief Communication Methylation of histone H4 at arginine 3 occurs in vivo and is mediated by the nuclear receptor coactivator PRMT1 Brian D. Strahl*, Scott D. Briggs*, Cynthia J. Brame, Jennifer A. Caldwell, Stephen S. Koh, Han Ma, Richard G. Cook, Jeffrey Shabanowitz, Donald F. Hunt, Michael R. Stallcup and C. David Allis* Posttranslational modifications of histone amino Results and discussion termini play an important role in modulating Recent reports showing that CARM1 and PRMT1 meth- chromatin structure and function [1 3]. Lysine ylate histones in vitro and contain transcriptional coactivator methylation of histones has been well documented function suggest that histones are potential physiologimethylation [4, 5], and recently this modification has been linked cal targets for arginine methylation [10, 12]. However, to cellular processes involving gene transcription clear evidence for the existence of arginine methylation and heterochromatin assembly [6 9]. However, the on histones has been lacking [13]. To identify sites of existence of arginine methylation on histones has arginine methylation on mammalian histones in vivo, we remained unclear. Recent discoveries of protein individually purified histones isolated from asynchronously arginine methyltransferases, CARM1 and PRMT1, growing human 293T cells by reverse-phase high-perfor- as transcriptional coactivators for nuclear receptors mance liquid chromatography (RP-HPLC), digested suggest that histones may be physiological targets them with chymotrypsin, and examined the resulting pep- of these enzymes as part of a poorly defined tides by nano-hplc microelectrospray ionization tandem transcriptional activation pathway [10 12]. Here we mass spectrometry [14]. Collision-activated dissociation show by using mass spectrometry that histone H4, (CAD) spectra of human H4 N-terminal chymotryptic isolated from asynchronously growing human 293T peptides revealed the addition of a methyl group to the cells, is methylated at arginine 3 (Arg-3) in vivo. In Arg-3 residue and indicated N G -monomethylation (Figure support, a novel antibody directed against histone 1a). The predicted b- and y-type ion series of the H4 H4 methylated at Arg-3 independently demonstrates peptide in which Arg-3 is monomethylated are shown, and the in vivo occurrence of this modification and underlined masses indicate those fragments identified in reveals that H4 Arg-3 methylation is highly the spectrum (Figure 1a). Furthermore, other CAD spectra conserved throughout eukaryotes. Finally, we show H4 N-terminal peptides lacking methylation at show that PRMT1 is the major, if not exclusive, H4 Arg-3 (data not shown); these results support the above Arg-3 methyltransfase in human 293T cells. These characterization and indicate that some but not all H4 findings suggest a role for arginine methylation of molecules are methylated at Arg-3. While arginine residues histones in the transcription process. are capable of being methylated at either or both of their two terminal guanidino nitrogen groups generating Addresses: *Department of Biochemistry and Molecular Genetics, N G -monomethylarginine, symmetric N G,N G -dimethylar- University of Virginia Health Science Center, and Department of Chemistry and Pathology, University of Virginia, Charlottesville, Virginia ginine, or asymmetric N G,N G -dimethylarginine [13], our 22908, USA. Department of Pathology, University of Southern analyses did not reveal the presence of dimethylarginine California, Los Angeles, California 90089, USA. Department of at H4 Arg-3. However, these data do not exclude the possi- Immunology, Baylor College of Medicine, Houston, Texas 77030, USA. bility that H4 Arg-3 dimethylation exists. Correspondence: C. David Allis allis@virginia.edu These authors contributed equally to this work. Received: 21 May 2001 Accepted: 23 May 2001 Published: 26 June 2001 Current Biology 2001, 11: We next sought to develop an antibody specific to this methylation site in histone H4. Rabbits were immunized with an H4 1 9 synthetic peptide in which Arg-3 was N G,N G -dimethylated. The specificity of this antiserum to methylated Arg-3 was verified by ELISA with N-terminal H4 1 9 peptides that were either unmodified or methylated at Arg-3 (Figure 1c). To examine the conservation of Arg-3 H4 methylation in vivo, we probed histones isolated from multiple eukaryotic organisms with the H4 Arg-3 methyl-specific antiserum (hereafter -H4 R3Me) /01/$ see front matter Immunoblot analyses revealed the presence of Arg Elsevier Science Ltd. All rights reserved. methylation on H4 in all histones tested with the exception of (1) Tetrahymena, which contains a divergent ex-

2 Brief Communication 997 Figure 1 Histone H4 is methylated at arginine 3 in vivo. (a) RP-HPLC-purified H4 isolated from 293T cells was digested with chymotrypsin, and the resulting peptides were examined by nanohplc microelectrospray ionization tandem mass spectrometry. The doubly charged Arg-3-methylated H4 peptide having a mass-to-charge (m/z) ratio of was subjected to collision-activated dissociation. The sequence of the peptide is inset; across the top are predicted b-type ions, which contain the amino terminus of the peptide, and across the bottom are predicted y-type ions, which contain the carboxyl terminus of the peptide. Those ions observed in the spectrum are underlined. Zeros indicate ions generated by the loss of water; asterisks indicate ions generated by the loss of ammonia. The addition of 14 mass units ( CH 3 H) to the Arg-3 residue, as indicated by the b 3,y 9,y 8 *, and y 9 * ions, indicates Arg-3 N G -monomethylation. No dimethylation of Arg-3 was observed (data not shown). The bracket indicates a region where intensity was multiplied five times for better visualization of identified ions. (b) Sequence alignment of the H4 N terminus from multiple eukaryotic species. The first residue in all species shown with the exception of Tetrahymena is known to be N-terminally blocked by acetylation (indicated by Ac-NH ). The asterisk indicates the arginine residue found to be methylated in vivo by mass spectrometry. (c) Unmodified or Arg-3 methylated H4 1 9 peptides were used for an ELISA analysis of an H4 Arg-3 methyl-specific rabbit antiserum ( -H4 R3Me). (d) -H4 R3Me rabbit antiserum was used for an immunoblot analysis of histones isolated from multiple eukaryotic species. Total core histones from the species indicated (5 g) along with 1 g of recombinant Xenopus H4 (a sequence that is identical to chicken and human H4; Figure 1b) were resolved on a 15% SDS-PAGE, transferred to a PVDF membrane support, and probed with the -H4 R3Me antibody (upper panel). We analyzed identical samples in parallel and examined them by Coomassie staining to monitor of histone loading (lower panel). treme amino-terminal H4 tail lacking Arg-3, and (2) recombinant Xenopus H4 (Figure 1d). Together with our mass spectrometric analyses, these data suggest that the -H4 R3Me antibody recognizes both mono- and dimethylarginine. These results clearly demonstrate the in vivo existence of arginine methylation in a broad range of eukaryotic histones. Given this conservation, H4 Arg-3 methylation may play an important role in histone metabolism. In an attempt to determine the identity of the H4 Arg-3 methyltransferase, we examined the substrate and site specificity of PRMT1, which was previously shown to methylate H4 in a mixture of free calf thymus histones [10]. Purified recombinant GST-PRMT1 was incubated with core histones isolated from chicken, Tetrahymena, and human 293T cells in the presence of S-Adenosyl-L- [methyl- 3 H] methionine ( 3 H-AdoMet), and reaction products were analyzed by SDS-PAGE and fluorography. The results showed that GST-PRMT1 efficiently methylates chicken and human 293T H4 from a mixture of core histones (Figure 2a). As expected, GST-PRMT1 was unable to methylate H4 from Tetrahymena (Figure 2a), and this finding suggests that Arg-3 is the major, if not exclusive, site of PRMT1 methylation under these assay conditions (see below). No methylation of histones was ob-

3 998 Current Biology Vol 11 No 12 Figure 2 (Figure 2b). In agreement, recombinant Xenopus H4 (rh4), incubated in the presence of GST-PRMT1, was strongly methylated at Arg-3 by immunoblot analysis with the -H4 R3Me antibody (Figure 2c). In contrast, rh4 from reactions lacking GST-PRMT1 was not immunoreactive (Figure 2c). Nano-HPLC microelectrospray ionization mass spectrometric analyses of the identical reactions above revealed that the exclusive methylation product at Arg-3 was N G -monomethylarginine (data not shown), a result consistent with that previously observed by mass spectrometry on H4 isolated from human 293T cells (see Figure 1a). No dimethylarginine was detected by mass spectrometry on rh4 after methylation in vitro by GST- PRMT1 under our assay conditions. PRMT1 selectively methylates arginine 3 of H4. (a) Histones isolated from cells of several eukaryotic species were reacted in the presence of purified GST-PRMT1 and 3 H-AdoMet. Reactions were analyzed on a 15% SDS-PAGE, and examination by Coomassie staining (lower panel) or fluorography (upper panel) followed. The asterisk indicates H2A methylation. (b) RP-HPLC-purified H4 isolated from GST-PRMT1- labeled chicken core histones was deblocked and subjected to N-terminal automated sequencing. A portion of each cycle was used for amino acid identification by RP-HPLC, while the remainder was counted for 3 H radioactivity. The arrow indicates the arginine residue found to be methylated. (c) Recombinant Xenopus H4 (rh4) reacted in the presence or absence of purified GST-PRMT1 along with AdoMet was immunoblotted as described in Figure 1 by the use of -H4 R3Me rabbit antiserum (upper panel). We performed identical reactions in parallel and examined them by Coomassie staining to monitor rh4 loading (lower panel). served in the absence of GST-PRMT1 (Figure 2a). Under these reaction conditions, mammalian H2A was also found to be weakly methylated (Figure 2a), a result consistent with earlier findings [10]. To determine definitively which arginine residue(s) in the H4 amino terminus is methylated by PRMT1, RP- HPLC-purified H4 from labeling reactions with purified GST-PRMT1 and chicken core histones was microsequenced, and 3 H incorporation associated with each cycle was determined. Microsequence analysis showed that Arg-3 is the exclusive site of methylation in the H4 tail Next, we sought to determine if PRMT1 is responsible for mediating H4 Arg-3 activity in human 293T cells. Initially, we asked if ectopically expressed PRMT1 isolated from these cells retained the same substrate specificity for H4 as seen with recombinant PRMT1. To that end, HA-tagged PRMT1 (HA-PRMT1 wt), HA-tagged PRMT1 mutant (HA-PRMT1 mut), which lacks the putative AdoMet binding site, or the parent vector (HA) were ectopically expressed in human 293T cells; nuclear isolation and DNase I extraction followed. We then used chicken core histones and either AdoMet or 3 H-AdoMet to perform histone methyltransferase (HMT) assays with these extracts. The assays revealed that nuclear extracts from HA- PRMT1-expressing cells incorporated more 3 H-AdoMet on H4 than extracts from either HA-PRMT1 mutant- or HA-expressing cells (Figure 3a). Furthermore, immu- noblots probed with the -H4 R3Me antibody showed that this activity is specific for histone H4 Arg-3 (Figure 3a). Immunoprecipitations of the same nuclear extracts with an HA-specific antibody directly demonstrate that the enhanced H4 Arg-3 methyl activity observed in Figure 3a is a result of HA-PRMT1 expression (Figure 3b). These results indicate that cellular-derived PRMT1 can directly and specifically methylate Arg-3 on H4. Further- more, given that expressed HA-PRMT1 is extracted by DNase I digestion, these data also suggest that PRMT1 is chromatin associated. In support, we note that the expression of HA-PRMT1 enhances nucleosomal H4 Arg-3 methylation activity that is detectable in 293T DNase I nuclear extracts (data not shown). Although recombinant or cellular PRMT1 can methylate H2A (as indicated by asterisks in Figures 2a and 3b,c), this methylation is not detected by the -H4 R3Me antibody (Figure 3b). This finding suggests that the -H4 R3Me antibody is selective for H4 Arg-3 methylation. Since H4 Arg-3 methylation activity was detected in 293T control DNase I extracts (see Figure 3a), we next wanted to determine if this activity was due to endogenous PRMT1. To directly test this idea, we immunodepleted endogenous PRMT1 from DNase I nuclear extracts and

4 Brief Communication 999 Figure 3 H4 Arg-3 methylation in human 293T cells is mediated by PRMT1. (a) 293T cells were transiently transfected with either parent vector (HA), HA-PRMT1 putative AdoMet binding mutant (HA-PRMT1 mut), or wild-type HA-PRMT1 (HA-PRMT1 wt) expression construct. This was followed by nuclear isolation and DNase I extraction. HMT assays with these DNase I extracts were performed with 2 g of chicken core histones in the presence of either radiolabeled or nonradiolabeled AdoMet and analyzed, as previously described, by fluorography or immunoblots, respectively. Expression of HA- PRMT1 mut and wild-type HA-PRMT1 was monitored by immunoblotting with a monoclonal -HA antibody. (b) DNase I extracts from transfected cells as described above were immunoprecipitated with an -HA antibody, and immunoprecipitations were assayed for 3 H-methyl and H4 Arg-3-specific HMT activity as described in Figure 2. (c) Immunodepletion of endogenous PRMT1 leads to the removal of H4 Arg-3 methylation activity. 293T DNase I extracts were incubated in the presence of protein G-sepharose with analyzed for both 3 H-methyl and H4 Arg-3- only versus beads plus addition of -PRMT1) or without -PRMT1 followed by specific HMT activity as described above. were assayed for 3 H-methyl HMT activity. immunoprecipitation. Supernatants were Pellets from these immunoprecipitates (beads Asterisks indicate H2A-specific methylation. assayed for any remaining H4 methylation activity. Immu- eral transcription-related histone acetyltransferases have nodepletion of PRMT1 resulted in a nearly complete been shown to bind selectively to acetylated histones abolishment of H4 Arg-3 methylation activity in these [17]. These data suggest that H4 Arg-3 methylation may extracts (Figure 3c) and indicated that PRMT1 is the function through selective recruitment of a yet-unidenti- major, if not exclusive, H4 Arg-3 HMT from human 293T fied factor(s). In addition, H4 Arg-3 methylation might cells. Furthermore, we show that immunoprecipitated also function in transcription by influencing adjacent mod- PRMT1 from the above immunodepleted assay retained ifications on the H4 amino terminus, such as serine 1 essentially all of the H4 HMT activity (Figure 3c). phosphorylation and/or lysines 5, 8, 12, and 16 acetylation [4, 18]. Recent studies on H3 suggest that adjacent modifications In sum, our data clearly establish the in vivo existence of can influence transcription [19] and other cellular arginine methylation on histone H4. Furthermore, we processes [20]. For example, serine 10 phosphorylation show, by using a novel Arg-3 H4 methylation-specific coexists and is synergistic with lysine 14 acetylation in antibody, that this modification is highly conserved among the mitogenic response [19, 21]. In contrast, serine 10 eukaryotes. Finally, strong evidence is provided that phosphorylation is inhibited by lysine 9 methylation in PRMT1 is the responsible activity for H4 Arg-3 methyla- heterochromatin [20]. Future studies, then, should shed tion in mammalian cells. Our data are consistent with light on how H4 Arg-3 methylation elicits meaningful recent reports showing that PRMT1 is responsible for the downstream responses. majority of nuclear arginine (type I) methylation activity found in mammalian cells [15, 16] Given its role as a Supplementary material nuclear receptor coactivator [12], our data suggest that Materials and methods with additional references are available at PRMT1 has an important role in regulating gene activity images.cellpress.com/supmat/supmatin.htm. through methylation of the H4 amino terminus. However, how methylation at this site might promote gene transcrip- Acknowledgements This research was supported by grants from the National Institutes of Health to tion remains an intriguing question. One possible explana- C.D.A. (GM53512), B.D.S. (GM20039), D.F.H. (GM ), and M.R.S. tion may be provided by the histone code hypothesis [1, (DK55274). S.D.B is a Leukemia and Lymphoma Society Fellow. We thank R. Rice, M.A. Jelinek, T. Jelinek, and S. Paschke at Upstate Biotechnology 2], which predicts that histone modifications selectively for their assistance with the development of the H4 Arg-3 methyl-specific recruit factors that contain modification-specific histone antibody. We thank K. Luger for providing recombinant histone H4, H. Hershman binding domains. Such methyl-specific recruitment on for the -PRMT1 polyclonal antibody, C. Mizzen for chicken histones and nucleosomes, and all current C.D.A. laboratory members for their helpful histones has already been described for lysine 9 methyladiscussions and technical advice. Also, we wish to thank Y. Zhang and D. tion of H3 [7 9]. Furthermore, the bromodomains of sev- Aswad for kindly sharing unpublished data.

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