a novel histone h4 arginine 3 methylation-sensitive histone h4

A Novel Histone H4 Arginine 3 Methylation-sensitive HistoneH4 Binding Activity and Transcriptional Regulatory Functionfor Signal Recognition Particle Subunits SRP68 and SRP72*□S

Received for publication, August 28, 2012, and in revised form, October 5, 2012 Published, JBC Papers in Press, October 8, 2012, DOI 10.1074/jbc.M112.414284

Jingjing Li‡, Fan Zhou§, Deguo Zhan‡, Qinqin Gao‡, Nan Cui‡, Jiwen Li‡, Elena Iakhiaeva¶1, Christian Zwieb¶,Biaoyang Lin§, and Jiemin Wong‡2

From the ‡Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East ChinaNormal University, Shanghai 200241, China, §Systems Biology Division, Zhejiang-California International Nanosystems Institute(ZCNI), Zhejiang University, Hangzhou, 310029, China, and the ¶Department of Biochemistry, The University of Texas HealthScience Center at San Antonio, San Antonio, Texas 78229

Background: Histone methylation is believed to recruit specific histone-binding proteins.Results:We identified SRP68/72 heterodimers as major nuclear proteins whose binding of histone H4 tail is inhibited by H4R3methylation.Conclusion: SRP68/72 are novel histone H4-binding proteins.Significance:Uncovers a novel chromatin regulatory function for SRP68/72 and suggests that histone argininemethylationmayfunction mainly in inhibiting rather than recruiting effector proteins.

Arginine methylation broadly occurs in the tails of core his-tones. However, the mechanisms by which histone argininemethylation regulates transcription remain poorly understood.In this study we attempted to identify nuclear proteins that spe-cifically recognize methylated arginine 3 in the histone H4(H4R3) tail using an unbiased proteomic approach. No majornuclear protein was observed to specifically bind to methylatedH4R3 peptides. However, H4R3 methylation markedly inhib-ited the binding of two proteins to H4 tail peptide. These pro-teins were identified as the SRP68 and SRP72 heterodimers(SRP68/72), the components of the signal recognition particle(SRP). Only SRP68/72, but not the SRP complex, bound the H4tail peptide. SRP68 and SRP72 bound the H4 tail in vitro andassociatedwith chromatin in vivo. The chromatin association ofSRP68 and SRP72 was regulated by PRMT5 and PRMT1. BothSRP68 and SRP72 activated transcription when tethered to areporter via a heterologous DNA binding domain. Analysis ofthe genome-wide occupancy of SRP68 identified target genesregulated by SRP68. Taken together, these results demonstratea role of H4R3 methylation in blocking the binding of effectorsto chromatin and reveal a novel role for the SRP68/SRP72 het-erodimer in the binding of chromatin and transcriptionalregulation.

Arginine methylation, a post-translational modification cat-alyzed by a family of protein arginine methyltransferases(PRMT),3 is commonly observed in cytoplasmic and nuclearproteins including core histones (1–3). Multiple arginine (Arg)residues in histone tails, including Arg-2, -8, -17, and -26 in H3and Arg-3 in H4 have been shown to be mono (me1)- or dim-ethylated (me2), with the latter in a symmetrical or a asymmet-rical configuration (me2s or me2a) (1, 4). For example, PRMT5has been shown to catalyze the symmetrical dimethylation ofthe Arg-3 residue in H4 N-terminal tail (H4R3me2s) (5, 6),whereas PRMT1 can catalyze the asymmetrical dimethylationof the same residue (H4R3me2a) (7). Extensive studies havecorrelated H4R3me2s catalyzed by PRMT5 with transcrip-tional repression of associated genes (8–10) and H4R3me2acatalyzed by PRMT1 with transcriptional activation (7, 11).Like histone lysine methylation (12–14), in principle histone

arginine methylation can regulate transcription either by effectin cis on other histone modifications and/or by serving as his-tone code to influence the binding of histone-interacting effec-tor proteins. In this regard,H4R3me2a catalyzed byPRMT1hasbeen shown to promote subsequent histone acetylation byCBP/p300 (7, 15); this in cis effect explains at least in part therole of H4R3me2a in transcriptional activation. In support ofthe histone code hypothesis, an increasingly large number ofproteins has been shown to specifically bind variousmethylatedlysine residues in histone N-terminal tails and plays diverseroles in epigenetic regulation (14, 16, 17). In contrast, so far onlya few proteins including Tudor domain-containing protein 3(TDRD3), DNA methyltransferase 3a (Dnmt3a), RNA polym-erase-associated protein 1 (PAF1) complex, and p300/CBP-as-sociated factor (PCAF) have been implicated in binding of

* This work was supported by Ministry of Science and Technology of ChinaGrants 2010CB944903, 2009CB918402, and 2009CB825601, National Nat-ural Science Foundation of China Grants 90919025 and 30871381, andScience Technology Commission of Shanghai Municipality Grants09DJ1400400 and 11DZ2260300.

□S This article contains supplemental Table S1 and Figs. S1 and S2.1 Present address: The University of Texas Health Science Center, Tyler, TX

75708.2 To whom correspondence should be addressed: Institute of Biomedical Sci-

ences and School of Life Sciences, East China Normal University, 500Dongchuan Rd., Shanghai 200241, China. E-mail: [email protected].

3 The abbreviations used are: PRMT, protein arginine methyltransferase; me1,monomethylated; me2, dimethylated; CBP, cAMP-response element-bind-ing protein (CREB)-binding protein; SRP, signal recognition particle; ER,endoplasmic reticulum; DBD, DNA binding domain; TK, thymidine kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 48, pp. 40641–40651, November 23, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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methylated arginine residues in histone tails (18–21), andamong them only the binding of H3R17me2a and H4R3me2aby TDRD3 is supported by biochemical and structural evi-dences (22). TDRD3 binds H3R17me2a and H4R3me2a via aTudor domain that has been recognized as a structuralmotif forbinding of arginine-methylated non-histone proteins (23). Thelimited number of arginine-methylated histone-binding pro-teins identified so far raises the possibility for the existence oflarge number of arginine-methylated histone-specific effectorsthat remain to be identified. Alternatively, it may underscore amajor mechanistic difference in the action of arginine andlysine methylation.Mammalian signal recognition particle (SRP) is a ribonucleo-

protein complex composed of six SRP proteins (SRP9, SRP14,SRP19, SRP54, SRP68, and SRP72) and a RNAmolecule knownas 7 S RNAor 7 SLRNA (24, 25). The SRP complex is conservedin evolution and plays a central role in the co-translational tar-geting of secretory and membrane proteins to the endoplasmicreticulum (ER). SRP binds nascent signal peptide sequences ofproteins as they emerge from the ribosome. The resulting tar-geting complex then docks to ER via interaction with the SRPreceptor in a GTP-dependent manner (26). Previous studieshave shown that SRP68 and SRP72 exist predominantly as astable SRP68/72 heterodimer that is essential for SRP-mediatedER-targeting of proteins (27).In this study we used an unbiased proteomic approach to

screen for proteins that bind specifically to H4R3me2s andH4R3me2a. Instead of identifying new methyl-H4R3-bindingproteins, we found two proteins, SRP68 and SRP72, whosebinding to the H4 tail was inhibited by arginine methylation.Our study illustrates a novel function of H4R3 methylation ininhibiting binding of chromatin effectors and reveals a noveltranscriptional function for SRP68 and SRP72.

EXPERIMENTAL PROCEDURES

Plasmids, Antibody, Cell Lines, Transfection, and LuciferaseAssay—The expression plasmids pcDNA3/SRP54, pcDNA3/SRP68, pcDNA3/SRP72, pGEX-4T-1/SRP68, and pGEX-4T-1/SRP72 were constructed by cloning the full-length humanSRP68 and SRP72 into pCDNA3.0 and pGEX4T-1 vectors,respectively. The CFP-Lac-H4t plasmid was generated by clon-ing 2 tandem copies of oligonucleotides encoding the first 20amino acids of human H4 N-terminal tail. The plasmids for invitro synthesis of [35S]Met-labeled SRP54, SRP68, and SRP68and their respective deletion mutants have been described pre-viously (27–29). To express SRP68 or SRP72 and their deletionmutants as Gal4(DBD) fusion proteins, the correspondingcDNAs were cloned into pCMV-Gal4(DBD) vector. The4xUAS-TK-luc luciferase reporter was as described (30). Com-mercially available antibodies directed toward H3, H4, andH4R3me2s were from Abcam (Cambridge, MA); HA was fromRoche Applied Science; FLAG was from Sigma; SRP54, SRP19,SRP14, and SRP9 were from eBiosciences (San Diego, CA).SRP68 and SRP72 antibodies were generated in the laboratoryby immunizing rabbits with GST-SRP68 and GST-SRP72.HeLa and 293T cell lines were maintained in Dulbecco’s

modified Eagle’s medium supplemented with 10% fetal bovineserum. Transient transfections in 293T and HeLa cells were

performed using Lipofectamine 2000 (Invitrogen) according tothe manufacturer’s instructions. Luciferase reporter assay wasessentially as described (30).Isolation of Binding Proteins from HeLa Nuclear Extracts

Using Biotinylated H4 Tail Peptides—Nuclear extracts wereprepared fromHeLa cells by the protocol of Dignam et al. (31).The C-terminal biotinylated H4 tail peptides (amino acids1–16) without or with either a H4R3me2a or H4R3me2s weresynthesized and purified by Beijing Scilight Biotechnology Ltd.Co. Purification of corresponding H4 peptide-binding proteinsfrom HeLa nuclear extracts was carried out essentially asdescribed (32).Mass Spectrometry and Western Blot Analysis—Both meth-

ods were performed as described previously (32).Pulldown Assay with in Vitro Synthesized Proteins and

Recombinant Proteins—Radiolabeled proteins were generatedwith the TNT coupled reticulocyte lysate system (Promega). Invitropulldown assayswere carried out by incubating the in vitrotranslated products with 1 �g of immobilized histone tail pep-tides as described (33). For binding of purified recombinantGST-SRP68, GST-SRP68-(436–620), GST-SRP72, and GST-SRP-(529–659), the recombinant proteins (10 �g) were incu-bated with immobilized H4 tail peptides in the binding buffer(20 mM HEPES, pH 7.9, 150 mM KCl, 1 mM dithiothreitol(DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10% glyc-erol, 0.1% Nonidet P-40, proteinase inhibitors) for 2 h at 4 °C.Unbound proteins were removed by washing the beads withwashing buffer (20mMHEPES, pH7.9, 200mMKCl, 1mMDTT,1 mM PMSF, 0.1% Nonidet P-40, proteinase inhibitors) 4 timesfor 5 min each. The proteins that remained bound to the pep-tides were separated by SDS-PAGE followed by CoomassieBlue staining.Preparation of Cytosol, Nuclear Extract, and Chromatin and

Chromatin Immunoprecipitation (ChIP)-Western Blot—Tofractionate cellular contents to cytosol and nuclear and chro-matin fractions, cultured cells were collected by centrifugationand washed twice with ice-cold PBS. The pellets were resus-pended in 2 packed cell volumes of solutionA (20mMTris-HCl,pH8.0, 50mMNaCl, 1%Nonidet P-40, 1mMDTT, and proteaseinhibitors), incubated on ice for 10min, and centrifuged at 4000rpm for 5 min at 4 °C. The resulting supernatants were collectedanddesignatedas cytosol.Thepelletswere resuspendedwith solu-tionA containing 0.4 MNaCl and incubated in ice for 20min. Thesampleswere centrifuged at 12,000 rpm for 10min, and the super-natants were designated as nuclear extracts. The pellets werewashedoncewithsolutionA, resuspended in2volumesof1�SDSloading buffer, and designated as chromatin fractions.For ChIP-Western blot analysis, HeLa or 293T cells were

treated with 1% formaldehyde for 15 min in culture medium.The cells were lysed as above, and the pellets containing nucleiwere resuspended in solution A plus 3 mM CaCl2 and 5 units ofmicrococcal nuclease. After incubation on ice for 1 h, the sam-ples were sonicated, and the soluble chromatin was prepared.Immunoprecipitationwas carried out with or without the addi-tion of anti-SRP68 antibody, and Western blot analyses wereperformed using the antibodies as indicated.

H4R3me2 Inhibits SRP68/72 Chromatin Association

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Immunofluorescent Staining and Cell Imaging with CFPFusion Proteins—Subcellular localization analyses for endoge-nous- and ectopic-expressed SRP proteins were performed inHeLa or 293T cells. The interaction between SRP68 and the H4tail by co-localization in DG44 CHO cells was assessed asdescribed previously (34).Knockdown of SRP68 by siRNA—The siRNAs against human

SRP68were synthesized and purified by Shanghai GenePharmaCo., Ltd. The sequences of three SRP68 siRNAs are as follow:GGCUGUGCUGUAUAACCAATT (siSRP-(68–722)), GAG-AUUCUUCAGAUUAUUATT (siSRP-(68–216)) and GCUUC-UGACCGAUAAUAGATT (siSRP-(68–383)). The knockdownof SRP68 in 293T cells for expression profiling was carried outusing 1:1mixture of siSRP-(68–722) and siSRP-(68–216) accord-ing to the manufacturer’s instructions. The sequence of controlsiRNA is UUCUCCGAACGUGUCACGUTT.Chromatin Immunoprecipitation and High-throughput

Sequencing—Chromatin immunoprecipitation assays wereperformed with or without immunoaffinity-purified SRP68antibody using chromatin prepared from 293T cells. Afterimmunoprecipitation, the purified DNAs were subjected tosequencing on a Illumina Genome analyzer. SOAP 2.20 wasused to align reads with two mismatches for each sample usingthe updated Human genome databases available on line atHuman (Homo sapiens) Genome Browser Gateway. Sequenceswith greater than 83% identity were used for further analyses.To identify ChIP peaks, ChIP seq data were analyzed using theMACS program available at with the SRP68 ChIP-Seq data asinput and the control ChIP-seq data as background. Defaultparameters were set for human genome, and the p value was setto p � 0.00001 or p � 0.0001 for the identification of positivepeaks. Cisgenome was used to annotate the peaks to their associ-ated genes and obtain the physical distribution in relation to pro-moters, exons, introns, and other features. The SRP68 associationwas confirmed using ChIP followed by quantitative PCR analysis.The ChIP primers used are shown in supplemental Table S1.

RESULTS

H4R3 Methylation Blocks the Binding of SRP68/72 to the H4Tail Peptide—In the attempt to identify proteins that specifi-cally recognize the H4R3me2s and H4R3me2a codes, weemployed an unbiased in vitro affinity purification approachusing immobilized histone tail peptides and HeLa nuclearextracts. We have previously used this approach to identifychromatin effectors specific for methylated H3K4 or H3K9 (32,35, 36). Three biotinylated H4 N-terminal tail peptides (aminoacids 1–16) containing either no modification or R3me2s orR3me2a were synthesized. These peptides were immobilizedon streptavidin-agarose beads through a C-terminal biotinmoiety and used for affinity purification of specific binding pro-teins obtained fromHeLa nuclear extracts. Bound polypeptideswere resolved by electrophoresis on a 4–20% SDS-PAGE geland visualized by silver staining (Fig. 1A). In multiple experi-ments no prominent polypeptides that bound specifically toeither the H4R3me2s or H4R3me2a peptides but not to the H4peptide was observed. Instead, two polypeptides with molecu-lar masses in the range of 70–75 kDa were reproduciblyobserved to be enriched in the control H4 peptide sample as

compared with the H4R3me2s and H4R3me2a peptides (Fig.1A). We sequenced the two protein bands by mass spectrome-try and determined their identities to be SRP68 and SRP72.SubsequentWestern blot analyses using antibodies specific forSRP68 and SRP72 confirmed that both proteins bound withhigher affinity to the H4 peptide than to the H4R3me2s andH4R3me2a peptides (Fig. 1B). Thus, we identified SRP68 andSRP72 as novel H4 tail-binding proteins whose binding activityis inhibited by H4R3 methylation.The SRP68/72 Heterodimer but Not the SRP Complex Binds to

the H4 Tail—The observed binding of SRP68 and SRP72 to theH4 N-terminal tail peptide raised the question if these proteinsbind to the H4 tail in the form of heterodimers or associatedwith the SRP complex (25, 27). Western blot analysis demon-strated that, unlike SRP68 and SRP72, SRP9, SRP14, SRP19, andSRP54 did not bind to the H4 peptide (Fig. 1B), suggesting thatSRP68 and SRP72 bind theH4 tail peptide independently of theother SRP proteins. To substantiate this further, we utilizedSuperose 6 gel filtration chromatography to separate SRP68and SRP72 inHeLa nuclear extract into a different complex(es).Western blot analysis revealed at least two protein complexesthat contained SRP68 and SRP72. The larger molecular weightcomplex cofractionated with SRP54 and probably representedthe fully assembled SRP (Fig. 1C, lanes 3–5). A smaller com-plex(es) also contained SRP68/72 but lacked SRP54 (Fig. 1C,lanes 6–9). When these fractions were assayed with regard totheir binding of H4 tail peptide, we observed that only SRP68 inthe smaller complex bound the H4 tail peptide (Fig. 1D). Inaddition, among all four histone tail peptides tested, SRP68 andSRP72 bound only the H4 tail peptide (Fig. 1E). The binding ofSRP68 and SRP72 to H4 peptide was insensitive to RNase Atreatment (Fig. 1F), further supporting the conclusion thatSRP68 and SRP72 bind in the form of heterodimers in theabsence of 7SL SRP RNA. Taken together, these data providecompelling evidence that SRP68/72 heterodimers, but not theSRP complex, are the histone H4 tail-specific binding proteins.SRP68 and SRP72 Associate with Chromatin in Vivo—SRP68

and SRP72 are well known for their exclusive protein targetingfunction within the SRP complex. Our finding that SRP68 andSRP72 bind specifically to the unmodified H4 tail peptide sug-gests a new function of SRP68 and SRP72 in chromatin regula-tion. In agreement with previous reports (37, 38), immuno-staining of HeLa cells revealed a predominantly cytoplasmiclocalization for SRP9, SRP19, and SRP54 (Fig. 2A and data notshown). However, the endogenous SRP68 in HeLa cells wasdetected predominantly in the nucleus, whereas SRP72 waspresent both in the nucleus and cytoplasm (Fig. 2A). Similarly,we observed that ectopically expressed SRP68 and SRP72 weremainly nuclear in HeLa cells (Fig. 2B).To investigate if the nuclear SRP68 and SRP72 associated

with chromatin, cytosol, nuclear, and chromatin fractions wereprepared from HeLa cells. A substantial amount of SRP68 andSRP72 was found to associate with chromatin (Fig. 2C). In con-trast, SRP54 did not associate with chromatin under the samecondition (Fig. 2C). As markers for appropriate cellular frac-tionation, �-actin was detected only in the cytosol, whereas thecore histone H3 was detected only in the chromatin fraction(Fig. 2C). To test further the chromatin association of SRP68




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and SRP72, HeLa cells were treated with 1% formaldehyde, andnuclei were prepared and subjected to chromatin digestionwith micrococcal nuclease. After centrifugation to removeinsoluble nuclei pellets, the soluble chromatin-containing frac-tion was immunoprecipitated with anti-SRP68 antibody andassayed for the presence of core histones byWestern blot anal-ysis. Fig. 2D shows the presence of core histones H3 and H4 inimmunoprecipitation of soluble chromatin with anti-SRP68antibody. Significantly, the chromatin that co-precipitatedwithSRP68 was devoid of H4R3me2s (Fig. 2D). This was in agree-ment with the in vitro peptide binding data that showed thatH4R3 methylation blocks the binding of SRP68/72 to the H4tail. Similar results were observed when immunoprecipitationwas performed with anti-SRP72 antibody and chromatinderived from HeLa cells (data not shown).To further demonstrate that SRP68 binds the H4 N-terminal

tail within cells, wemade use of a CHO cell line, which contains alarge number of Lac operator sequences stably integrated in a sin-gle chromosomal site (39). Expression of control CFP-tagged Lacproteins or CFP-Lac fused with a tandem H4 tail peptide (desig-nated as CFP-Lac-H4t) in these cells generated bright foci due tothe binding of integrated Lac sequences by CFP-Lac fusion pro-teins. We observed co-localization of ectopically expressed HA-

SRP68 with CFP-Lac-H4t but not the control CFP-Lac (Fig. 2E),indicating a tandem H4 tail dependent interaction with SRP68.Similar results were observed for SRP72 (data not shown). Takentogether these data demonstrate that SRP68 binds H4 tail peptidein cells and that a portion of the SRP68 and SRP72 proteins isintracellularly associated with chromatin.SRP68 and SRP72 Directly Interact with the H4 Tail in a

H4R3 Methylation-sensitive Manner—To investigate if SRP68and SRP72 directly interact with the H4 tail, in vitro translatedpolypeptides were assayed for binding to the four histone tailpeptides using pulldown assays. In vitro synthesized SRP68 andSRP72 bound H4 but not other histone tail peptides (Fig. 2F).Under the same conditions, SRP54 did not bind to the H4 tailpeptide (Fig. 2F). Using a series of SRP68 deletion mutants, wefurther mapped the H4 tail binding activity to the C-terminalregion (amino acids 436–620) of SRP68 that is known to alsobind to SRP72 (Fig. 2G). We observed that both the N-terminalregion (amino acids 1–356) and C-terminal region (amino acids529–659) of SRP72were able to bind theH4 tail peptide (Fig. 2H).To test if SRP68 and SRP72 bind directly the H4 tail peptide,

we expressed andpurifiedGST fusions of full-length SRP68 andSRP72 and their C-terminal H4 binding domains. In pulldownassays, these recombinant proteins bound specifically the H4

FIGURE 1. H4R3 methylation inhibits the binding of SRP68 and SRP72 heterodimers to the H4 tail peptide. A, an unbiased peptide pulldown assayrevealed SRP68 and SRP72 as major nuclear proteins whose binding to the H4 tail peptide was inhibited by either H4R3me2a or H4R3me2s. The identities of thepolypeptides were determined by mass spectrometry. B, HeLa nuclear extracts were subjected to pulldown assay as in A and analyzed by Western blot analysisusing antibodies as indicated. Western blot analyses confirmed that SRP68 and SRP72 bound to the H4 tail peptide but not H4R3me2a and H4R3me2s peptides.In addition, the binding of H4 tail peptide was detected for SRP68 and SRP72 but not other SRP proteins. The amounts of immobilized peptides employed inthe pulldown reactions were shown by Coomassie Blue staining. C, HeLa nuclear extracts were fractionated with a Superose 6 gel filtration column, and theindicated fractions were analyzed by Western blot. Brg1, a subunit of human SWI/SNF complex with molecular masses of �2 MDa, was served as a control.D, the fractions of HeLa nuclear extracts derived from Superose 6 gel filtration were tested for binding to the H4 tail peptide by pulldown assay. The amountsof immobilized peptides in the pulldown reactions were shown by Coomassie Blue staining. E, the SRP proteins in HeLa nuclear extracts were tested for bindingof all four histone tail peptides by in vitro pulldown and analyzed by Western blot using antibodies as indicated. F, the HeLa nuclear extracts were treated withor without RNase A first and then assayed for binding of H4 tail peptide by in vitro pulldown assay.




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tail peptide (Fig. 2I). Furthermore, H3R4methylation abolishedthe binding of recombinant SRP68 and SRP72 to the H4 tail(Fig. 2I, compare lane 5with lane 4). Together these data dem-onstrate that both SRP68 and SRP72 bind directly to the H4 tailin a Arg-3 methylation-sensitive manner.

PRMT5 Regulates SRP68/72 Chromatin Association andSubcellular Localization—Having established thatH4R3meth-ylation blocks the binding of SRP68 and SRP72 to the H4 tailpeptide and that SRP68 and SRP72 associate with chromatin incells, we next investigated the effect of H4R3 methylation on

FIGURE 2. SRP68 and SRP72 associate with chromatin in cells and directly bind to the H4 tail peptide in vivo and in vitro. A, the subcellular localizationof SRP subunits in HeLa cells were analyzed by immunofluorescent staining using antibody specific for each subunit. The nuclei were revealed by DAPI staining.Note that a predominant nuclear localization of endogenous SRP68 and SRP72 was observed. In contrast, SRP54 was found primarily in the cytoplasm.B, HA-tagged SRP68 and SRP72 were transfected into HeLa cells, and the subcellular localization of these proteins was revealed by immunostaining usinganti-HA antibody. Note both HA-SRP68 and HA-SRP72 expressed in HeLa cells were predominantly a nuclear localization. C, cellular fractionation of HeLa cellsrevealed the presence of SRP68 and SRP72 but not SRP54 in chromatin fraction. HeLa cells were fractionated into cytosol (cyto), nuclear (Nucl), and chromatin(chro) as described under “Experimental Procedures,” and the presence of SRP subunits in each fraction was determined by Western blot analysis. �-Actinserved as a cytosol marker, and core histone H3 served as a chromatin marker. D, ChIP followed by Western blot analysis revealed the association of SRP68 withsoluble chromatin. HeLa cells were treated with 1% formaldehyde to cross-link the chromatin-associated proteins with chromatin. The nuclei were prepared,and chromatin was released into soluble fraction by micrococcal nuclease digestion. The soluble chromatin fraction was then subjected to immunoprecipi-tation with anti-SRP68 antibody followed by Western blot (WB) analysis using antibodies as indicated. Note that the chromatin co-precipitated with SRP68 wasdevoid of H4R3me2s. E, colocalization of HA-SRP68 with CFP-Lac-H4t but not the control CFP-Lac in DG44 CHO cells is shown. CFP-Lac-H4t contains a tandemH4 tail (amino acids 1–20) peptide. The bright foci with colocalization of CFP-Lac-H4t and HA-SRP68 was marked by an arrow. F, in vitro synthesized, [35S]Met-labeled SRP68 and SRP72 bound to the H4 but not other histone tails in in vitro pulldown assays. As a control of binding specificity, no binding was detectedfor SRP54 under the same condition. The binding was revealed by autoradiography. G, mapping the H4 tail binding domain of SRP68 by using a series of SRP68deletion mutants and peptide pulldown assay is shown. The top panel illustrates the domain structure of SRP68. The SRP68 mutants used for peptide pulldownassay were in vitro synthesized, and [35S]Met was labeled. The binding was revealed by autoradiography. aa, amino acids; FL, full-length. H, mapping the H4 tailbinding domain of SRP72 by using a series of SRP72 deletion mutants and peptide pulldown assay was as above. I, the recombinant SRP68 and SRP72 boundpreferentially to the H4 tail peptide. GST fusion proteins were purified and subjected to in vitro binding with various histone tail peptides as indicated. Thebinding of recombinant proteins were revealed by Coomassie Blue staining.




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the intracellular association of SRP68 and SRP72 with chroma-tin. For this purpose, we overexpressed FLAG-PRMT5 and itsenzymatic defective mutant in 293T cells and analyzed theeffect on SRP68 and SRP72 subcellular localization and chro-matin association by cellular fractionation.We found that over-expression of wild type (Fig. 3A, compare lanes 5 and 6 withlanes 2 and 3) but not the mutant PRMT5 (Fig. 3A, comparelanes 8 and 9 with lanes 2 and 3) reduced the levels of nuclearand chromatin-associated SRP68 and SRP72. Western blotanalysis also detected increased levels of H4R3me2s in chroma-tin derived from FLAG-PRMT5 but not FLAG-PRMT5m-ex-pressed cells (see Fig. 5A, compare lane 6with lanes 3 and 9). Ascontrols for proper cellular fractionation, �-actin was detectedmainly in the cytosol and core histone H3 in the chromatin.These results indicate that PRMT5 regulates SRP68 and SRP72chromatin association in an enzymatic activity-dependentmanner. Furthermore, PRMT5 appears to promote nuclear tocytoplasmic translocation of SRP68 and SRP72.To further examine the effect of PRMT5 on SRP68 and SRP72

subcellular localization and chromatin association, we cotrans-

fected FLAG-PRMT5 with HA-SRP68 or HA-SRP72 into HeLacells and analyzed the subcellular localization of HA-SRP68 andHA-SRP72 by immunofluorescent staining. AlthoughHA-SRP68was primarily nuclear in cells expressing HA-SRP68 alone, itwas predominantly localized in the cytoplasm in cells that co-expressed wild type but not mutant PRMT5 (Fig. 3B). Similarresults were observed for SRP72 (Fig. 3C). Together with thecellular fractionation experiments described above, theseresults suggest that PRMT5 inhibits the binding of SRP68/72 tochromatin and sequesters SRP68 and SRP72 from the nucleustoward the cytosol in an enzymatic activity-dependentmanner.In our in vitro binding assays both H4R3me2a and

H4R3me2s modifications inhibited the binding of SRP68 andSRP72 to H4 tail peptide (Fig. 1, A and B). As H4R3me2aand H4R3me2s are known to have distinct transcriptional reg-ulatory functions, wewere eager to determine if overexpressionof PRMT1, the enzyme that catalyzes H4R3me2amodification,also influences the chromatin association and subcellular local-ization of SRP68 and SRP72.We thus overexpressed PRMT1 in293T cells and carried out cellular fractionation experiments to

FIGURE 3. PRMT5 and PRMT1 regulate SRP68 and SRP72 chromatin association. A, the 293T cells were transfected with or without FLAG-tagged wild-typePRMT5 (F-PRMT5) or enzymatic inactive PRMT5 (F-PRMT5m), and 2 days after transfection the cells were collected and fractionated into cytoplasm (Cyto),nuclear (Nucl), and chromatin (Chro). The expression of F-PRMT5 and F-PRMT5m was confirmed by Western blot analysis. The presence of SRP68 and SRP72 ineach fraction was determined by Western blot analysis. Note that expression of F-PRMT5 but not F-PRMT5m led to an �2-fold increase of H4R3me2s. �-Actinserved as a cytosol marker, and core histone served H3 as a chromatin marker. B, HeLa cells were cotransfected with F-PRMT5 or F-PRMT5m together withHA-SRP68. The effect of ectopic expressed PRMT5 on HA-SRP68 subcellular localization was analyzed by immunofluorescent staining. Note that expression ofF-PRMT5 but not F-PRMT5m resulted in export of transfected HA-SRP68 from the nucleus to cytoplasm. C, the effect of ectopic expressed PRMT5 on HA-SRP72subcellular localization was analyzed by immunofluorescent staining as in B. Again, expression of F-PRMT5 but not F-PRMT5m resulted in export of transfectedHA-SRP72 from the nucleus to cytoplasm. D, the 293T cells were transfected with or without HA-tagged PRMT1 (HA-PRMT1), and 2 days after transfection thecells were collected and fractionated into cytoplasm, nuclear, and chromatin. The effect of ectopic expression of HA-PRMT1 on SRP68 and SRP72 distributionwas analyzed by Western blot analysis. Note that expression of HA-PRMT1 diminished the level of SRP68 and SRP72 in chromatin but not nuclear fraction. Alsonote that expression of HA-PRMT1 led to increased levels of H4R3me2a. E, HeLa cells were cotransfected with FLAG-tagged PRMT1 and HA-SRP68 or SRP72, andthe subcellular localization was revealed by immunofluorescent staining. Note that expression of F-PRMT1 did not affect the nuclear localization of HA-SRP68and HA-SRP72.




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determine the effect on SRP68 and SRP72 chromatin associa-tion and subcellular fractionation. As shown in Fig. 3D, wefound that overexpression of PRMT1 led to the dissociation ofSRP68 and SRP72 from chromatin. Western blot analysis con-firmed an increased H4R3me2a level upon ectopic expressionof HA-PRMT1. This result is in agreement with our in vitroH4tail peptide binding data showing that H4R3me2amodificationalso inhibits the binding of H4 tail by SRP68 and SRP72. Unlikethe case of PRMT5 overexpression, PRMT1 overexpression didnot appear to reduce the nuclear fraction of SRP68 and SRP72(Fig. 3D, compare lane 5 and lane 2). Indeed, unlike PRMT5,ectopic expression of PRMT1 did not affect the nuclear local-ization of SRP68 and SRP72 as shown by immunofluorescentstaining (Fig. 3E). Together these results show that bothPRMT5 and PRMT1 regulate SRP68/72 chromatin association,presumably through its ability to catalyze H4R3me2s andH4R3me2a, respectively, which in turn interfereswith the bind-ing of SRP68/72 to histone H4 tail in chromatin. The mecha-nismbywhichPRMT5andPRMT1differentially affect the sub-cellular localization of SRP68/72 remains to be investigated.Both SRP68 and SRP72 Appear to Possess a Transcriptional

Activation Activity—The above findings that both SRP68 andSRP72 associate with chromatin in cells and that their chroma-tin association is regulated by H4R3 methylation raise the pos-sibility that SRP68 and SRP72 are involved in transcriptionalregulation. To this end, we investigated if SRP68 and SRP72possess transcriptional activity. We generated fusion proteins

of SRP68 and SRP72 with a heterologous DNA binding domain(DBD amino acids 1–147) from yeast transcription factor Gal4.When cotransfected with aminimal TK promoter-driven lucif-erase reporter containing four tandemGal4 binding sites (UAS)upstream of the TK promoter (4xUAS-TK-luc) into 293T cells,we found that expression of Gal-SRP68 or Gal-SRP72 led totranscriptional activation in a dose-dependent manner (Fig.4A). The correct expression of Gal-SRP68 and Gal-SRP72 wasverified by Western blot analysis using a Gal4(DBD)-specificantibody (Fig. 4A, lower panel). These results suggest thatSRP68 and SRP72 have a transcriptional activation function.To map the potential transcriptional activation domain(s) in

SRP68 and SRP72, we fused the different regions of SRP68 andSRP72 to Gal4(DBD) and tested their ability to activate tran-scription in luciferase reporter assay as above. We found thatthe transcriptional activation activity of SRP68 mainly residedin the C-terminal region amino acids 436–620 (Fig. 4B). Thedifferences in the transcriptional activity for different regions ofSRP68 were not due to variation in protein expression, becauseWestern blot analysis revealed a similar expression level forvarious Gal-SRP68 fusion proteins (Fig. 4B, lower panel). ForSRP72, the major transcriptional activation domain wasmapped to the N-terminal region, amino acids 1–356. Giventhat Gal-(1–356) exhibited only half of the transcriptionalactivity of the full-length SRP68, the contribution of the addi-tional C-terminal region to the transcriptional activity couldnot be excluded. Fig. 4D summaries the transcriptional activa-

FIGURE 4. Tethering SRP68 or SRP72 to a reporter gene results in transcriptional activation. A, 293T cells were co-transfected with 4xUAS-TK-Luc reporter(100 ng) and control Gal(DBD) vector, Gal-SRP68, and Gal-SRP72 constructs as indicated. Two days after the transfection, the cells were collected, and therelative luciferase activities were determined. The amounts of expression plasmids used were: �, 100 ng, ��, 200 ng. The samples were also analyzed forexpression of Gal-SRP68 and Gal-SRP72 by Western blot (WB). The control Gal(DBD) was not detected due to small size (running out of gel). B, mapping thepotential transcriptional activation domain of SRP68 is shown. 293T cells were transfected with 4xUAS-TK-Luc reporter and SRP68 mutants as indicated, andluciferase activities were assayed as above. Amount of DNA used: 100 ng each. C, mapping the potential transcriptional activation domain of SRP72 is shown.The experiments were performed as in B except various SRP72 mutants as indicated were used. The amount of DNA used was 100 ng each. D, shown is asummary of the transcriptional activity for SRP68 and SRP72 and their deletion mutants.




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tion domain mapping results of SRP68 and SRP72. Althoughthe precise transcriptional activation domain(s) and mecha-nism(s) by which they activate transcription remains to bedetermined, these data nevertheless support a transcriptionalregulatory function for both SRP68 and SRP72.Identification of Potential SRP68 Target Genes by ChIP-Seq

Analysis—Wenext attempted to identify potential endogenousSRP68 target genes using ChIP followed by high throughputsequencing (ChIP-seq). 293T cells were fixed by formaldehyde,and the chromatin was fragmented by sonication and immuno-precipitated using purified specific anti-SRP68 antibodies or noantibodies as the ChIP negative control group. A total of 1166SRP68 binding peaks with a p value 1.00e-004 was identified,and 681 of the 1166 peaks can be annotated to 638 unique genesusing a parameter of maximum distance of 100 kb up- anddownstream of the transcription starting sites (TSS) (Fig. 5, Aand B). The representative SRP68 binding profiles were shownin Fig. 5C for the CD1E and TCTA genes. The SRP68 bindingsites were not enriched in particular genomic regions (e.g. pro-moters or introns) (supplemental Fig. S1A) but interestinglywere enriched in chromosomes 1, 3, 13, and X (supplementalFig. S1B). We randomly selected 18 SRP68 peaks and validatedthe binding of SRP68 to these regions by ChIP followed byquantitative PCR analysis (Fig. 5D). As negative controls, thebinding of SRP68 was not observed in the promoter regions ofNKX3.1 and PSA genes (data not shown). The results suggestedthatmost if not all peaks identified by our ChIP-seq analysis areauthentic SRP68 binding sites. GO analysis revealed that the638 unique SRP68 binding site-containing genes are slightlyenriched for cytoskeleton organization, cell adhesion, DNAcatabolic, and apoptosis processes (supplemental Fig. S2).

SRP68MayRegulate TargetGene Expression in aContext-de-pendentManner—Having identified the potential SRP68 targetgenes, we next investigated if SRP68 had a role in their expres-sion. We knocked down SRP68 in 293T cells by RNAi and ver-ified the efficient down-regulation of SRP68 protein by West-ern blotting (Fig. 5E). We then analyzed the effect of SRP68knockdown on mRNA levels of the 18 genes that had beenverified for binding of SRP68 by quantitative RT-PCR. Wefound that knockdown of SRP68 resulted in the substantial up-regulation of DDIT3, DIDO1, CUL1, HNRNPA3, SLC37A3,YEATS4, and DRD2, in a significant down-regulation ofTMEM110, TCTA, NF1, OCDC43, CD1E, and CDKN1A, andin insignificant changes of the remaining genes. This effect ontarget gene expression was reproducible in three independentexperiments. Thus, although the reporter assays clearly sug-gested a transcriptional activation function for SRP68, theknockdownof SRP68 differentially affected the expression of itsassociated target genes, suggesting a context-dependent tran-scriptional function for SRP68.

DISCUSSION

In this study we attempted to use an unbiased proteomicapproach to identify nuclear proteins that selectively bind histoneH4 N-terminal tail peptides with H4R3me2a or H4R3me2s.Despite extensive effort, we did not observe any prominentnuclear protein that binds theH4 tail peptide in anH4R3me2a-or H4R3me2s-dependent manner (Fig. 1A). Instead, we consis-tently observed that both H4R3me2a and H4R3me2s inhibitedthe binding of two nuclear proteins that were subsequentlyidentified as the SRP68/72 heterodimers. It is noteworthy thatthe same experimental approach has permitted us previously

FIGURE 5. Genome-wide SRP68 distribution determined by ChIP-seq analysis and effect of SRP68 on target gene expression. A, the total numbers ofpeaks and annotated peaks enriched by anti-SRP68 antibody are shown. ChIP-seq analysis was performed with 293T cells. B, shown is the distance of SRP68peaks relative to the transcriptional start site (TSS) of the genes. C, the SRP68 binding profiling for two representative genes is shown. SRP68 is in black, and thecontrol is in purple. D, the validation of SRP68 binding for 18 annotated SRP68 peaks (target genes) by quantitative PCR ChIP analysis is shown. E, Western blotanalysis confirmed the efficient knockdown of SRP68 in 293T cells by siRNA. F, 293T cells were treated with siSRP68, and the effect on the transcription of 18SRP68 target genes was assessed quantitative RT-PCR. The relative levels of transcription to that of the control siRNA treated sample were calculated as log 2.




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the identification of multiple H3K4me2- and H3K9me2-bind-ing proteins (36). Thus, the failure of identification of methy-lated H4R3-specific binding proteins in this study is unlikelydue to technical reasons but more likely reflects the generalnature of histone arginine methylation. In contrast to the situ-ation that an increasingly large number of methylated lysine-specific histone-binding proteins have been identified, so faronly limited proteins have been reported for binding of argin-ine-methylated histones. On the basis of our study and others(see next), we therefore propose that histone arginine methyla-tion is more likely employed to inhibit than to recruit effectorproteins (Fig. 6).The role of histone arginine methylation in inhibiting rather

than recruiting effector proteins is not unique to H4R3 methy-lation. It was shown previously that H3R2me2a catalyzed byPRMT6 antagonizes H3K4 methylation by interfering with thebinding of H3K4 methyltransferase mixed lineage leukemia(MLL) complexes and other proteins to chromatin (40–42).More recently, H3R2methylation has been shown to inhibit thebinding of UHRF1/ICBP90 to histone H3 tail (43). In addition,by using the same unbiased peptide pulldown approach asemployed in this study, we also failed to detect any prominentnuclear proteins that bind theH3 tail peptides containing eitherH3R17me2a and/or H3R26me2a (44). Instead, we uncovered arole of H3R17me2a and H3R26me2a in conjunction with his-tone acetylation in inhibiting the binding of corepressors suchasMi-2/NURD/NuRD complex to H3 tail in vitro and chroma-tin in vivo (44). Together these data support that, unlike histonelysine methylation, histone arginine methylation includingH4R3 methylation may mainly act to inhibit or repel ratherthan recruit histone effector proteins. In this regard, arginineresidues are often involved in protein-protein interactionbecause it contains five potential hydrogen bond donors thatcan form favorable interaction with biological hydrogen bondacceptors (4).Methylation on argininewould affect not only thenumber of available hydrogen bond donors but also alters itsconformation, which in turn may inhibit the arginine-engagedprotein-protein interaction. We want to emphasize that our

data do not exclude the existence of methylated arginine-spe-cific binding proteins such as survival motor neuron protein(SMN) and TDRD3. The Tudor domain-containing proteinsare known to engage hydrophobic and hydrogen-bond interac-tions with methylated arginine residues in histones and non-histone proteins through an aromatic cage. Thus, histone argi-nine methylation is likely to have a combinatorial effect onbinding of chromatin effector proteins; on one hand by inhib-iting the binding of proteins such asMLL (40–42), UHRF1(43)and SRP68/72 and on the other hand by facilitating the chro-matin association of proteins such as TDRD3 (19). Neverthe-less, the limited number of arginine-methylated histone-bind-ing proteins identified so far raises the possibility that this typeof histone modification mainly functions to inhibit rather thanto recruit the effector proteins.H4R3me2a and H4R3me2s are catalyzed, respectively, by

PRMT1 and PRMT5 and have been linked to transcriptionalactivation and repression, respectively (7, 9–11). Given theiropposite roles in transcription, we initially expected to identifydistinct sets of proteins that bind H4R3me2a or H4R3me2s,respectively, or whose binding to H4 tail are differentiallyaffected. Although we could not rule out the possibility that wefailed to observe these proteins due to technical limitations inour experiments, it is equally possible that H4R3me2a orH4R3me2s may exert opposite effect on transcription throughtheir distinct cis-effect on other histone modifications. Forexample, the presence of H4R3me2a has been shown to facili-tate in cis histone acetylation catalyzed by CBP/p300 (7) andPCAF (20). On the other hand,H4R3me2s catalyzed by PRMT5has not been shown to enhance in cis histone acetylation. Thus,the effect of histone arginine methylation on transcription islikely due to the combinatorial effect of histone arginine meth-ylation on the binding of effectors and/or other histone modi-fications in cis.A surprising finding in this study is the identification of

SRP68 and SRP72 as major H4-binding proteins. SRP68 andSRP72 are part of the SRP, a particle critically important fortargeting secretory and membrane proteins to ER. SRP68 andSRP72 were previously shown to form heterodimers independ-ent of the SRP complex and are released from the SRP as a stableSRP68/72 that is essential for SRP-mediated protein targeting(27, 29). Our pulldown and gel filtration assays provide compel-ling evidence that the SRP68/72 heterodimers, but not the SRPcomplex, binds the H4 tail. Multiple lines of evidence support adirect binding of SRP68/72 to H4 tails, including a H4 tail-de-pendent recruitment (co-localization) of SRP68 in CHO cells(Fig. 2E) and binding of H4 tail peptide in vitro by recombinantSRP68 and SRP72 (Fig. 2I). The ability for both SRP68 andSRP72 to bind H4 tail may allow SRP68/72 heterodimers tobind chromatin with high affinity. It is noteworthy that SRP68and SRP72 do not share sequence similarity. Exactly how theseproteins bind the H4 tail peptide remains to be determined.Consistent with an inhibitory role of H4R3me2s and

H4R3me2a on binding of SRP68/72 to H4 tail peptide, ectopicexpression of PRMT5 or PRMT1 all resulted in the dissociationof SRP68/72 from chromatin. Interestingly, ectopic expressionof PRMT5 also drives SRP68/72 out of the nucleus, whereasectopic expression of PRMT1 does not. At this stage we do not

FIGURE 6. A model of H4R3 methylation in regulating chromatin associa-tion of SRP68/72. SRP68/72 has dual roles either in the SRP complex partic-ipating in protein targeting (left) or as heterodimer involving in transcription(right). Indicated are the six SRP proteins (SRP9 to SRP72) in relation to thefolded SRP RNA (black line) as well as the signal peptide of the secretory pro-tein (sp). The methylated H4 tail (H4R3me2, red triangle) is highlighted in thechromatin complex. Binding of SRP68/72 to H4 tails of chromatin is inhibitedby H4R3 methylation as shown by the X. The association of SRP68/72 withchromatin is likely to influence transcription either directly and/or indirectly.




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know how PRMT1 and PRMT5 differentially regulate SRP68/72subcellular localization. One possibility is that PRMT5 not onlymethylates H4R3, which leads to dissociation of SRP68/72 fromchromatin, but alsomethylates SRP68/72,which results innuclearexport of SRP68/72. On the other hand, PRMT1may only meth-ylate H4R3 to affect SRP68/72 chromatin association. Furtherwork is needed to elucidate the mechanisms by which PRMT5regulates SRP68/72 subcellular localization.By tethering SRP68 and SRP72 to a luciferase reporter

through a heterologousGal4DNAbinding domain, we demon-strated that both SRP68 and SRP72 possess a transcriptionalactivation activity (Fig. 4A). The transcriptional activity can bemappedprimarily to theC-terminal region of SRP68 andN-ter-minal region of SRP72 (Fig. 4, B and C). Although the mecha-nism by which tethering SRP68 or SRP72 to DNA leads to tran-scriptional activation remains to be investigated, it neverthelesssuggests that the chromatin-associated SRP68 and SRP72 mayhave a transcriptional regulatory function. In support of thisnotion, we carried out ChIP-seq analysis and identified 1166SRP68 associated regions and 638 potential SRP68 target genesusing a parameter of maximum SRP68 peak distance of 100 kbup- and downstream of the transcription starting sites (TSS)(Fig. 5, A and B). As the enrichment of SRP68 in the SRP68peaks was confirmed by ChIP-quantitative PCR analysis for 18randomly selected genes (Fig. 5D), most of the SRP68 peaksidentified in this study are likely the authentic SRP68-associ-ated regions in 293T cells. Given that SRP68 and SRP72 formheterodimers, the SRP68-associated regions are most likely theSRP68/72-associated regions, although this remains to betested experimentally. Interestingly, although SRP68 possessesa transcriptional activation activity in the reporter assay,knockdownof SRP68 affects positively or negatively the expres-sion of its directly associated genes (Fig. 5E), suggesting that theeffect of SRP68 on target gene expression is likely context-de-pendent. Although the underlying mechanism remains to befully investigated, many transcription factors or epigenetic reg-ulators possess a context-dependent transcriptional function.For example, stem cell factor Oct4 can activate or repress itstarget gene expression (45), in part depending on the coregula-tors it interacts with (46, 47).Taken together, our study has identified SRP68 and SRP72 as

novel H4 tail-binding proteins whose binding of H4 tails isinhibited by H4R3methylation, and thus we uncovered a noveltranscriptional regulatory function for SRP68/72 (Fig. 6). Theidentification of potential SRP68/72 target genes by ChIP-seqsubstantiates the histone binding activity of SRP68/72 and setsup the stage for characterization of their transcription andpotentially other chromatin-related function.

Acknowledgments—We thank Drs. M. David Stewart and BingdingHuang for critical reading of the manuscript. We are very grateful tomembers of the Wong laboratory for valuable discussions.

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Christian Zwieb, Biaoyang Lin and Jiemin WongJingjing Li, Fan Zhou, Deguo Zhan, Qinqin Gao, Nan Cui, Jiwen Li, Elena Iakhiaeva,

SRP68 and SRP72and Transcriptional Regulatory Function for Signal Recognition Particle Subunits A Novel Histone H4 Arginine 3 Methylation-sensitive Histone H4 Binding Activity

doi: 10.1074/jbc.M112.414284 originally published online October 8, 20122012, 287:40641-40651.J. Biol. Chem.

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a novel histone h4 arginine 3 methylation-sensitive histone h4

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