prmt5-mediated histone h4 arginine-3 symmetrical ... · or suv420h1/h2 (kmt5b/c) affected h3k9me3...

PRMT5-mediated histone H4 arginine-3 symmetricaldimethylation marks chromatin at G+C-rich regionsof the mouse genomeMichael Girardot1 Ryutaro Hirasawa1 Salim Kacem1 Lauriane Fritsch2 Julien Pontis2

Satya K Kota1 Doria Filipponi1 Eric Fabbrizio1 Claude Sardet1 Felix Lohmann3

Shilpa Kadam3 Slimane Ait-Si-Ali2 and Robert Feil1

1Institute of Molecular Genetics (IGMM) CNRS UMR 5535 University of Montpellier 1919 route de Mende34293 Montpellier 2Laboratoire Epigenetique et Destin Cellulaire UMR7216 CNRS Universite Paris Diderot35 rue Helene Brion 75013 Paris France and 3Developmental and Molecular Pathways Novartis Institutes forBioMedical Research 250 Massachusetts Avenue Cambridge MA 02139 USA

Received June 17 2013 Revised August 14 2013 Accepted August 31 2013

ABSTRACT

Symmetrical dimethylation on arginine-3 of histoneH4 (H4R3me2s) has been reported to occur atseveral repressed genes but its specific regulationand genomic distribution remained unclear Here weshow that the type-II protein arginine methyl-transferase PRMT5 controls H4R3me2s in mouseembryonic fibroblasts (MEFs) In these differentiatedcells we find that the genome-wide pattern ofH4R3me2s is highly similar to that in embryonicstem cells In both the cell types H4R3me2s peaksare detected predominantly at G+C-rich regionsPromoters are consistently marked by H4R3me2sindependently of transcriptional activity RemarkablyH4R3me2s is mono-allelic at imprinting controlregions (ICRs) at which it marks the same parentalallele as H3K9me3 H4K20me3 and DNA methylationThese repressive chromatin modifications areregulated independently however since PRMT5-de-pletion in MEFs resulted in loss of H4R3me2swithout affecting H3K9me3 H4K20me3 or DNAmethylation Conversely depletion of ESET (KMT1E)or SUV420H1H2 (KMT5BC) affected H3K9me3 andH4K20me3 respectively without altering H4R3me2sat ICRs Combined our data indicate that PRMT5-mediated H4R3me2s uniquely marks the mammaliangenome mostly at G+C-rich regions and independ-ently from transcriptional activity or chromatin

repression Furthermore comparative bioinformaticsanalyses suggest a putative role of PRMT5-mediatedH4R3me2s in chromatin configuration in the nucleus

INTRODUCTION

DNA methylation is involved in diverse epigeneticphenomena in mammalian development includingX-chromosome inactivation and genomic imprinting(1ndash3) Similarly histone lysine methylation plays diverseroles in the establishment and maintenance of functionalchromatin states during development (4) Different arginineresidues on histones H2A H3 and H4 can also be mono- ordimethylated (5) Previously we and others have shown theassociation of H4 arginine-3 symmetrical dimethylation(H4R3me2s) with specific repressed loci in mammals(5ndash11) These findings evoked possible roles of H4R3me2sin gene regulation but also emphasized the need to furtherexplore its regulation and genome-wide distributionImprinted gene expression in mammals is mediated by

lsquoimprinting control regionsrsquo (ICRs) CpG-rich regulatorysequences that are marked by DNA methylation on one ofthe two parental alleles only (312) Besides differentialhistone lysine methylation and acetylation we andothers detected H4R3me2s at ICRs and at intracisternalA particles (IAPs) in mouse embryos (71314) This ob-servation suggested that H4R3me2s could be linked togene repression Consistent with this model earlierreports had shown enrichment of H4R3me2s at differentrepressed genes including the rDNA hemoglobin beta

To whom correspondence should be addressed Tel +33 43 435 9663 Fax +33 43 435 9634 Email robertfeiligmmcnrsfr

The authors wish it to be known that in their opinion the first two authors should be regarded as Joint First Authors

Present addressesRyutaro Hirasawa RIKEN BioResource Center 3-1-1 Koyadai Tsukuba Ibaraki 305-0074 JapanDoria Filipponi Institute of Molecular and Cellular Biology 61 Biopolis Drive Singapore 138673

Published online 3 October 2013 Nucleic Acids Research 2014 Vol 42 No 1 235ndash248doi101093nargkt884

The Author(s) 2013 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby30) whichpermits unrestricted reuse distribution and reproduction in any medium provided the original work is properly cited

(HBB) and Cyclin E1 genes (6811) However genome-wide analyses of the distribution of this covalent histonemark were missing to test this idea It also remainedunclear which protein arginine methyltransferase(s)(PRMT) could control the H4R3me2s mark in mamma-lian somatic cells In the current study we explored thesekey questions in mouse embryonic fibroblasts (MEFs) andembryonic stem (ES) cells Using an shRNA-mediatedgene knock-down approach we demonstrate that thetype-II protein arginine methyltransferase PRMT5controls the bulk of H4R3me2s in MEFs Chromatinimmunoprecipitation (ChIP) followed by next-generationDNA sequencing (ChIP-seq) was used to delineate thegenome-wide distribution of H4R3me2s in both ES andMEF cells The latter analysis revealed that rather unex-pectedly H4R3me2s is enriched preferentially at DNAsequences with a high G+C content including ICRsUsing imprinted gene loci as a model system we carefullyexplored the relationship of H4R3me2s with other histonemodifications in somatic cells Finally using comparativebioinformatics analyses we found interesting correlationswith factors involved in higher order chromatin contacts

MATERIALS AND METHODS

Primary cells immuno-cytochemistry and RT-polymerasechain reaction analysis

Primary MEFs used for Prmt5 and Eset knockdown wereestablished from E135 embryos obtained by crossingC57BL6J (Mus musculus domesticus) females with JF1(Mus musculus molossinus) males (15) The ES cells (lineBJ1) used for immunoblotting and immunocytochemistrywere newly established in serum-free 2i medium fromblastocysts obtained by mating a C57BL6J Oct4-GFPtransgenic female (16) with a JF1 male Their pluripotencyis described elsewhere (Kota et al in preparation) Esetknockout ES cells and Suv4-20h1h2 double-knockoutMEFs were derived by Lohmann et al (17) and Schottaet al (18) respectively Total RNA was isolated usingTRIzol reagent (Invitrogen) and cDNA was synthesizedusing SuperScript-2 reverse transcriptase (Invitrogen) andrandom oligonucleotides Primers used for polymerasechain reaction (PCR) are described in SupplementaryTable S2 For immunocytochemistry cells were seededon gelatin-coated cover slips fixed for 10min in 4paraformaldehyde in phosphate buffered saline (PBS) atroom temperature and washed with PBS After 30-minincubation with pre-treatment buffer (1 horse serum2 Triton-X100 in PBS) cells were incubated for 3 hwith specific antisera at RT After extensive washes theywere incubated for 30min at RT with an appropriateAlexa488-conjugated second antibody Stained cells weremounted in Vectorshield mounting medium (VectorLaboratory) containing DAPI for counterstaining ofDNA and were microscopically analysed

Western blotting

Whole cell lysates or protein extracts were resolved onsodium dodecyl sulphatendashpolyacrylamide gel electrophor-esis (SDSndashPAGE) gels or on pre-cast NuPage 4ndash12

acrylamide gradient SDSndashPAGE gels (Invitrogen) andtransferred onto nitrocellulose membrane Membraneswere blocked and incubated overnight at 4C withprimary antibodies After incubation with appropriatesecondary antibodies coupled to horseradish peroxidaseimages were developed using the West Dura kit andChemiSmart 5000 system (Vilber Laurmat) Forproducing PRMT5- and PRMT7 over-expressing 293Tcells full-length cDNAs cloned into the vector pCMV-SPORT6 (Open Bioscience) were used

ChIP and PCR analysis of precipitated chromatin

ChIP on native chromatin was performed following a pre-viously described protocol (19) with the following modifi-cations The purified nuclei were incubated for 15min at37C with 60 units of Micrococcal nuclease (S7 Micro-coccal nuclease from Staphylococcus aureus Roche) toproduce monodi-nucleosomes as verified by agarose gelelectrophoresis Nucleosomes were precipitated with theH4R3me2s antiserum-2 (Supplementary Table S1) andDNA was purified from precipitated chromatin fractionswith the lsquoChIP DNA purification kitrsquo (Zymo Research)For ChIP on cross-linked chromatin cells were fixed with1 formaldehyde for 10min and quenched with 125mMof glycine Subsequently cells were washed in PBS col-lected in 15-ml tubes and centrifuged to remove the super-natant Cells were re-suspended in 100 ml of SDS lysisbuffer (100mM NaCl 1 SDS 5mM ethylenediaminete-traacetic acid (EDTA) 50mM TrisndashCl and lsquoCompleteprotease inhibitor cocktailrsquo from Roche) After 10minon ice with occasional vortexing cells were diluted in200 ml of ice-cold ChIP dilution buffer (001 SDS11 Triton X-100 12mM EDTA 167mM TrisndashCl167mM NaCl and lsquoComplete protease inhibitor cocktailrsquofrom Roche) and sonicated twice at high power for 30cycles of 30 s ON 30 s OFF in a BioRuptor twin appar-atus (Diagenode) The average DNA fragmentsrsquo lengthfrom ES and MEF cells was 200 bp as estimated byagarose gel electrophoresis Q-PCR on purified DNAwas performed using the LightCycler 480 SYBR GreenI Master Mix (Roche) and LigthCycler 480 apparatus(Roche) Antisera and oligonucleotides used are listed inSupplementary Tables S1 and S2

High-throughput sequencing and bioinformatics

Input and ChIPed DNAs were sequenced on an IlluminaHi-seq 2000 instrument (Fasteris Switzerland) using theTruSeqTM SBS v3 kit The sequences produced from theinput chromatin and H4R3me2s ChIPed DNA from ESand MEF cells are detailed in the Supplementary Table S3ChIP-seq raw data (fastq) were aligned on theM musculusreference genome (mm9) using the Bowie 0127 softwareSmoothed tag density compared to input tag density werecomputed with the Sequence processing pipeline (20) andvisualized with the integrative Genomics Viewer (21)Median tag density around the defined peak positionswere calculated using an in-house designed R-scriptBriefly peaksrsquo summits coordinates were grouped accord-ing to the level of enrichment for the mark examined andmedian tag densities were calculated every 100bp across

236 Nucleic Acids Research 2014 Vol 42 No 1

5 kb windows around the peaksrsquo summits Heatmaps ofH4R3me2s were clustered and represented using theseqMINER 133 program (22) H3K4me3 andH3K4me1 peaks generated by Dr Bing Renrsquos laboratorywere downloaded from the UCSC genome browser(httpgenomeucsceducgi-binhgFileUidb=mm9ampg=wgEncodeLicrHistone) Peak predictions for H4R3me2sand PRMT5 in the two cell types were performed withthe Model-based Analysis for ChIP-seq (MACS) 142 al-gorithm with the default parameters (Plt 1e-5) (23) 100-bpwindows with the same H4R3me2s enrichment in MEFand ES cells were grouped together and represented as adensity array using the hexbin package The genome-wideG+C frequency was calculated using the LetterFrequencyInSlidingView function from the Biostrings package(httpwwwbioconductororgpackages211biochtmlBiostringshtml) in R 2130 (httpwwwR-projectorg)The MeDIP and hMeDIP-seq in ES cells were fromFicz et al (24) (httpwwwebiacukenadataviewPRJEB2462) Published ChIP-seq profiles for H3K4me3H3K27me3 and RNA-seq from ES cells cultivated in 2iserum-free medium (25) were retrieved from the GEOdatabase (GSE23943) Genomic coordinates for repeatelements were retrieved from the RepeatMasker database(httpwwwrepeatmaskerorg)

shRNA preparation and transfection

Prmt5 shRNAs were designed against unique sequencessuch as to ensure that only the Prmt5 mRNA wastargeted 50-GAG GGA GTT CAT TCA GGA A-30

(shPrmt5-1) and 50-GGA TGT GGT GGC ATA ACTT-30 (shPrmt5-2) The sh-Ctrl targets a non-genomicfirefly luciferase sequence (26) Similarly sh-Eset-1targets a unique sequence in Eset mRNA 50-CAGTTCTCAAGATCTACAT-30 shRNA sequences were clonedinto the retroviral vector RNAi Ready pSiren (BDBiosciences) according to the manufacturerrsquos instructionsRetroviral vector and packaging vectors (gag-pol andVSV-G) were co-transfected into 293T cells using thecalcium phosphate method After 3 days incubationsupernatants were collected filtrated and used for infec-tion Primary MEFs at passages 2-3 were incubated for48 h with virus particles and polybrene (Millipore) Aftermedium change cells were selected in 20 mgml puromycinfor 48 h Selected cells were incubated with fresh mediumfor 3 days and then used for analyses

Data access

The ChIP-seq data from this publication have beensubmitted to the GEO database httpwwwncbinlmnihgovgeo and assigned the identifier GSE37604

RESULTS

PRMT5 controls arginine-3 symmetrical dimethylationon H4 in embryonic cells

First we sought to determine which enzyme(s) control(s)H4R3me2s (and H2AR3me2s whose first 8 residues are88 identical) in primary MEFs We explored the type-IIPRMTs candidate for this type of symmetrical

dimethylation PRMT5 and PRMT7 (5) Since PRMT5had been reported to control H2AR3me2s in ES cells(27) we also studied ES cells that were newly derived in2i-medium (28) and which were of the same genotype asthe primary MEFs Prmt5 mRNA expression was readilydetected in both MEFs and ES cells and in all somatictissues analysed (Figure 1A Supplementary Figure S1A)suggestive of ubiquitous expression Prmt7 in contrast waspoorly expressed in MEFs but showed relatively high ex-pression in ES cells and in male and female gonads(Supplementary Figure S1A) Similarly western analysesshowed high expression of PRMT5 protein in MEFs andES cells whereas PRMT7 was barely detectable in MEFsbut was highly expressed in ES cells (Figure 1B) Thesedata pinpoint PRMT5 as the main type-II PRMT inMEFsPRMT5 had been reported to be mostly cytoplasmic in

ES cells (27) We confirm this finding by immunostainingof fixed cells and western blot analyses of cytoplasmic andnuclear protein fractions (Figure 1C and D) In MEFs incontrast PRMT5 is readily detectable in the nucleus(Figure 1C and D) In both cell types H2AR3me2 andH4R3me2s are detected in the nucleus In ES cellshowever part of the H2AR3me2s signal is present in thecytoplasm as well a finding which agrees with the prop-osition that in undifferentiated cells PRMT5 may pre-modify this core histone already in the cytoplasm (27)In MEFs in contrast H2AR3me2 and H4R3me2s areexclusively nuclear suggesting that in differentiated cellsPRMT5 targets histones H2A and H4 predominantly inthe nucleus Combined our data pinpoint PRMT5 as thecandidate type-II enzyme that modifies nuclear histones inMEFsNext to explore the importance of PRMT5 in histone

arginine methylation we knocked down its expression inprimary cells in culture In an earlier study it had beenfound that complete genetic ablation of Prmt5 in themouse was not compatible with cell survival during earlydevelopment (27) This deleterious knockout phenotypecould be related to multiple known roles of PRMT5 un-related to chromatin including the biogenesis and actionof SMNndashSm protein complex RNA splicing andsignalling (2930) To reduce rather than completelyabrogate expression we designed retroviral shRNA con-structs directed against Prmt5 Two lentiviral constructssh-Prmt5-1 and sh-Prmt5-2 were stably introduced intoprimary MEFs with gt80 of cells surviving 48 h afterviral infection and selection for shRNA expression (datanot shown) A construct with a scrambled DNA sequenceSh-Ctrl was used as a negative control in these experi-ments Both sh-Prmt5-1 and sh-Prmt5-2 led to reducedPRMT5 mRNA and protein expression after 72 h ofgrowth in selective medium (to ablate non-infected cellsnot expressing the shRNA) (Figure 1E and F) The ex-pression of the other type-II PRMT Prmt7 was un-affected (Figure 1F) In agreement with the essentialroles of PRMT5 we observed reduced cellular prolifer-ation in PRMT5-depleted cells (Supplementary FigureS1B) Significantly the reduction in PRMT5 proteinlevels led to a global reduction in H4R3me2s andH2AR3me2s (85 reduction for Sh-Prmt5-1) as

Nucleic Acids Research 2014 Vol 42 No 1 237

detected by western blotting with a specific antiserum thatrecognizes both histones (Figure 1E lsquoantiserum 1rsquo) Thisfinding was confirmed with an independent second anti-serum (Figure 1E lsquoantiserum 2rsquo) which only recognizedH4R3me2s again showing 85 reduction for thishistone modification Importantly in the PRMT5-depleted cells overall histone H4 levels and histone H3lysine-9 trimethylation (H3K9me3) appeared unaffected(Figure 1E) We conclude from these data that PRMT5controls H4R3me2s in MEF cells

H4 arginine-3 symmetrical dimethylation is enriched atG+C-rich sequences of the genome

We next performed ChIP-seq assays to identify the globalgenomic distribution of H4R3me2s Before performingChIP-sequencing however we further assessed the

quality of lsquoantiserum 2rsquo directed against H4R3me2s(Supplementary Table S1) This antiserum did notrecognize H2AR3me2s (Figure 1E) nor asymmetricaldimethylation or monomethylation of H4R3 (31) Theimmunofluorescence staining on fixed cells with this anti-serum was mostly nuclear in MEF cells (SupplementaryFigure S2A) Using a peptide array of histone modifica-tions we confirmed that antiserum 2 was highly specificfor the H4(1-19)R3me2s modification (SupplementaryFigure S2B) and that its binding was not altered by thepresence of neighbouring modifications (namely S1pK5ac K8ac and K12ac of H4) (Supplementary FigureS2C) Treatment of cells with the HDAC inhibitorTrichostatin-A (TSA) did not change the antibodybinding of H4R3me2s in western blotting analyses(Supplementary Figure S2D) confirming that gain of

AMEF

ES PRMT5

PRMT7

PRMT5

PRMT7

ACTB

BRT

MEF ES

+ - + -

Prmt5

Prmt7

Gapdh

PRMT5

DAPI

Merged

MEF ESC

F

D

Cyt Nuc

PRMT5

MEFCyt Nuc

ES

Tubulin

H3K9me3

H2AR3me2sH4R3me2s

E

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MEFsh

-Ctrl

sh-P

rmt5-

1

sh-P

rmt5-

2

ACTB

H4H3K9me3

H2AR3me2sH4R3me2s

H4R3me2s

anti-serum 1

anti-serum 1

anti-serum 2

Prmt5 Prmt7R

elat

ive

expr

essi

on (

)

sh-C

trl

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rmt5-

1

sh-P

rmt5-

2

sh-C

trl

sh-P

rmt5-

1

sh-P

rmt5-

2

100

120140

8060

40

20

0

100

60

80

120140

20

40

0

Figure 1 PRMT5 expression localization and histone methylation activity in embryonic cells (A) RT-PCR analysis of Prmt5 and Prmt7 expressionin primary MEFs and ES cells RT+and RTndash indicate presence and absence of reverse transcriptase (RT) respectively (B) Western blot analysis oftotal protein extracts Lanes lsquoPRMT5rsquo and lsquoPRMT7rsquo were loaded with diluted protein samples of 293T cells in which the PRMT5 and PRMT7proteins were over-expressed b-Actin (ACTB) is used as a loading control (C) Immunofluorescence staining of PRMT5 in MEFs and ES cells(upper panels) Nuclei were counter-stained with DAPI (middle panels) (D) Western blot analysis of cytoplasmic (Cyt) and nuclear (Nuc) proteinfractions of MEFs and ES cells Tubulin and H3K9me3 constitute cytoplasmic and nuclear controls respectively (E) Strongly reduced PRMT5protein expression in MEFs stably infected with lentiviral shRNA constructs (sh-Prmt5-1 and sh-Prmt5-2) directed against Prmt5 As a negativecontrol MEFs stably expressing a scrambled shRNA (Sh-Ctrl) were analysed PRMT5 H4R3me2s (anti-serum 2) and H2AH4R3me2s levels (anti-serum 1) were assessed by western blotting of total protein extracts ACTB provides a loading control (F) Strongly reduced Prmt5 gene expression insh-Prmt5-1 and sh-Prmt5-2 cells cDNA was made from total RNAs using random oligonucleotides Expression levels were determined relative toGapdh by real-time PCR amplification and were put arbitrarily at 100 in the control cells (Sh-Ctrl)


acetylation at nearby residues (H4K5 H4K8) in livingcells did not significantly impair recognition ofH4R3me2s by the lsquoantiserum 2rsquo Importantly the Prmt5knock-down in MEF cells led to an almost complete lossof H4R3me2s detection with this antiserum (Figure 1E)providing another indication of its specificity Finally sixpromoters were chosen randomly (Supplementary FigureS2E) to perform ChIP assay in the absence or in thepresence of an H4R3me2s-blocking peptide (Supplemen-tary Figure S2F) We observed strongly reduced immuno-precipitation of H4R3me2s in the presence of anH4R3me2s-blocking peptide (Supplementary FigureS2F) which further validates the choice of lsquoantiserum 2rsquofor subsequent ChIP-seq studies

Whereas H3K9me3 showed a punctuated stainingpattern mostly confined to heterochromatic foci thestaining of H4R3me2s on fixed MEF cells was homogen-ous in the nucleus (Supplementary Figure S2A) suggest-ing that this histone arginine methylation could be broadlypresent along the genome To explore the genomic distri-bution pattern of H4R3me2s we performed ChIP-seq onnative chromatin from mouse embryonic cells using theantiserum 2 Indeed we found that H4R3me2s displayed abroad pattern of precipitation with many peaks along thegenome similarly in ES and MEF cells As an example ofthis unique epigenomic pattern a representative 65-kbgenomic window is shown in Figure 2A Significantlythe majority of the H4R3me2s-marked regions werecommon between MEFs and ES cells (Figure 2B)(Pearsonrsquos r=098) Next we defined the peaks ofH4R3me2s enrichment over input chromatin that had ahigh statistical probability (P-value cut-off for peak detec-tion by MACS Plt 1e-5) No fewer than 250 349 lsquohigh-probability peaksrsquo were thus identified in ES cells and131 856 in MEFs cells Interestingly the vast majority ofthe high probability peaks in MEFs were also present inES cells (Figure 2C) and their genomic localization issimilar in the two cell types (Figure 2D) with a predom-inant accumulation at genes Noteworthy we did notdetect enrichment in sequence reads for satellite sequencesin H4R3me2s ChIP compared with input chromatin(Supplementary Table S3) However we detected signifi-cant H4R3me2s enrichments in repetitive elements such asLINEs (long interspersed nuclear elements) SINEs (shortinterspersed nuclear elements) and LTRs (long terminalrepeats including IAPs)

We next asked whether H4R3me2s enrichmentcorrelated with either gene expression or gene repressionRemarkably 29 539 (872 of total) and 22 741 (67 oftotal) promoters in ES and MEF cells respectively wereenriched in H4R3me2s This indicates that the majority ofall annotated promoters are marked by H4R3me2s(Figure 3A) Importantly promoters that are enrichedfor H3K4me3 and that are actively transcribed (RNA-seq) (Figure 3A group 1) are similarly enriched forH4R3me2s as transcriptionally inactive promoters(group 2) that show low H3K4me3 and high H4K27me3enrichments Promoters that were statistically enriched inH4R3me2s in ES cells but not in MEFs included genesexpressed in undifferentiated ES cells (and the earlyembryo) but not in differentiated cells such as Dnmt3L

Dnmt3A PiwiL1 and Tdrd1 Also the Prmt7 gene washighly marked by H4R3me2s in ES cells but much lessso in MEFs The silenced gene promoters that showedlittle H4R3me2s enrichment (marked by an asterisk inFigure 3A) in ES cells included immune response genessuch as the CD4 antigen (Cd4) and Interferon beta 1(Ifnb1) genesInterestingly we found that the H4R3me2s-negative

inactive promoter sequences (group 3 n=5871) were sig-nificantly less G+C-rich (mean G+C ratio=043) thanthe H4R3me2s-positive inactive promoters (group 2n=7303) (mean G+C ratio=050) (Wilcoxon ranksum test with a Plt 22e-16) (Figure 3B) This findingprovided a first indication that sequence context couldbe determinant in guiding H4R3me2s levels (see below)Importantly we did not observe differences in H4R3me2senrichment between four different classes of expressedgenes that were grouped according to their expressionlevels in ES cells (Figure 3C) Thus gene promoterswere generally marked independently of their levels of ex-pression Concordantly H4R3me2s co-localized withH3K4me3-enriched regions in the genome which aremostly confined to active promoters (EncodeLICRdataset with the UCSC reference numberwgEncodeEM001455) (Figure 3D) In contrast no co-localization was observed with H3K4me1-enrichedregions (Figure 3D) which include enhancers nor withH3K9me3-marked regions (data not shown) whichinclude heterochromatin and stably repressed genesExamples of the lack of correlation between transcrip-tional activity and H4R3me2s enrichment are providedby the Oct4 (Pou5f1) and Nanog genes which are ex-pressed in undifferentiated ES cells but not in MEFs(Supplementary Figure S2G-H) In both the cell typeshowever the promoters of these pluripotency genes werestrongly marked by H4R3me2s Similar examples areprovided by the Col1a1 and Actb genes (SupplementaryFigure S2I-J)Most promoter regions have a high G+C content and

many correspond to CpG islands (32) We therefore askedwhether H4R3me2s could be enriched at G+C-richsequence elements in general Specifically we evaluatedH4R3me2s levels of 100-bp windows along the mousegenome ranked according to their G+C contentRemarkably enrichment of H4R3me2s was confined toregions with a G+C content of more than 50Regions of low G+C content did not show anyH4R3me2s This strong link between G+C content andH4R3me2s enrichment was observed both in MEFs andES cells (Figure 3E)G+C-rich sequences comprise CpG dinucleotides at

which the cytosine can be methylated or not Thereforewe explored whether levels of 5m-Cytosine methylation or5-hydroxymethylation in ES cells correlated with theobserved enrichment levels of H4R3me2s We found aweak correlation between the level of CpG methylationand H4R3me2s (Pearsonrsquos r=03) A stronger correlationwas found with 5-hydroxymethylation of Cytosines(Pearsonrsquos r=033) which is associated with increasedtranscriptional activity (24) (Figure 3F) Howeverstrong H4R3me2 enrichment was detected both at


DNA-methylated and at unmethylated G+C-rich se-quences Accordingly we observed H4R3me2s enrichmentin ES and MEFs at the Oct4 and Nanog promoters whichare unmethylated in ES cells and methylated in the E145MEFs indicating that H4R3me2s is independent of 5m-Cytosine at these genes (Supplementary Figure S2G-H)Similar findings were reported in resting human Blymphocytes at rDNA genes at which H4R3me2s isenriched both at DNA-methylated and unmethylatedcopies (6) Combined these data indicate thatH4R3me2s is a hallmark of G+C-rich sequenceelements but is generally independent of transcriptionallevels or DNA methylation

ICRs are marked by parental allele-specific H4R3me2s

CpG islands are particularly rich in G+C nucleotides(G+C content gt50 length gt200 bp and observedexpected CpG gt06) and they are all marked byH4R3me2s (Figure 4A) One class of CpG islands corres-ponds to the ICRs the regulatory elements which mediateimprinted mono-allelic gene expression in mammals (33)Imprinted CpG islands are exceptional in that they areDNA-methylated on one of the two parental allelesonly and this differential DNA methylation is maintainedthroughout development We find that both in ES cellsand MEFs ICRs are marked by peaks of H4R3me2s

(Figure 4BndashF) Previously we reported for wholeembryos that H4R3me2s was present preferentially onthe DNA-methylated alleles of ICRs (7) To determinewhether this is also the case in primary MEFs ChIPwas performed on MEFs that were generated fromembryos that were hybrid between M m domesticusstrain C57BL6 and M m molussinus strain JF1 Forthis allele-specific ChIP assay we generated larger nativechromatin fragments of 2ndash7 nucleosomes in length Singlenucleotide polymorphisms (SNPs) allowed us to directlydistinguish the parental chromosomes in the precipitatedchromatin fractions (see Supplementary Table S2) Atfour different ICRs analysed PCR amplification ofChIPed DNA followed by DNA sequencing clearlyshowed that H4R3me2s was precipitated on the DNA-methylated allele predominantly The oppositeunmethylated allele was largely devoid of H4R3me2s(Figure 4G) These results were confirmed by PCR amp-lification followed by electrophoretic detection of single-strand conformation polymorphisms (SSCPs) (data notshown) We conclude from these data that H4R3me2smarks the DNA-methylated alleles of ICRs in MEFsSince chromatin on the unmethylated alleles of ICRs isenriched in histone H4 acetylation this finding alsoconfirms that the antiserum used does not cross-reactwith acetylated H4

C DES

111719

MEFChIP - H4R3me2s

Troap C1ql4 Dnajc22 Spats2

H4R3me2s ES

H4R3me2s MEF

9891

0 kb

9892

0 kb

9893

0 kb

9894

0 kb

9895

0 kb

9896

0 kb

chr15

A

B

131856 peaks250349 peaks

H4R3me2s enrichment in MEF cells

H4R

3me2

s en

richm

ent

in E

S c

ells

0 1 2 3 4 5

0

1

2

3

4

5

6Counts

10 E

80 E

15 E

20 E

0

10

20

30

40

50

60

70

LINESIN

ELT

R

Satellit

es

Promote

rs

(-25k

b +25

kb)CpG

GenesExo

ns

Intron

s

pe

aks

ES (250349 peaks)MEF (131856 peaks)

Figure 2 Genome-wide analyses of H4R3me2s in mouse ES cells and MEFs (A) Representative ChIP-Seq data along a 65-kb window on mousechromosome 15 normalized tag densities for H4R3me2s in ES and MEF are represented Gene positions are indicated below (B) Genome-widecorrelation between H4R3me2s-enriched regions in ES (y-axis) and MEF (x-axis) cells The scales of the x- and y-axis are transformed by thefollowing formula Log10(1+x) Genomic regions (100-bp windows) with defined degrees of ES and MEF H4R3me2s enrichments are plotted ashexagonal tiles The number of genomic regions in each hexagon is depicted by a grey colour scale of increasing darkness according to the countscale reported on the right (C) Venn diagram representing the number of MACS-predicted peaks (Plt 1e-5) of H4R3me2s in ES and MEF cells Theintersection of the areas representing ES and MEF cells indicates peaks common to both cell types (D) Comparisons of the localizations of ES andMEFrsquos H4R3me2s enrichments peaks In both cell types most peaks are located at genes


PRMT5 controls the parental allele-specific H4R3me2sat ICRs

PRMT5 is the main type-II PRMT in the nucleus ofMEFs that targets histone H4 and is responsible forH4R3me2s We asked whether allele-specific recruitmentof PRMT5 to the chromatin could be responsible for theobserved allelic H4R3me2s at ICRs Real-time PCR amp-lifications revealed a degree of PRMT5 binding to ICRs

that was comparable to that observed at the CyclinE1promoter (Figure 5A and B) at which we reportedbinding previously (10) At two different ICRs(KvDMR1 and Snrpn) we sequenced the PRMT5ChIPed DNA and SNPs showed that PRMT5 was pref-erentially precipitated on the DNA-methylated allele(Figure 5C) Furthermore H4R3me2s levels werereduced at all ICRs tested in the Prmt5 knock-down

A

E

100 20 30 40 50 60 70 80 90 100

minus50

050

100

G+C

H4R

3me2

s E

nric

hmen

t

ES cells

100 20 30 40 50 60 70 80 90 100

minus10

010

2030

G+C

H4R

3me2

s E

nric

hmen

t

MEF cells

B F

ES cells

-2 -1 0 1 2-10

0

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60

H4R

3me2

sen

richm

ent

Distance fromTSS (kb)

246

Log

expr

essi

on

5940

Numberof genes

1096999477014

C

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60

-2 -1 0 1 2

H4R

3me2

sen

richm

ent

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High H3K4me3(9076 peaks)Low H3K4me3(10371 peaks)High H3K4me1(88478 peaks)Low H3K4me1(43472 peaks)

D

Distance from TSS(kb)[0-60] reads100pb

-5

TSS +5

H3K4me3 RNA

-5

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H3K27me3

-5

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-5

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ress

edN

ot e

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ssed

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sity

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3me2

s en

richm

ent

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00

02

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00 01 02 03 04

Counts

00

01

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00 E

26 E

10 E

23 E

Figure 3 H4R3me2s accumulates at promoters and other G+C-rich regions generally irrespective of transcriptional activity (A) The unsupervisedclustering of transcriptional start sites (TSSs) is represented as a heatmap of tag densities (0ndash60) per 100-bp bins from 5 kb upstream to 5 kbdownstream of mouse TSS (n=33876) for the indicated ChIP-seqRNA-seq experiments in ES cells ChIP-seq profiles for H3K4me3 H3K27me3and RNA-seq from ES are from Marks et al (25) The non-expressed class of TSS marked by an asterisk contains genes expressed in differentiatedcells such as genes belonging to the immune response inflammatory response or locomotory behaviour Gene Ontology terms (B) The two class ofTSS (groups 2 and 3) indicated in (A) were analysed for their G+C ratio The two classes of promoters are represented as density plot and box-and-whisker plots in red and blue for the groups 2 and 3 respectively () Indicates Wilcoxon rank sum test with a Plt 22e-16 (C) H4R3me2senrichment around TSSs grouped into four different classes according to the genesrsquo log expression values (D) Mean H4R3me2s enrichment levels inES cells are represented as a function of their distance from the peak summits of H3K4me3 or H3K4me1 peaks H4R3me2s is enriched aroundH3K4me3 peaks but not around H3K4me1 peaks However H4R3me2s enrichment occurs both at H3K4me3-enriched regions with high H3K4me3levels (high H3K4me3) and those with lower H3K4me3 enrichment (low H3K4me2) (E) H4R3me2s enrichment according to the G+C content of100-bp genomic windows in ES and MEF cells showing the absence of H4R3me2s in regions with less than 50 G+C ratio (F) Hexagonal binningof H4R3me2s-enriched regions according to their DNA methylation level assessed by MeDIP or hydroxymethylation level assessed byhydroxyMeDIP (hMeDIP) in ES cells (24) The scales of the x- and y-axis are transformed by the following formula asinh-1(x) Darker shadesof grey indicate higher densities of bins as reported on the count scale


MEFs and a marked reduction was observed at IAPelements as well (Figure 5D) However imprinted geneexpression was retained in the Prmt5 knock-down MEFs(Supplementary Figure S3A) arguing against a role ofH4R3me2s in the allelic repression of imprinted genesWe conclude from these data that PRMT5 is recruitedto the DNA methylated allele of ICRs on which it methy-lates H4R3me2sNext we asked whether a PRMT5 partner protein

called COPR5 previously reported to recruit PRMT5 tothe Cyclin E1 promoter (34) could recruit PRMT5 ontothe chromatin of ICRs as well We found that in newly

derived Copr5 knock-out MEF cells (E Fabbrizio unpub-lished results) the overall PRMT5 levels were decreased inthe nucleus In the COPR5-deficient cells however theglobal histone H4R3me2s levels remained unaffectedThis indicates that COPPR5 levels do not impact signifi-cantly on the bulk of PRMT5-mediated H4R3me2s on thechromatin (Supplementary Figure S3B) However con-sistent with our previous data (34) PRMT5 recruitmentwas decreased at specific promoters such as the Cyclin E1promoter Interestingly decreased PRMT5 binding wasnot observed at the KvDMR1 ICR region(Supplementary Figure S3C) arguing against a role of

Kcnq1ot1

MEF

Mat

erna

l IC

Rs

CpG

isla

nds

(160

26)

Pat

erna

l IC

Rs

ESES

Peg3Gnas

Peg10

Nespas-Gnasxl

DMR-Zdbf2

Peg1ImpactZac1

Snrpn

Igf2rMeg1Grb10

H19IG-DMR

Rasgrf1

[0 - 60] Mean density (tags50bp)A

G

B

-25 0 +25 -25 0 +25 -25 0 +25Distance from

center (kb)Distance from center (kb)

C

D

E

Kcnq1Kcnq1ot1

GC KvDMR1

ES

MEF

1504

80 kb

1504

90 kb

chr7

Snrpn-ICRGC

ES

MEF

chr7

Snrpn

6714

5 kb

6715

5 kb

IG-DMRGC

ES

MEF

chr12

Meg3

1107

65 kb

1107

75 kb

InputA A T T G

ChIPH3K4me3

A A T G

ChIP H4R3me2s

A A T G

ChIPH3K9me3

A A T T G

C G G

PaternalC G G

MaternalT G G

MaternalG G

KvDMR1

T G A G

T G A G

T G A G

T G A G

G A T

G A T

G G T

G GTA

Snrpn-ICR

Maternal

Maternal

Paternal

G GC G G G

G GC G GG

GGC G GG

G GC G G G

CT

C

C

T

IG-DMR

Paternal

Paternal

Maternal

T T

T T

T T

T T

T T C T T

T T C T T

T C T T T

T TC

TC

T T

Rasgrf1-ICR

Paternal

Paternal

Maternal

F

Rasgrf1

Rasgrf1-ICRGC

ES

MEF

8978

0 kb

8980

0 kb

chr9

Figure 4 Allelic H4R3me2s enrichment at ICRs (A) The unsupervised clustering of non-oriented CpG islands (n=16026) is represented as aheatmap of H4R3me2s enrichment levels in mouse ES cells in a window of 5 kb around the centre of each CGIs (the minimum to maximum lengthsof CGIs are 201ndash5129 bp with an average length of 655 bp) (B) Heatmap representation of H4R3me2s enrichments in MEF and ES cells at ICRswhich are DNA-methylated on the maternal (lsquomaternal ICRsrsquo) or the paternal (lsquopaternal ICRsrsquo) chromosome (CndashF) ChIP-seq profiles forH4R3me2s in ES cells and MEFs at the KvDMR1 (C) Snrpn (D) IG-DMR (E) and Rasgrf1 (F) ICRs (G) Sequence profiles around singlenucleotide polymorphisms (SNPs) at the indicated ICRs The parental alleles are equally present in the input chromatin used for ChIP (Input)H4R3me2s H3K9me3 and H3K4me3 are preferentially enriched at one parental allele indicated by an arrow and the identified parental allele atICRs H3K4me3 is present on non-methylated alleles while H4R3me2s and H3K9me3 are present on DNA-methylated alleles of ICRs


COPR5 in the recruitment of PRMT5 to this ICRPossibly another partner protein could be involved inthe recruitment of PRMT5 to the DNA-methylatedalleles of ICRs

PRMT5-mediated H4R3me2s is independent fromH3K9me3 H4K20me3 and DNA methylation at imprintedgene loci

At ICRs H4R3me2s was consistently detected on thesame parental allele as H3K9me3 (Figure 4G) We there-fore explored whether H3K9me3 could contribute to thesomatic maintenance of H4R3me2s ESET (also calledSetDB1 KMT1E) is a candidate lysine-methyltransferase(KMT) for the allelic H3K9me3 at ICRs In a recentgenome-wide ESET binding assay this SET domain-

containing protein had been reported to bind to severalICRs including the H19 ICR (35) Because complete lossof Eset (Setdb1) expression induces death in differentiatedcells (36) we used an shRNA-based approach to stronglybut not completely reduce ESET levels (35) One out ofseveral retroviral constructs tested sh-Eset-1 induced amarked depletion of ESET in the MEFs (Figure 6A) Asexpected this led to a partial but significant reduction inoverall H3K9me3 levels as detected by western blottingIn contrast the overall levels of H4R3me2s remained un-altered in the knock-down cells At two ICRs analysedKvDMR1 and H19 ICR there was a marked reduction inH3K9me3 but not in H4R3me2s levels in the sh-Eset-1cells compared to control MEFs (sh-Ctrl) (Figure 6B)Next we analysed mouse ES cells with a conditional Esetallele for which we showed previously that ESET controlsH3K9me3 at lineage differentiation markers (17)Similarly as in the knock-down MEFs inducible CRE-mediated loss of ESET in ES cells (Figure 6D) led toreduced levels of H3K9me3 at the KvDMR1 and H19ICRs but again without a noticeable loss of H4R3me2s(Figure 6E) Combined these data indicate that ESETcontrols H3K9me3 at ICRs in MEFs and ES cells andthat its depletion does not affect H4R3me2s at ICRsNext we addressed the opposite question namely

whether H3K9me3 is affected by the loss of PRMT5 Asreported by others (37) we observed by western blottingthat there was no overall reduction in H3K9me3 levels inthe PRMT5-depleted MEF cells (Figure 1E) Similarly wedid not observe reduced H3K9me3 precipitation at theH19 and KvDMR1 ICRs (Figure 6C) These datasuggest that PRMT5 and H4R3me2s are not essentialfor the somatic maintenance of H3K9me3 in MEFsChromatin at the repressed alleles of ICRs is also con-

sistently marked by H4 lysine-20 trimethylation(H4K20me3) (38) Previously we showed that the main-tenance of H4K20me3 at the KvDMR1 and H19 ICRs iscontrolled by the combined action of SUV4-20H1 andSUV4-20H2 (Suv4-20H1H2 also called KMT5BC)These KMTs are recruited to the chromatin on the re-pressed alleles of ICRs (26) Suv4-20h1h2 double-knockout cells (18) are viable and do not show loss ofimprinting (26) Absence of SUV4-20H1 and SUV4-20H2 had also no apparent impact on global H4R3me2slevels and did not alter H4R3me2s at the KvDMR1 andH19 ICRs either (Figure 6F) This result indicates thatSUV4-20H1 and SUV4-20H2 do not interfere with thePRMT5-mediated H4R3me2s at imprinted target lociConversely in the sh-Prmt5-1 knock-down cells therewas no apparent reduction in H4K20me3 at ICRs(Figure 6G)Finally we determined whether the allelic DNA methy-

lation patterns at ICRs were altered in Prmt5 knock-downcells There was no evidence for altered DNA methylationat the H19 ICR IG-DMR Peg3 ICR and Snrnp ICRs(Supplementary Figure S4) CpG methylation at IAPelements was not altered either Thus the loss ofH4R3me2s in the experimental MEFs had not affectedthe maintenance of CpG methylation at ICRs nor theH3K9me3 and H4K20me3 chromatin marks associatedwith this DNA methylation

sh-Ctrl sh-Prmt5-1

H4R3m

e2s

H4R3m

e2s

In

put

0

10

20

30

40

50

H4R3m

e2s

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H19 ICR

H4R3m

e2s

Mock

0

10

20

30

40

50

60KvDMR1

Mock

0

10

20

30

40

Rasgrf1

H4R3m

e2s

Mock

010203040506070

Snrpn

Mock

010203040506070

IAP

D

B C

Input

ChIP PRMT5

KvDMR1

Maternal

A A T G G G

A A T T G G G

TC

Snrpn ICR

Maternal

A

T G G A T A G

T G G G T A G

CCNE1

PRMT5ChIP

Mock

KvDMR1

IAP0

002

004

006

008

In

put

A chr738886000-38896000

GC MEF

H4R3me2s

Ccne1

1 kb

Figure 5 PRMT5 recruitment correlates with H4R3me2s enrichmentat specific target loci (A) ChIP of PRMT5 on cross-linked chromatinfrom MEFs The CyclinE1 (Ccne1) promoter is bound by PRMT5 andenriched for H4R3me2s in MEF cells in agreement with previousstudies (10) Shown are real-time PCR quantifications of PRMT5 as-sociation at Cyclin E1 (Ccne1) the KvDMR1 ICR and IAP elements(B) Direct DNA sequencing of ChIP samples shows PRMT5 binding tothe H4R3me2s-marked (and DNA-methylated) allele of the KvDMR1and Snrpn ICRs (C) Real-time PCR amplification shows reducedH4R3me2s at ICRs and at IAP elements in PRMT5-depleted MEFs(sh-Prmt5-1 cells) ChIP was performed on native chromatin In amock ChIP experiment (Mock) an unrelated control IgG (againstchicken IgY) was used Experiments were performed in triplicatebars indicate standard deviation


Our combined knock-down and knockout studiessuggest that PRMT5-mediated H4R3me2s does notdepend on ESET-mediated H3K9me3 or SUV4-20H1H2-mediated H4K20me3 at imprinted target lociConversely ESET-mediated H3K9me3 SUV4-20H1H2-mediated H4K20me3 and DNA methylation seem not todepend on PRMT5-mediated H4R3me2s Though the

three chromatin-modifying systems act on identicaltarget regions such as ICRs and IAP elements PRMT5-mediated H4R3me2s seems to be regulated independentlyfrom the two repressive lysine methylation pathways

Although our data provide novel insights into the regu-lation and epigenomic distribution of H4R3me2s theyleave open the question as to what could be the function

H19 ICR

02468

10121416

H3K9m

e3

Mock

H3K9m

e3

Mock

KvDMR1

02468

1012141618 KvDMR1H19 ICR

H19 ICR KvDMR1 KvDMR1H19 ICR

Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

10

20

30

40

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10

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sh-Ctrl sh-Eset-1A

D

F G

B C

ES Eset ++ ES Eset --E

PRMT5

ACTB

H4R3me2s

Histone H4

H3K9me3

ESET

ES

0 2 3 4

H3K9m

e3

Mock

H3K9m

e3

Mock

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H4R3m

e2s

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H4R3m

e2s

0

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10

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5

10

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10

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30

40

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put

In

put

CRE induction (Days)

PRMT5

ACTB

H4R3me2s

Histone H4

ESET

MEFsh

-Ctrl

sh-E

set-1

H3K9me3

sh-Ctrl sh-Prmt5-1

In

put

H19 ICR

H3K9m

e3

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H3K9m

e3

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KvDMR1

0

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sh-Ctrl MEF (sh-Prmt5-1)MEF (WT)

In

put

In

put

Suv420h12 double KOMEF ( )

H4R3m

e2s

0

10

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30

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70

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H19 ICR

H4R3m

e2s

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KvDMR1

0

05

1

15

2

H4K20

me3Moc

k

H19 ICR

0

05

1

15

2

H4K20

me3Moc

k

KvDMR1

Figure 6 H4R3me2s is regulated independently from H3K9me3 and H4K20me3 at imprinted loci (A) Knockdown of ESET by retroviral shRNA inMEF cells (line sh-Eset-1) Levels of ESET H3K9me3 PRMT5 and H4R3me2s were assessed by western blotting ACTB and H4 are included asinternal controls As a negative control MEFs stably expressing a scrambled shRNA were analysed (line sh-Ctrl) (B) ChIP-qPCR shows loss ofH3K9me3 at the H19 and KvDMR1 ICRs in the sh-Eset-1 MEFs H4R3me2s levels are unaltered Experiments were performed in triplicate barsindicate standard deviation (C) In PRMT5-depleted MEFs (line sh-Prmt5-1) H3K9me3 levels are unaltered at H19 and KvDMR1 (D) Conditionalknockout of Eset in ES cells through a Cre-recombinase and loxP system (17) Levels of ESET H3K9me3 PRMT5 and H4R3me2s were assessed bywestern blotting (E) ChIP-qPCR shows loss of H3K9me3 at the H19 and KvDMR1 ICRs in ESET knockout ES cells H4R3me2s levels areunaffected Mock ChIPs were as described for Figure 5G (F) In Suv4-20h1h2 double knockout MEFs H4R3me2s levels assessed by ChIP-qPCR arenot affected at the H19 and KvDMR1 ICRs (G) H4K20me3 levels are unaltered at ICRs in PRMT5-depleted MEFs (line sh-Prmt5-1) Experimentswere performed in triplicate bars indicate standard deviation Mock ChIPs were as described for Figure 5G


of this covalent histone modification in chromatinHowever there seems not to be a general link with generepression or gene expression As a first step to addressthis question we re-analysed 29 published ChIP-seq datasets together with our ChIP-seq data for H4R3me2s in EScells and evaluated the genome-wide correlations betweenthem (see Supplementary Table S4 for the list of dataused and Figure S6 for the analysis) This confirmed thegenome-wide correlation of H4R3me2s with methylatedCpG dinucleotides and with hydroxymethylated CpGs(Pearsonrsquos r=03 033 respectively see alsoFigure 3F) Interestingly besides a link with H3K4me3at promoter regions (r=029) we also detected a signifi-cant correlation with the binding of two proteins of theMediator complex (Med1 and Med12) (r=029 and 028)and with the cohesin loading protein Nipbl (r=031) andwith two cohesin proteins (Smc1 and Smc3) (r= 025 and024) Interestingly the latter factors are all involved inDNA configuration and looping such as betweenenhancer and promoters (39) suggesting that in partH4R3me2s is enriched at regions involved in chromatincontacts

DISCUSSION

The main findings of this study is that PRMT5 controlsH4R3me2s in embryonic fibroblasts and that H4R3me2speaks are largely confined to G+C-rich regions includingmost of the genomersquos promotersCpG islands Our bio-chemical studies identify PRMT5 as the main type-IIenzyme for H4R3 and H2AR3 in embryonic fibroblastsThis function of PRMT5 complements the earlier demon-stration that PRMT5 methylates H3R8 and H4R3 atspecific target loci including repressed tumour suppressorgenes (940) The pronounced nuclear presence of PRMT5in fibroblasts compared to ES cells seemed unrelated tothe observed patterns of H4R3me2s enrichment along thegenome which were similar between MEFs and ES cellsHowever some statistically defined peaks were identifiedin ES cells but not in MEFs This difference between un-differentiated and differentiated cells may come from theexpression of PRMT7 in ES cells which could potentiallyalso regulate H4R3me2s either directly (4142) or indir-ectly via the production of H4R3me1 (43) This possibilitywould need to be addressed in future studies

Unexpectedly our data did not reveal a general correl-ation between gene expression and H4R3me2s enrich-ment Indeed the vast majority of annotated mousepromoters are marked by H4R3me2s in both ES cellsand MEFs including actively transcribed and repressedgenes This novel insight changes our view on thepossible role(s) of H4R3me2s which in earlier studies onestablished human cell lines was found to be associatedwith gene repression (6811) One characteristic of chro-matin surrounding active promoters is that manycomprise the non-canonical H3 variant H33 (44)whereas the H2A variant H2AZ is present at mitoticallysilenced genes (45) In future research it should berelevant to explore whether a non-canonical nucleosomalorganization possibly in combination with specific histone

modifications interferes with the acquisition andor main-tenance of H4R3me2s One modification that could inter-fere with H4R3me2s acquisition would be asymmetricaldimethylation of H4R3 (H4R3me2a) Recent in vitrostudies on the transcriptional co-activator TDRD3mdashaTudor-domain protein that binds to H4R3me2amdashshowthat this protein binds to the TSS regions of certaingenes in human breast cancer cells (46) however no sig-nificant enrichment was detected on the non-methylatedalleles of human ICRs For technical reasons so far nostudies have reported the genome-wide patterns ofH4R3me2a but such data should be interesting in com-parison to the H4R3me2s profiles obtained in the currentstudy Recently an inverse correlation betweenH4R3me2s and MLL4-mediated H3K4me3 was reportedat the HOXA and HOXB promoters in human cells (47)Although we found H4R3me2s enrichment at the Hoxaand Hoxb promoters in mouse ES cells as well it isunlikely that H4R3me2s and H3K4me3s are mutually ex-clusive since we detected co-enrichment of the two marksat all active promoters in ES cells Another recent studyreported H2AH4R3me2s association with satelliterepeats based on a rather limited filtered ChIP-seq dataset (only 1845 nucleosomes were analysed in total) (48) Inour ChIP-seq experiments we did not observe significantH4R3me2s enrichment at satellite-II or III sequenceswhich are G+C-poor repeats (Supplementary Table S3and Figure S5) In contrast there was a generalH4R3me2s enrichment at IAP elements which are longtandem repeats of higher G+C content and this findingconfirms our earlier findings in embryos and primordialgerm cells (49)Although H4R3me2s did not generally correlate with

transcriptional activity or DNA methylation levels itwas consistently linked to the presence of CpG methyla-tion at specific loci These included IAP retro-transposons(Supplementary Figure S4) and ICRs at imprinted genedomains We used the latter allelic model system to care-fully explore the link between H4R3me2s and repressivehistone lysine methylation PRMT5-mediated H4R3me2swas detected on the DNA-methylated alleles of ICRs onlyand was found to be independent from the repressivehistone lysine methylation controlled by the SET-domain proteins ESET and SUV4-20H1H2 Whatdictates the allelic PRMT5-mediated H4R3me2s at ICRsremains unclear It should be interesting to explorewhether this process could be linked to other histonemodifications or to allele-specific higher order chromatinfeatures at these unusual regulatory elementsPRMT5 and H4R3me2s were shown previously to be

associated with methylated CpG dinucleotides in culturedhuman cells through interaction of PRMT5 with MBD2(Methyl CpG Binding Domain 2) (MCF7) (50) Anotherstudy reported that H4R3me2s serves as a direct bindingtarget for DNA methyltransferase DNMT3A (8) suggest-ing that PRMT5-mediated methylation of H4-arginine3could be necessary for the establishment of de novoDNA methylation In agreement with this hypothesiswe and others reported that ICRs acquire high levels ofH4R3me2s in developing male germ cells prior to andduring the acquisition of DNA methylation (4149)


In another study by Otani et al (51) however noevidence was obtained for interaction of the ADD(ATRX-DNMT3A-DNMT3L) domain of DNMT3Awith an H4R3me2s peptide These different observationsprompted us to ask whether H4R3me2s could be neces-sary for DNA methylation at ICRs in somatic cellsHowever no loss of CpG methylation was observed atICRs or at IAPs in the PRMT5 knock-down cells sug-gesting that DNA methylation maintenance might be in-dependent from H4R3me2s Altogether our data indicatethat H4R3me2s is a PRMT5-controlled histone markwhich accumulates at G+C-rich sequence elements andat repressed alleles of ICRs in somatic cells This processis generally independent from the transcriptional activityor the occurrence of repressive chromatin marksFinally our preliminary bioinformatic comparison with

published ChIP-seq data reveals an intriguing linkbetween H4R3me2s-enrichment and recruitment of corecomponents of the Mediator complex and cohesinproteins at gene regions As concerns the Mediatorcomplex one way this has been reported to interact withPRMT5 is through its associated cyclin-dependent kinasesubunits (52) Although this needs to be explored in futurestudies the observed link with cohesins and mediatorcomplexes suggests that PRMT5-mediated H4R3me2smay directly or indirectly facilitate higher order chromatincontacts

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Onlineincluding [53ndash65]

ACKNOWLEDGEMENTS

We thank members of our teams for discussion andhelpful comments and Thomas Jenuwein and GunnarSchotta for kindly providing the Suv420h1h2 double-knockout cells

FUNDING

Agence Nationale de la Recherche (ANR) the InstitutNational Contre le Cancer (INCa) La Ligue Contre leCancer and the Agency for International CancerResearch (AICR UK) We acknowledge post-doctoralfellowships from the Japanese Society for the Promotionof Science (JSPS) (to RH) the lsquoLigue Contre le Cancerrsquo(France) (to MG) and the University of Montpellier-I (toMG) affiliated to the European network EpiGeneSysand the LABEX EpiGenMed (to RF) Funding foropen access charge INCA (French National CancerInstitute)

Conflict of interest statement None declared

REFERENCES

1 LawJA and JacobsenSE (2010) Establishing maintaining andmodifying DNA methylation patterns in plants and animals NatRev Genet 11 204ndash220

2 JaenischR and BirdA (2003) Epigenetic regulation of geneexpression how the genome integrates intrinsic and environmentalsignals Nat Genet 33(Suppl) 245ndash254

3 BarlowDP (2011) Genomic imprinting a mammalian epigeneticdiscovery model Ann Rev Genet 45 379ndash403

4 DambacherS HahnM and SchottaG (2010) Epigeneticregulation of development by histone lysine methylationHeredity 105 24ndash37

5 Di LorenzoA and BedfordMT (2011) Histone argininemethylation FEBS Lett 585 2024ndash2031

6 MajumderS AlinariL RoyS MillerT DattaJ SifSBaiocchiR and JacobST (2010) Methylation of histone H3 andH4 by PRMT5 regulates ribosomal RNA gene transcriptionJ Cell Biochem 109 553ndash563

7 HenckelA NakabayashiK SanzLA FeilR HataK andArnaudP (2009) Histone methylation is mechanistically linked toDNA methylation at imprinting control regions in mammalsHum Mol Genet 18 3375ndash3383

8 ZhaoQ RankG TanYT LiH MoritzRL SimpsonRJCerrutiL CurtisDJ PatelDJ AllisCD et al (2009)PRMT5-mediated methylation of histone H4R3 recruitsDNMT3A coupling histone and DNA methylation in genesilencing Nat Struct Mol Biol 16 304ndash311

9 WangL PalS and SifS (2008) Protein argininemethyltransferase 5 suppresses the transcription of the RB familyof tumor suppressors in leukemia and lymphoma cells Mol CellBiol 28 6262ndash6277

10 FabbrizioE El MessaoudiS PolanowskaJ PaulC CookJRLeeJH NegreV RoussetM PestkaS Le CamA et al(2002) Negative regulation of transcription by the type II argininemethyltransferase PRMT5 EMBO Rep 3 641ndash645

11 El MessaoudiS FabbrizioE RodriguezC ChuchanaPFauquierL ChengD TheilletC VandelL BedfordMT andSardetC (2006) Coactivator-associated arginine methyltransferase1 (CARM1) is a positive regulator of the Cyclin E1 gene ProcNatl Acad Sci USA 103 13351ndash13356

12 KotaSK and FeilR (2010) Epigenetic transitions in germ celldevelopment and meiosis Dev Cell 19 675ndash686

13 VeronaRI ThorvaldsenJL ReeseKJ and BartolomeiMS(2008) The transcriptional status but not the imprinting controlregion determines allele-specific histone modifications at theimprinted H19 locus Mol Cell Biol 28 71ndash82

14 SinghP ChoJ TsaiSY RivasGE LarsonGP andSzaboPE (2010) Coordinated allele-specific histone acetylation atthe differentially methylated regions of imprinted genes NucleicAcids Res 38 7974ndash7990

15 KoideT MoriwakiK UchidaK MitaA SagaiTYonekawaH KatohH MiyashitaN TsuchiyaK NielsenTJet al (1998) A new inbred strain JF1 established from Japanesefancy mouse carrying the classic piebald allele Mamm Genome9 15ndash19

16 YoshimizuT SugiyamaN De FeliceM YeomYI OhboKMasukoK ObinataM AbeK ScholerHR and MatsuiY(1999) Germline-specific expression of the Oct-4green fluorescentprotein (GFP) transgene in mice Dev Growth Differ 41675ndash684

17 LohmannF LoureiroJ SuH FangQ LeiH LewisTYangY LabowM LiE ChenT et al (2010) KMT1Emediated H3K9 methylation is required for the maintenance ofembryonic stem cells by repressing trophectoderm differentiationStem Cells 28 201ndash212

18 SchottaG SenguptaR KubicekS MalinS KauerMCallenE CelesteA PaganiM OpravilS De La Rosa-VelazquezIA et al (2008) A chromatin-wide transition toH4K20 monomethylation impairs genome integrity andprogrammed DNA rearrangements in the mouse Genes Dev 222048ndash2061

19 WagschalA DelavalK PannetierM ArnaudP and FeilR(2007) Chromatin immunoprecipitation (chip) on unfixedchromatin from cells and tissues to analyze histone modificationsCSH Protoc 2007 pdb prot4767

20 KharchenkoPV TolstorukovMY and ParkPJ (2008) Designand analysis of ChIP-seq experiments for DNA-binding proteinsNat Biotechnol 26 1351ndash1359


21 RobinsonJT ThorvaldsdottirH WincklerW GuttmanMLanderES GetzG and MesirovJP (2011) Integrativegenomics viewer Nat Biotechnol 29 24ndash26

22 YeT KrebsAR ChoukrallahMA KeimeC PlewniakFDavidsonI and ToraL (2011) seqMINER an integrated ChIP-seq data interpretation platform Nucleic Acids Res 39 e35

23 ZhangY LiuT MeyerCA EeckhouteJ JohnsonDSBernsteinBE NusbaumC MyersRM BrownM LiW et al(2008) Model-based analysis of ChIP-Seq (MACS) Genome Biol9 R137

24 FiczG BrancoMR SeisenbergerS SantosF KruegerFHoreTA MarquesCJ AndrewsS and ReikW (2011)Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cellsand during differentiation Nature 473 398ndash402

25 MarksH KalkanT MenafraR DenissovS JonesKHofemeisterH NicholsJ KranzA StewartAF SmithAet al (2012) The transcriptional and epigenomic foundations ofground state pluripotency Cell 149 590ndash604

26 PannetierM JulienE SchottaG TardatM SardetCJenuweinT and FeilR (2008) PR-SET7 and SUV4-20H regulateH4 lysine-20 methylation at imprinting control regions in themouse EMBO Rep 9 998ndash1005

27 TeeWW PardoM TheunissenTW YuL ChoudharyJSHajkovaP and SuraniMA (2010) Prmt5 is essential for earlymouse development and acts in the cytoplasm to maintain ES cellpluripotency Genes Dev 24 2772ndash2777

28 YingQL WrayJ NicholsJ Batlle-MoreraL DobleBWoodgettJ CohenP and SmithA (2008) The ground state ofembryonic stem cell self-renewal Nature 453 519ndash523

29 BoisvertFM CoteJ BoulangerMC ClerouxP BachandFAutexierC and RichardS (2002) Symmetrical dimethylargininemethylation is required for the localization of SMN in Cajalbodies and pre-mRNA splicing J Cell Biol 159 957ndash969

30 LemmI GirardC KuhnAN WatkinsNJ SchneiderMBordonneR and LuhrmannR (2006) Ongoing U snRNPbiogenesis is required for the integrity of Cajal bodies Mol BiolCell 17 3221ndash3231

31 AncelinK LangeUC HajkovaP SchneiderR BannisterAJKouzaridesT and SuraniMA (2006) Blimp1 associates withPrmt5 and directs histone arginine methylation in mouse germcells Nat Cell Biol 8 623ndash630

32 Gardiner-GardenM and FrommerM (1987) CpG islands invertebrate genomes J Mol Biol 196 261ndash282

33 StrogantsevR and Ferguson-SmithAC (2012) Proteins involvedin establishment and maintenance of imprinted methylationmarks Brief Funct Genomics 11 227ndash239

34 LacroixM El MessaoudiS RodierG Le CamA SardetCand FabbrizioE (2008) The histone-binding protein COPR5 isrequired for nuclear functions of the protein argininemethyltransferase PRMT5 EMBO Rep 9 452ndash458

35 YuanP HanJ GuoG OrlovYL HussM LohYHYawLP RobsonP LimB and NgHH (2009) Eset partnerswith Oct4 to restrict extraembryonic trophoblast lineage potentialin embryonic stem cells Genes Dev 23 2507ndash2520

36 DodgeJE KangYK BeppuH LeiH and LiE (2004)Histone H3-K9 methyltransferase ESET is essential for earlydevelopment Mol Cell Biol 24 2478ndash2486

37 MiglioriV MullerJ PhalkeS LowD BezziM MokWCSahuSK GunaratneJ CapassoP BassiC et al (2012)Symmetric dimethylation of H3R2 is a newly identified histonemark that supports euchromatin maintenance Nat Struct MolBiol 19 136ndash144

38 DelavalK GovinJ CerqueiraF RousseauxS KhochbinSand FeilR (2007) Differential histone modifications mark mouseimprinting control regions during spermatogenesis EMBO J 26720ndash729

39 KageyMH NewmanJJ BilodeauS ZhanY OrlandoDAvan BerkumNL EbmeierCC GoossensJ RahlPBLevineSS et al (2010) Mediator and cohesin connect geneexpression and chromatin architecture Nature 467 430ndash435

40 PalS VishwanathSN Erdjument-BromageH TempstP andSifS (2004) Human SWISNF-associated PRMT5 methylates

histone H3 arginine 8 and negatively regulates expression of ST7

and NM23 tumor suppressor genes Mol Cell Biol 249630ndash9645

41 JelinicP StehleJC and ShawP (2006) The testis-specific factorCTCFL cooperates with the protein methyltransferase PRMT7 inH19 imprinting control region methylation PLoS Biol 4 e355

42 LeeJH CookJR YangZH MirochnitchenkoOGundersonSI FelixAM HerthN HoffmannR and PestkaS(2005) PRMT7 a new protein arginine methyltransferase thatsynthesizes symmetric dimethylarginine J Biol Chem 2803656ndash3664

43 Zurita-LopezCI SandbergT KellyR and ClarkeSG (2012)Human protein arginine methyltransferase 7 (PRMT7) is a typeIII enzyme forming omega-NG-monomethylated arginine residuesJ Biol Chem 287 7859ndash7870

44 JinC ZangC WeiG CuiK PengW ZhaoK andFelsenfeldG (2009) H33H2AZ double variant-containingnucleosomes mark lsquonucleosome-free regionsrsquo of active promotersand other regulatory regions Nat Genet 41 941ndash945

45 KellyTK MirandaTB LiangG BermanBP LinJCTanayA and JonesPA (2010) H2AZ maintenance duringmitosis reveals nucleosome shifting on mitotically silenced genesMol Cell 39 901ndash911

46 YangY LuY EspejoA WuJ XuW LiangS andBedfordMT (2010) TDRD3 is an effector molecule for arginine-methylated histone marks Mol Cell 40 1016ndash1023

47 DharSS LeeSH KanPY VoigtP MaL ShiXReinbergD and LeeMG (2012) Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation ismediated by a tandem PHD of MLL4 Genes Dev 262749ndash2762

48 CapursoD XiongH and SegalMR (2012) A histone argininemethylation localizes to nucleosomes in satellite II and III DNAsequences in the human genome BMC Genomics 13 630

49 HenckelA ChebliK KotaSK ArnaudP and FeilR (2012)Transcription and histone methylation changes correlate withimprint acquisition in male germ cells EMBO J 31 606ndash615

50 Le GuezennecX VermeulenM BrinkmanABHoeijmakersWA CohenA LasonderE and StunnenbergHG(2006) MBD2NuRD and MBD3NuRD two distinct complexeswith different biochemical and functional properties Mol CellBiol 26 843ndash851

51 OtaniJ NankumoT AritaK InamotoS AriyoshiM andShirakawaM (2009) Structural basis for recognition of H3K4methylation status by the DNA methyltransferase 3A ATRX-DNMT3-DNMT3L domain EMBO Rep 10 1235ndash1241

52 TsutsuiT FukasawaR ShinmyouzuK NakagawaR TobeKTanakaA and OhkumaY (2013) Mediator complex recruitsepigenetic regulators via its two cyclin-dependent kinase subunitsto repress transcription of immune-response genes J Biol Chem288 20955ndash20965

53 GregoryRI OrsquoNeillLP RandallTE FournierC KhoslaSTurnerBM and FeilR (2002) Inhibition of histone deacetylasesalters allelic chromatin conformation at the imprinted U2af1-rs1locus in mouse embryonic stem cells J Biol Chem 27711728ndash11734

54 ShiraishiM and HayatsuH (2004) High-speed conversion ofcytosine to uracil in bisulfite genomic sequencing analysis ofDNA methylation DNA Res 11 409ndash415

55 KanedaM HirasawaR ChibaH OkanoM LiE andSasakiH (2010) Genetic evidence for Dnmt3a-dependentimprinting during oocyte growth obtained by conditionalknockout with Zp3-Cre and complete exclusion of Dnmt3b bychimera formation Genes Cells 3 169ndash179

56 HirasawaR ChibaH KanedaM TajimaS LiE JaenischRand SasakiH (2008) Maternal and zygotic Dnmt1 are necessaryand sufficient for the maintenance of DNA methylation imprintsduring preimplantation development Genes Dev 22 1607ndash1616

57 MarsonA LevineSS ColeMF FramptonGMBrambrinkT JohnstoneS GuentherMG JohnstonWKWernigM NewmanJ et al (2008) Connecting microRNA genesto the core transcriptional regulatory circuitry of embryonic stemcells Cell 134 521ndash533

58 HandokoL XuH LiG NganCY ChewE SchnappMLeeCW YeC PingJL MulawadiF et al (2011)


CTCF-mediated functional chromatin interactome in pluripotentcells Nat Genet 43 630ndash638

59 MikkelsenTS KuM JaffeDB IssacB LiebermanEGiannoukosG AlvarezP BrockmanW KimTK KocheRPet al (2007) Genome-wide maps of chromatin state in pluripotentand lineage-committed cells Nature 448 553ndash560


61 KuM KocheRP RheinbayE MendenhallEM EndohMMikkelsenTS PresserA NusbaumC XieX ChiAS et al(2008) Genomewide analysis of PRC1 and PRC2 occupancyidentifies two classes of bivalent domains PLoS Genet 4e1000242

62 MeissnerA MikkelsenTS GuH WernigM HannaJSivachenkoA ZhangX BernsteinBE NusbaumC JaffeDB

et al (2008) Genome-scale DNA methylation maps of pluripotentand differentiated cells Nature 454 766ndash770

63 AngYS TsaiSY LeeDF MonkJ SuJ RatnakumarKDingJ GeY DarrH ChangB et al (2011) Wdr5 mediatesself-renewal and reprogramming via the embryonic stem cell coretranscriptional network Cell 145 183ndash197

64 NitzscheA Paszkowski-RogaczM MatareseF Janssen-MegensEM HubnerNC SchulzH de VriesI DingLHuebnerN MannM et al (2011) RAD21 cooperates withpluripotency transcription factors in the maintenance ofembryonic stem cell identity PLoS One 6 e19470



(HBB) and Cyclin E1 genes (6811) However genome-wide analyses of the distribution of this covalent histonemark were missing to test this idea It also remainedunclear which protein arginine methyltransferase(s)(PRMT) could control the H4R3me2s mark in mamma-lian somatic cells In the current study we explored thesekey questions in mouse embryonic fibroblasts (MEFs) andembryonic stem (ES) cells Using an shRNA-mediatedgene knock-down approach we demonstrate that thetype-II protein arginine methyltransferase PRMT5controls the bulk of H4R3me2s in MEFs Chromatinimmunoprecipitation (ChIP) followed by next-generationDNA sequencing (ChIP-seq) was used to delineate thegenome-wide distribution of H4R3me2s in both ES andMEF cells The latter analysis revealed that rather unex-pectedly H4R3me2s is enriched preferentially at DNAsequences with a high G+C content including ICRsUsing imprinted gene loci as a model system we carefullyexplored the relationship of H4R3me2s with other histonemodifications in somatic cells Finally using comparativebioinformatics analyses we found interesting correlationswith factors involved in higher order chromatin contacts

MATERIALS AND METHODS

Primary cells immuno-cytochemistry and RT-polymerasechain reaction analysis

Primary MEFs used for Prmt5 and Eset knockdown wereestablished from E135 embryos obtained by crossingC57BL6J (Mus musculus domesticus) females with JF1(Mus musculus molossinus) males (15) The ES cells (lineBJ1) used for immunoblotting and immunocytochemistrywere newly established in serum-free 2i medium fromblastocysts obtained by mating a C57BL6J Oct4-GFPtransgenic female (16) with a JF1 male Their pluripotencyis described elsewhere (Kota et al in preparation) Esetknockout ES cells and Suv4-20h1h2 double-knockoutMEFs were derived by Lohmann et al (17) and Schottaet al (18) respectively Total RNA was isolated usingTRIzol reagent (Invitrogen) and cDNA was synthesizedusing SuperScript-2 reverse transcriptase (Invitrogen) andrandom oligonucleotides Primers used for polymerasechain reaction (PCR) are described in SupplementaryTable S2 For immunocytochemistry cells were seededon gelatin-coated cover slips fixed for 10min in 4paraformaldehyde in phosphate buffered saline (PBS) atroom temperature and washed with PBS After 30-minincubation with pre-treatment buffer (1 horse serum2 Triton-X100 in PBS) cells were incubated for 3 hwith specific antisera at RT After extensive washes theywere incubated for 30min at RT with an appropriateAlexa488-conjugated second antibody Stained cells weremounted in Vectorshield mounting medium (VectorLaboratory) containing DAPI for counterstaining ofDNA and were microscopically analysed

Western blotting

Whole cell lysates or protein extracts were resolved onsodium dodecyl sulphatendashpolyacrylamide gel electrophor-esis (SDSndashPAGE) gels or on pre-cast NuPage 4ndash12

acrylamide gradient SDSndashPAGE gels (Invitrogen) andtransferred onto nitrocellulose membrane Membraneswere blocked and incubated overnight at 4C withprimary antibodies After incubation with appropriatesecondary antibodies coupled to horseradish peroxidaseimages were developed using the West Dura kit andChemiSmart 5000 system (Vilber Laurmat) Forproducing PRMT5- and PRMT7 over-expressing 293Tcells full-length cDNAs cloned into the vector pCMV-SPORT6 (Open Bioscience) were used

ChIP and PCR analysis of precipitated chromatin

ChIP on native chromatin was performed following a pre-viously described protocol (19) with the following modifi-cations The purified nuclei were incubated for 15min at37C with 60 units of Micrococcal nuclease (S7 Micro-coccal nuclease from Staphylococcus aureus Roche) toproduce monodi-nucleosomes as verified by agarose gelelectrophoresis Nucleosomes were precipitated with theH4R3me2s antiserum-2 (Supplementary Table S1) andDNA was purified from precipitated chromatin fractionswith the lsquoChIP DNA purification kitrsquo (Zymo Research)For ChIP on cross-linked chromatin cells were fixed with1 formaldehyde for 10min and quenched with 125mMof glycine Subsequently cells were washed in PBS col-lected in 15-ml tubes and centrifuged to remove the super-natant Cells were re-suspended in 100 ml of SDS lysisbuffer (100mM NaCl 1 SDS 5mM ethylenediaminete-traacetic acid (EDTA) 50mM TrisndashCl and lsquoCompleteprotease inhibitor cocktailrsquo from Roche) After 10minon ice with occasional vortexing cells were diluted in200 ml of ice-cold ChIP dilution buffer (001 SDS11 Triton X-100 12mM EDTA 167mM TrisndashCl167mM NaCl and lsquoComplete protease inhibitor cocktailrsquofrom Roche) and sonicated twice at high power for 30cycles of 30 s ON 30 s OFF in a BioRuptor twin appar-atus (Diagenode) The average DNA fragmentsrsquo lengthfrom ES and MEF cells was 200 bp as estimated byagarose gel electrophoresis Q-PCR on purified DNAwas performed using the LightCycler 480 SYBR GreenI Master Mix (Roche) and LigthCycler 480 apparatus(Roche) Antisera and oligonucleotides used are listed inSupplementary Tables S1 and S2

High-throughput sequencing and bioinformatics

Input and ChIPed DNAs were sequenced on an IlluminaHi-seq 2000 instrument (Fasteris Switzerland) using theTruSeqTM SBS v3 kit The sequences produced from theinput chromatin and H4R3me2s ChIPed DNA from ESand MEF cells are detailed in the Supplementary Table S3ChIP-seq raw data (fastq) were aligned on theM musculusreference genome (mm9) using the Bowie 0127 softwareSmoothed tag density compared to input tag density werecomputed with the Sequence processing pipeline (20) andvisualized with the integrative Genomics Viewer (21)Median tag density around the defined peak positionswere calculated using an in-house designed R-scriptBriefly peaksrsquo summits coordinates were grouped accord-ing to the level of enrichment for the mark examined andmedian tag densities were calculated every 100bp across






Data access


RESULTS











AMEF

ES PRMT5

PRMT7

PRMT5

PRMT7

ACTB

BRT

MEF ES

+ - + -

Prmt5

Prmt7

Gapdh

PRMT5

DAPI

Merged

MEF ESC

F

D

Cyt Nuc

PRMT5

MEFCyt Nuc

ES

Tubulin

H3K9me3

H2AR3me2sH4R3me2s

E

PRMT5

MEFsh

-Ctrl

sh-P

rmt5-

1

sh-P

rmt5-

2

ACTB

H4H3K9me3

H2AR3me2sH4R3me2s

H4R3me2s

anti-serum 1

anti-serum 1

anti-serum 2

Prmt5 Prmt7R

elat

ive

expr

essi

on (

)

sh-C

trl

sh-P

rmt5-

1

sh-P

rmt5-

2

sh-C

trl

sh-P

rmt5-

1

sh-P

rmt5-

2

100

120140

8060

40

20

0

100

60

80

120140

20

40

0















C DES

111719

MEFChIP - H4R3me2s


H4R3me2s ES

H4R3me2s MEF

9891

0 kb

9892

0 kb

9893

0 kb

9894

0 kb

9895

0 kb

9896

0 kb

chr15

A

B



H4R

3me2

s en

richm

ent

in E

S c

ells

0 1 2 3 4 5

0

1

2

3

4

5

6Counts

10 E

80 E

15 E

20 E

0

10

20

30

40

50

60

70

LINESIN

ELT

R

Satellit

es

Promote

rs

(-25k

b +25

kb)CpG

GenesExo

ns

Intron

s

pe

aks







A

E

100 20 30 40 50 60 70 80 90 100

minus50

050

100

G+C

H4R

3me2

s E

nric

hmen

t

ES cells

100 20 30 40 50 60 70 80 90 100

minus10

010

2030

G+C

H4R

3me2

s E

nric

hmen

t

MEF cells

B F

ES cells

-2 -1 0 1 2-10

0

10

20

30

40

50

60

H4R

3me2

sen

richm

ent


246

Log

expr

essi

on

5940

Numberof genes

1096999477014

C

-10

0

10

20

30

40

50

60

-2 -1 0 1 2

H4R

3me2

sen

richm

ent



D


-5

TSS +5

H3K4me3 RNA

-5

TSS +5

H3K27me3

-5

TSS +5

H4R3me2s

-5

TSS +5

Exp

ress

edN

ot e

xpre

ssed

1

2

3

035

0

2

4

6

8

10

040 045 050 055 060 065

Den

sity


H4R

3me2

s en

richm

ent


00

02

04

06

08

00 01 02 03 04

Counts

00

01

02

03

04

05

00 01 02 03 04

00 E

26 E

10 E

23 E






Kcnq1ot1

MEF

Mat

erna

l IC

Rs

CpG

isla

nds

(160

26)

Pat

erna

l IC

Rs

ESES

Peg3Gnas

Peg10

Nespas-Gnasxl

DMR-Zdbf2

Peg1ImpactZac1

Snrpn

Igf2rMeg1Grb10

H19IG-DMR

Rasgrf1


G

B

-25 0 +25 -25 0 +25 -25 0 +25Distance from


C

D

E

Kcnq1Kcnq1ot1

GC KvDMR1

ES

MEF

1504

80 kb

1504

90 kb

chr7

Snrpn-ICRGC

ES

MEF

chr7

Snrpn

6714

5 kb

6715

5 kb

IG-DMRGC

ES

MEF

chr12

Meg3

1107

65 kb

1107

75 kb

InputA A T T G

ChIPH3K4me3

A A T G

ChIP H4R3me2s

A A T G

ChIPH3K9me3

A A T T G

C G G

PaternalC G G

MaternalT G G

MaternalG G

KvDMR1

T G A G

T G A G

T G A G

T G A G

G A T

G A T

G G T

G GTA

Snrpn-ICR

Maternal

Maternal

Paternal

G GC G G G

G GC G GG

GGC G GG

G GC G G G

CT

C

C

T

IG-DMR

Paternal

Paternal

Maternal

T T

T T

T T

T T

T T C T T

T T C T T

T C T T T

T TC

TC

T T

Rasgrf1-ICR

Paternal

Paternal

Maternal

F

Rasgrf1

Rasgrf1-ICRGC

ES

MEF

8978

0 kb

8980

0 kb

chr9










sh-Ctrl sh-Prmt5-1

H4R3m

e2s

H4R3m

e2s

In

put

0

10

20

30

40

50

H4R3m

e2s

Mock

H19 ICR

H4R3m

e2s

Mock

0

10

20

30

40

50

60KvDMR1

Mock

0

10

20

30

40

Rasgrf1

H4R3m

e2s

Mock

010203040506070

Snrpn

Mock

010203040506070

IAP

D

B C

Input

ChIP PRMT5

KvDMR1

Maternal

A A T G G G

A A T T G G G

TC

Snrpn ICR

Maternal

A

T G G A T A G

T G G G T A G

CCNE1

PRMT5ChIP

Mock

KvDMR1

IAP0

002

004

006

008

In

put

A chr738886000-38896000

GC MEF

H4R3me2s

Ccne1

1 kb






H19 ICR

02468

10121416

H3K9m

e3

Mock

H3K9m

e3

Mock

KvDMR1

02468

1012141618 KvDMR1H19 ICR


Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

10

20

30

40

50

60

0

10

20

30

40

50

60

sh-Ctrl sh-Eset-1A

D

F G

B C


PRMT5

ACTB

H4R3me2s

Histone H4

H3K9me3

ESET

ES

0 2 3 4

H3K9m

e3

Mock

H3K9m

e3

Mock

Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

In

put

In

put


PRMT5

ACTB

H4R3me2s

Histone H4

ESET

MEFsh

-Ctrl

sh-E

set-1

H3K9me3

sh-Ctrl sh-Prmt5-1

In

put

H19 ICR

H3K9m

e3

Mock

0

5

10

15

20

25

30

35

H3K9m

e3

Mock

KvDMR1

0

5

10

15

20

25

30

35


In

put

In

put


H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

H19 ICR

H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

KvDMR1

0

05

1

15

2

H4K20

me3Moc

k

H19 ICR

0

05

1

15

2

H4K20

me3Moc

k

KvDMR1




DISCUSSION









SUPPLEMENTARY DATA


ACKNOWLEDGEMENTS


FUNDING



REFERENCES













































































Data access


RESULTS











AMEF

ES PRMT5

PRMT7

PRMT5

PRMT7

ACTB

BRT

MEF ES

+ - + -

Prmt5

Prmt7

Gapdh

PRMT5

DAPI

Merged

MEF ESC

F

D

Cyt Nuc

PRMT5

MEFCyt Nuc

ES

Tubulin

H3K9me3

H2AR3me2sH4R3me2s

E

PRMT5

MEFsh

-Ctrl

sh-P

rmt5-

1

sh-P

rmt5-

2

ACTB

H4H3K9me3

H2AR3me2sH4R3me2s

H4R3me2s

anti-serum 1

anti-serum 1

anti-serum 2

Prmt5 Prmt7R

elat

ive

expr

essi

on (

)

sh-C

trl

sh-P

rmt5-

1

sh-P

rmt5-

2

sh-C

trl

sh-P

rmt5-

1

sh-P

rmt5-

2

100

120140

8060

40

20

0

100

60

80

120140

20

40

0















C DES

111719

MEFChIP - H4R3me2s


H4R3me2s ES

H4R3me2s MEF

9891

0 kb

9892

0 kb

9893

0 kb

9894

0 kb

9895

0 kb

9896

0 kb

chr15

A

B



H4R

3me2

s en

richm

ent

in E

S c

ells

0 1 2 3 4 5

0

1

2

3

4

5

6Counts

10 E

80 E

15 E

20 E

0

10

20

30

40

50

60

70

LINESIN

ELT

R

Satellit

es

Promote

rs

(-25k

b +25

kb)CpG

GenesExo

ns

Intron

s

pe

aks







A

E

100 20 30 40 50 60 70 80 90 100

minus50

050

100

G+C

H4R

3me2

s E

nric

hmen

t

ES cells

100 20 30 40 50 60 70 80 90 100

minus10

010

2030

G+C

H4R

3me2

s E

nric

hmen

t

MEF cells

B F

ES cells

-2 -1 0 1 2-10

0

10

20

30

40

50

60

H4R

3me2

sen

richm

ent


246

Log

expr

essi

on

5940

Numberof genes

1096999477014

C

-10

0

10

20

30

40

50

60

-2 -1 0 1 2

H4R

3me2

sen

richm

ent



D


-5

TSS +5

H3K4me3 RNA

-5

TSS +5

H3K27me3

-5

TSS +5

H4R3me2s

-5

TSS +5

Exp

ress

edN

ot e

xpre

ssed

1

2

3

035

0

2

4

6

8

10

040 045 050 055 060 065

Den

sity


H4R

3me2

s en

richm

ent


00

02

04

06

08

00 01 02 03 04

Counts

00

01

02

03

04

05

00 01 02 03 04

00 E

26 E

10 E

23 E






Kcnq1ot1

MEF

Mat

erna

l IC

Rs

CpG

isla

nds

(160

26)

Pat

erna

l IC

Rs

ESES

Peg3Gnas

Peg10

Nespas-Gnasxl

DMR-Zdbf2

Peg1ImpactZac1

Snrpn

Igf2rMeg1Grb10

H19IG-DMR

Rasgrf1


G

B

-25 0 +25 -25 0 +25 -25 0 +25Distance from


C

D

E

Kcnq1Kcnq1ot1

GC KvDMR1

ES

MEF

1504

80 kb

1504

90 kb

chr7

Snrpn-ICRGC

ES

MEF

chr7

Snrpn

6714

5 kb

6715

5 kb

IG-DMRGC

ES

MEF

chr12

Meg3

1107

65 kb

1107

75 kb

InputA A T T G

ChIPH3K4me3

A A T G

ChIP H4R3me2s

A A T G

ChIPH3K9me3

A A T T G

C G G

PaternalC G G

MaternalT G G

MaternalG G

KvDMR1

T G A G

T G A G

T G A G

T G A G

G A T

G A T

G G T

G GTA

Snrpn-ICR

Maternal

Maternal

Paternal

G GC G G G

G GC G GG

GGC G GG

G GC G G G

CT

C

C

T

IG-DMR

Paternal

Paternal

Maternal

T T

T T

T T

T T

T T C T T

T T C T T

T C T T T

T TC

TC

T T

Rasgrf1-ICR

Paternal

Paternal

Maternal

F

Rasgrf1

Rasgrf1-ICRGC

ES

MEF

8978

0 kb

8980

0 kb

chr9










sh-Ctrl sh-Prmt5-1

H4R3m

e2s

H4R3m

e2s

In

put

0

10

20

30

40

50

H4R3m

e2s

Mock

H19 ICR

H4R3m

e2s

Mock

0

10

20

30

40

50

60KvDMR1

Mock

0

10

20

30

40

Rasgrf1

H4R3m

e2s

Mock

010203040506070

Snrpn

Mock

010203040506070

IAP

D

B C

Input

ChIP PRMT5

KvDMR1

Maternal

A A T G G G

A A T T G G G

TC

Snrpn ICR

Maternal

A

T G G A T A G

T G G G T A G

CCNE1

PRMT5ChIP

Mock

KvDMR1

IAP0

002

004

006

008

In

put

A chr738886000-38896000

GC MEF

H4R3me2s

Ccne1

1 kb






H19 ICR

02468

10121416

H3K9m

e3

Mock

H3K9m

e3

Mock

KvDMR1

02468

1012141618 KvDMR1H19 ICR


Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

10

20

30

40

50

60

0

10

20

30

40

50

60

sh-Ctrl sh-Eset-1A

D

F G

B C


PRMT5

ACTB

H4R3me2s

Histone H4

H3K9me3

ESET

ES

0 2 3 4

H3K9m

e3

Mock

H3K9m

e3

Mock

Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

In

put

In

put


PRMT5

ACTB

H4R3me2s

Histone H4

ESET

MEFsh

-Ctrl

sh-E

set-1

H3K9me3

sh-Ctrl sh-Prmt5-1

In

put

H19 ICR

H3K9m

e3

Mock

0

5

10

15

20

25

30

35

H3K9m

e3

Mock

KvDMR1

0

5

10

15

20

25

30

35


In

put

In

put


H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

H19 ICR

H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

KvDMR1

0

05

1

15

2

H4K20

me3Moc

k

H19 ICR

0

05

1

15

2

H4K20

me3Moc

k

KvDMR1




DISCUSSION









SUPPLEMENTARY DATA


ACKNOWLEDGEMENTS


FUNDING



REFERENCES













































































AMEF

ES PRMT5

PRMT7

PRMT5

PRMT7

ACTB

BRT

MEF ES

+ - + -

Prmt5

Prmt7

Gapdh

PRMT5

DAPI

Merged

MEF ESC

F

D

Cyt Nuc

PRMT5

MEFCyt Nuc

ES

Tubulin

H3K9me3

H2AR3me2sH4R3me2s

E

PRMT5

MEFsh

-Ctrl

sh-P

rmt5-

1

sh-P

rmt5-

2

ACTB

H4H3K9me3

H2AR3me2sH4R3me2s

H4R3me2s

anti-serum 1

anti-serum 1

anti-serum 2

Prmt5 Prmt7R

elat

ive

expr

essi

on (

)

sh-C

trl

sh-P

rmt5-

1

sh-P

rmt5-

2

sh-C

trl

sh-P

rmt5-

1

sh-P

rmt5-

2

100

120140

8060

40

20

0

100

60

80

120140

20

40

0















C DES

111719

MEFChIP - H4R3me2s


H4R3me2s ES

H4R3me2s MEF

9891

0 kb

9892

0 kb

9893

0 kb

9894

0 kb

9895

0 kb

9896

0 kb

chr15

A

B



H4R

3me2

s en

richm

ent

in E

S c

ells

0 1 2 3 4 5

0

1

2

3

4

5

6Counts

10 E

80 E

15 E

20 E

0

10

20

30

40

50

60

70

LINESIN

ELT

R

Satellit

es

Promote

rs

(-25k

b +25

kb)CpG

GenesExo

ns

Intron

s

pe

aks







A

E

100 20 30 40 50 60 70 80 90 100

minus50

050

100

G+C

H4R

3me2

s E

nric

hmen

t

ES cells

100 20 30 40 50 60 70 80 90 100

minus10

010

2030

G+C

H4R

3me2

s E

nric

hmen

t

MEF cells

B F

ES cells

-2 -1 0 1 2-10

0

10

20

30

40

50

60

H4R

3me2

sen

richm

ent


246

Log

expr

essi

on

5940

Numberof genes

1096999477014

C

-10

0

10

20

30

40

50

60

-2 -1 0 1 2

H4R

3me2

sen

richm

ent



D


-5

TSS +5

H3K4me3 RNA

-5

TSS +5

H3K27me3

-5

TSS +5

H4R3me2s

-5

TSS +5

Exp

ress

edN

ot e

xpre

ssed

1

2

3

035

0

2

4

6

8

10

040 045 050 055 060 065

Den

sity


H4R

3me2

s en

richm

ent


00

02

04

06

08

00 01 02 03 04

Counts

00

01

02

03

04

05

00 01 02 03 04

00 E

26 E

10 E

23 E






Kcnq1ot1

MEF

Mat

erna

l IC

Rs

CpG

isla

nds

(160

26)

Pat

erna

l IC

Rs

ESES

Peg3Gnas

Peg10

Nespas-Gnasxl

DMR-Zdbf2

Peg1ImpactZac1

Snrpn

Igf2rMeg1Grb10

H19IG-DMR

Rasgrf1


G

B

-25 0 +25 -25 0 +25 -25 0 +25Distance from


C

D

E

Kcnq1Kcnq1ot1

GC KvDMR1

ES

MEF

1504

80 kb

1504

90 kb

chr7

Snrpn-ICRGC

ES

MEF

chr7

Snrpn

6714

5 kb

6715

5 kb

IG-DMRGC

ES

MEF

chr12

Meg3

1107

65 kb

1107

75 kb

InputA A T T G

ChIPH3K4me3

A A T G

ChIP H4R3me2s

A A T G

ChIPH3K9me3

A A T T G

C G G

PaternalC G G

MaternalT G G

MaternalG G

KvDMR1

T G A G

T G A G

T G A G

T G A G

G A T

G A T

G G T

G GTA

Snrpn-ICR

Maternal

Maternal

Paternal

G GC G G G

G GC G GG

GGC G GG

G GC G G G

CT

C

C

T

IG-DMR

Paternal

Paternal

Maternal

T T

T T

T T

T T

T T C T T

T T C T T

T C T T T

T TC

TC

T T

Rasgrf1-ICR

Paternal

Paternal

Maternal

F

Rasgrf1

Rasgrf1-ICRGC

ES

MEF

8978

0 kb

8980

0 kb

chr9










sh-Ctrl sh-Prmt5-1

H4R3m

e2s

H4R3m

e2s

In

put

0

10

20

30

40

50

H4R3m

e2s

Mock

H19 ICR

H4R3m

e2s

Mock

0

10

20

30

40

50

60KvDMR1

Mock

0

10

20

30

40

Rasgrf1

H4R3m

e2s

Mock

010203040506070

Snrpn

Mock

010203040506070

IAP

D

B C

Input

ChIP PRMT5

KvDMR1

Maternal

A A T G G G

A A T T G G G

TC

Snrpn ICR

Maternal

A

T G G A T A G

T G G G T A G

CCNE1

PRMT5ChIP

Mock

KvDMR1

IAP0

002

004

006

008

In

put

A chr738886000-38896000

GC MEF

H4R3me2s

Ccne1

1 kb






H19 ICR

02468

10121416

H3K9m

e3

Mock

H3K9m

e3

Mock

KvDMR1

02468

1012141618 KvDMR1H19 ICR


Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

10

20

30

40

50

60

0

10

20

30

40

50

60

sh-Ctrl sh-Eset-1A

D

F G

B C


PRMT5

ACTB

H4R3me2s

Histone H4

H3K9me3

ESET

ES

0 2 3 4

H3K9m

e3

Mock

H3K9m

e3

Mock

Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

In

put

In

put


PRMT5

ACTB

H4R3me2s

Histone H4

ESET

MEFsh

-Ctrl

sh-E

set-1

H3K9me3

sh-Ctrl sh-Prmt5-1

In

put

H19 ICR

H3K9m

e3

Mock

0

5

10

15

20

25

30

35

H3K9m

e3

Mock

KvDMR1

0

5

10

15

20

25

30

35


In

put

In

put


H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

H19 ICR

H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

KvDMR1

0

05

1

15

2

H4K20

me3Moc

k

H19 ICR

0

05

1

15

2

H4K20

me3Moc

k

KvDMR1




DISCUSSION









SUPPLEMENTARY DATA


ACKNOWLEDGEMENTS


FUNDING



REFERENCES





















































































C DES

111719

MEFChIP - H4R3me2s


H4R3me2s ES

H4R3me2s MEF

9891

0 kb

9892

0 kb

9893

0 kb

9894

0 kb

9895

0 kb

9896

0 kb

chr15

A

B



H4R

3me2

s en

richm

ent

in E

S c

ells

0 1 2 3 4 5

0

1

2

3

4

5

6Counts

10 E

80 E

15 E

20 E

0

10

20

30

40

50

60

70

LINESIN

ELT

R

Satellit

es

Promote

rs

(-25k

b +25

kb)CpG

GenesExo

ns

Intron

s

pe

aks







A

E

100 20 30 40 50 60 70 80 90 100

minus50

050

100

G+C

H4R

3me2

s E

nric

hmen

t

ES cells

100 20 30 40 50 60 70 80 90 100

minus10

010

2030

G+C

H4R

3me2

s E

nric

hmen

t

MEF cells

B F

ES cells

-2 -1 0 1 2-10

0

10

20

30

40

50

60

H4R

3me2

sen

richm

ent


246

Log

expr

essi

on

5940

Numberof genes

1096999477014

C

-10

0

10

20

30

40

50

60

-2 -1 0 1 2

H4R

3me2

sen

richm

ent



D


-5

TSS +5

H3K4me3 RNA

-5

TSS +5

H3K27me3

-5

TSS +5

H4R3me2s

-5

TSS +5

Exp

ress

edN

ot e

xpre

ssed

1

2

3

035

0

2

4

6

8

10

040 045 050 055 060 065

Den

sity


H4R

3me2

s en

richm

ent


00

02

04

06

08

00 01 02 03 04

Counts

00

01

02

03

04

05

00 01 02 03 04

00 E

26 E

10 E

23 E






Kcnq1ot1

MEF

Mat

erna

l IC

Rs

CpG

isla

nds

(160

26)

Pat

erna

l IC

Rs

ESES

Peg3Gnas

Peg10

Nespas-Gnasxl

DMR-Zdbf2

Peg1ImpactZac1

Snrpn

Igf2rMeg1Grb10

H19IG-DMR

Rasgrf1


G

B

-25 0 +25 -25 0 +25 -25 0 +25Distance from


C

D

E

Kcnq1Kcnq1ot1

GC KvDMR1

ES

MEF

1504

80 kb

1504

90 kb

chr7

Snrpn-ICRGC

ES

MEF

chr7

Snrpn

6714

5 kb

6715

5 kb

IG-DMRGC

ES

MEF

chr12

Meg3

1107

65 kb

1107

75 kb

InputA A T T G

ChIPH3K4me3

A A T G

ChIP H4R3me2s

A A T G

ChIPH3K9me3

A A T T G

C G G

PaternalC G G

MaternalT G G

MaternalG G

KvDMR1

T G A G

T G A G

T G A G

T G A G

G A T

G A T

G G T

G GTA

Snrpn-ICR

Maternal

Maternal

Paternal

G GC G G G

G GC G GG

GGC G GG

G GC G G G

CT

C

C

T

IG-DMR

Paternal

Paternal

Maternal

T T

T T

T T

T T

T T C T T

T T C T T

T C T T T

T TC

TC

T T

Rasgrf1-ICR

Paternal

Paternal

Maternal

F

Rasgrf1

Rasgrf1-ICRGC

ES

MEF

8978

0 kb

8980

0 kb

chr9










sh-Ctrl sh-Prmt5-1

H4R3m

e2s

H4R3m

e2s

In

put

0

10

20

30

40

50

H4R3m

e2s

Mock

H19 ICR

H4R3m

e2s

Mock

0

10

20

30

40

50

60KvDMR1

Mock

0

10

20

30

40

Rasgrf1

H4R3m

e2s

Mock

010203040506070

Snrpn

Mock

010203040506070

IAP

D

B C

Input

ChIP PRMT5

KvDMR1

Maternal

A A T G G G

A A T T G G G

TC

Snrpn ICR

Maternal

A

T G G A T A G

T G G G T A G

CCNE1

PRMT5ChIP

Mock

KvDMR1

IAP0

002

004

006

008

In

put

A chr738886000-38896000

GC MEF

H4R3me2s

Ccne1

1 kb






H19 ICR

02468

10121416

H3K9m

e3

Mock

H3K9m

e3

Mock

KvDMR1

02468

1012141618 KvDMR1H19 ICR


Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

10

20

30

40

50

60

0

10

20

30

40

50

60

sh-Ctrl sh-Eset-1A

D

F G

B C


PRMT5

ACTB

H4R3me2s

Histone H4

H3K9me3

ESET

ES

0 2 3 4

H3K9m

e3

Mock

H3K9m

e3

Mock

Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

In

put

In

put


PRMT5

ACTB

H4R3me2s

Histone H4

ESET

MEFsh

-Ctrl

sh-E

set-1

H3K9me3

sh-Ctrl sh-Prmt5-1

In

put

H19 ICR

H3K9m

e3

Mock

0

5

10

15

20

25

30

35

H3K9m

e3

Mock

KvDMR1

0

5

10

15

20

25

30

35


In

put

In

put


H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

H19 ICR

H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

KvDMR1

0

05

1

15

2

H4K20

me3Moc

k

H19 ICR

0

05

1

15

2

H4K20

me3Moc

k

KvDMR1




DISCUSSION









SUPPLEMENTARY DATA


ACKNOWLEDGEMENTS


FUNDING



REFERENCES












































































A

E

100 20 30 40 50 60 70 80 90 100

minus50

050

100

G+C

H4R

3me2

s E

nric

hmen

t

ES cells

100 20 30 40 50 60 70 80 90 100

minus10

010

2030

G+C

H4R

3me2

s E

nric

hmen

t

MEF cells

B F

ES cells

-2 -1 0 1 2-10

0

10

20

30

40

50

60

H4R

3me2

sen

richm

ent


246

Log

expr

essi

on

5940

Numberof genes

1096999477014

C

-10

0

10

20

30

40

50

60

-2 -1 0 1 2

H4R

3me2

sen

richm

ent



D


-5

TSS +5

H3K4me3 RNA

-5

TSS +5

H3K27me3

-5

TSS +5

H4R3me2s

-5

TSS +5

Exp

ress

edN

ot e

xpre

ssed

1

2

3

035

0

2

4

6

8

10

040 045 050 055 060 065

Den

sity


H4R

3me2

s en

richm

ent


00

02

04

06

08

00 01 02 03 04

Counts

00

01

02

03

04

05

00 01 02 03 04

00 E

26 E

10 E

23 E






Kcnq1ot1

MEF

Mat

erna

l IC

Rs

CpG

isla

nds

(160

26)

Pat

erna

l IC

Rs

ESES

Peg3Gnas

Peg10

Nespas-Gnasxl

DMR-Zdbf2

Peg1ImpactZac1

Snrpn

Igf2rMeg1Grb10

H19IG-DMR

Rasgrf1


G

B

-25 0 +25 -25 0 +25 -25 0 +25Distance from


C

D

E

Kcnq1Kcnq1ot1

GC KvDMR1

ES

MEF

1504

80 kb

1504

90 kb

chr7

Snrpn-ICRGC

ES

MEF

chr7

Snrpn

6714

5 kb

6715

5 kb

IG-DMRGC

ES

MEF

chr12

Meg3

1107

65 kb

1107

75 kb

InputA A T T G

ChIPH3K4me3

A A T G

ChIP H4R3me2s

A A T G

ChIPH3K9me3

A A T T G

C G G

PaternalC G G

MaternalT G G

MaternalG G

KvDMR1

T G A G

T G A G

T G A G

T G A G

G A T

G A T

G G T

G GTA

Snrpn-ICR

Maternal

Maternal

Paternal

G GC G G G

G GC G GG

GGC G GG

G GC G G G

CT

C

C

T

IG-DMR

Paternal

Paternal

Maternal

T T

T T

T T

T T

T T C T T

T T C T T

T C T T T

T TC

TC

T T

Rasgrf1-ICR

Paternal

Paternal

Maternal

F

Rasgrf1

Rasgrf1-ICRGC

ES

MEF

8978

0 kb

8980

0 kb

chr9










sh-Ctrl sh-Prmt5-1

H4R3m

e2s

H4R3m

e2s

In

put

0

10

20

30

40

50

H4R3m

e2s

Mock

H19 ICR

H4R3m

e2s

Mock

0

10

20

30

40

50

60KvDMR1

Mock

0

10

20

30

40

Rasgrf1

H4R3m

e2s

Mock

010203040506070

Snrpn

Mock

010203040506070

IAP

D

B C

Input

ChIP PRMT5

KvDMR1

Maternal

A A T G G G

A A T T G G G

TC

Snrpn ICR

Maternal

A

T G G A T A G

T G G G T A G

CCNE1

PRMT5ChIP

Mock

KvDMR1

IAP0

002

004

006

008

In

put

A chr738886000-38896000

GC MEF

H4R3me2s

Ccne1

1 kb






H19 ICR

02468

10121416

H3K9m

e3

Mock

H3K9m

e3

Mock

KvDMR1

02468

1012141618 KvDMR1H19 ICR


Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

10

20

30

40

50

60

0

10

20

30

40

50

60

sh-Ctrl sh-Eset-1A

D

F G

B C


PRMT5

ACTB

H4R3me2s

Histone H4

H3K9me3

ESET

ES

0 2 3 4

H3K9m

e3

Mock

H3K9m

e3

Mock

Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

In

put

In

put


PRMT5

ACTB

H4R3me2s

Histone H4

ESET

MEFsh

-Ctrl

sh-E

set-1

H3K9me3

sh-Ctrl sh-Prmt5-1

In

put

H19 ICR

H3K9m

e3

Mock

0

5

10

15

20

25

30

35

H3K9m

e3

Mock

KvDMR1

0

5

10

15

20

25

30

35


In

put

In

put


H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

H19 ICR

H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

KvDMR1

0

05

1

15

2

H4K20

me3Moc

k

H19 ICR

0

05

1

15

2

H4K20

me3Moc

k

KvDMR1




DISCUSSION









SUPPLEMENTARY DATA


ACKNOWLEDGEMENTS


FUNDING



REFERENCES












































































Kcnq1ot1

MEF

Mat

erna

l IC

Rs

CpG

isla

nds

(160

26)

Pat

erna

l IC

Rs

ESES

Peg3Gnas

Peg10

Nespas-Gnasxl

DMR-Zdbf2

Peg1ImpactZac1

Snrpn

Igf2rMeg1Grb10

H19IG-DMR

Rasgrf1


G

B

-25 0 +25 -25 0 +25 -25 0 +25Distance from


C

D

E

Kcnq1Kcnq1ot1

GC KvDMR1

ES

MEF

1504

80 kb

1504

90 kb

chr7

Snrpn-ICRGC

ES

MEF

chr7

Snrpn

6714

5 kb

6715

5 kb

IG-DMRGC

ES

MEF

chr12

Meg3

1107

65 kb

1107

75 kb

InputA A T T G

ChIPH3K4me3

A A T G

ChIP H4R3me2s

A A T G

ChIPH3K9me3

A A T T G

C G G

PaternalC G G

MaternalT G G

MaternalG G

KvDMR1

T G A G

T G A G

T G A G

T G A G

G A T

G A T

G G T

G GTA

Snrpn-ICR

Maternal

Maternal

Paternal

G GC G G G

G GC G GG

GGC G GG

G GC G G G

CT

C

C

T

IG-DMR

Paternal

Paternal

Maternal

T T

T T

T T

T T

T T C T T

T T C T T

T C T T T

T TC

TC

T T

Rasgrf1-ICR

Paternal

Paternal

Maternal

F

Rasgrf1

Rasgrf1-ICRGC

ES

MEF

8978

0 kb

8980

0 kb

chr9










sh-Ctrl sh-Prmt5-1

H4R3m

e2s

H4R3m

e2s

In

put

0

10

20

30

40

50

H4R3m

e2s

Mock

H19 ICR

H4R3m

e2s

Mock

0

10

20

30

40

50

60KvDMR1

Mock

0

10

20

30

40

Rasgrf1

H4R3m

e2s

Mock

010203040506070

Snrpn

Mock

010203040506070

IAP

D

B C

Input

ChIP PRMT5

KvDMR1

Maternal

A A T G G G

A A T T G G G

TC

Snrpn ICR

Maternal

A

T G G A T A G

T G G G T A G

CCNE1

PRMT5ChIP

Mock

KvDMR1

IAP0

002

004

006

008

In

put

A chr738886000-38896000

GC MEF

H4R3me2s

Ccne1

1 kb






H19 ICR

02468

10121416

H3K9m

e3

Mock

H3K9m

e3

Mock

KvDMR1

02468

1012141618 KvDMR1H19 ICR


Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

10

20

30

40

50

60

0

10

20

30

40

50

60

sh-Ctrl sh-Eset-1A

D

F G

B C


PRMT5

ACTB

H4R3me2s

Histone H4

H3K9me3

ESET

ES

0 2 3 4

H3K9m

e3

Mock

H3K9m

e3

Mock

Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

In

put

In

put


PRMT5

ACTB

H4R3me2s

Histone H4

ESET

MEFsh

-Ctrl

sh-E

set-1

H3K9me3

sh-Ctrl sh-Prmt5-1

In

put

H19 ICR

H3K9m

e3

Mock

0

5

10

15

20

25

30

35

H3K9m

e3

Mock

KvDMR1

0

5

10

15

20

25

30

35


In

put

In

put


H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

H19 ICR

H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

KvDMR1

0

05

1

15

2

H4K20

me3Moc

k

H19 ICR

0

05

1

15

2

H4K20

me3Moc

k

KvDMR1




DISCUSSION









SUPPLEMENTARY DATA


ACKNOWLEDGEMENTS


FUNDING



REFERENCES
















































































sh-Ctrl sh-Prmt5-1

H4R3m

e2s

H4R3m

e2s

In

put

0

10

20

30

40

50

H4R3m

e2s

Mock

H19 ICR

H4R3m

e2s

Mock

0

10

20

30

40

50

60KvDMR1

Mock

0

10

20

30

40

Rasgrf1

H4R3m

e2s

Mock

010203040506070

Snrpn

Mock

010203040506070

IAP

D

B C

Input

ChIP PRMT5

KvDMR1

Maternal

A A T G G G

A A T T G G G

TC

Snrpn ICR

Maternal

A

T G G A T A G

T G G G T A G

CCNE1

PRMT5ChIP

Mock

KvDMR1

IAP0

002

004

006

008

In

put

A chr738886000-38896000

GC MEF

H4R3me2s

Ccne1

1 kb






H19 ICR

02468

10121416

H3K9m

e3

Mock

H3K9m

e3

Mock

KvDMR1

02468

1012141618 KvDMR1H19 ICR


Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

10

20

30

40

50

60

0

10

20

30

40

50

60

sh-Ctrl sh-Eset-1A

D

F G

B C


PRMT5

ACTB

H4R3me2s

Histone H4

H3K9me3

ESET

ES

0 2 3 4

H3K9m

e3

Mock

H3K9m

e3

Mock

Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

In

put

In

put


PRMT5

ACTB

H4R3me2s

Histone H4

ESET

MEFsh

-Ctrl

sh-E

set-1

H3K9me3

sh-Ctrl sh-Prmt5-1

In

put

H19 ICR

H3K9m

e3

Mock

0

5

10

15

20

25

30

35

H3K9m

e3

Mock

KvDMR1

0

5

10

15

20

25

30

35


In

put

In

put


H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

H19 ICR

H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

KvDMR1

0

05

1

15

2

H4K20

me3Moc

k

H19 ICR

0

05

1

15

2

H4K20

me3Moc

k

KvDMR1




DISCUSSION









SUPPLEMENTARY DATA


ACKNOWLEDGEMENTS


FUNDING



REFERENCES












































































H19 ICR

02468

10121416

H3K9m

e3

Mock

H3K9m

e3

Mock

KvDMR1

02468

1012141618 KvDMR1H19 ICR


Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

10

20

30

40

50

60

0

10

20

30

40

50

60

sh-Ctrl sh-Eset-1A

D

F G

B C


PRMT5

ACTB

H4R3me2s

Histone H4

H3K9me3

ESET

ES

0 2 3 4

H3K9m

e3

Mock

H3K9m

e3

Mock

Mock

H4R3m

e2s

Mock

H4R3m

e2s

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

In

put

In

put


PRMT5

ACTB

H4R3me2s

Histone H4

ESET

MEFsh

-Ctrl

sh-E

set-1

H3K9me3

sh-Ctrl sh-Prmt5-1

In

put

H19 ICR

H3K9m

e3

Mock

0

5

10

15

20

25

30

35

H3K9m

e3

Mock

KvDMR1

0

5

10

15

20

25

30

35


In

put

In

put


H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

H19 ICR

H4R3m

e2s

0

10

20

30

40

50

60

70

Mock

KvDMR1

0

05

1

15

2

H4K20

me3Moc

k

H19 ICR

0

05

1

15

2

H4K20

me3Moc

k

KvDMR1




DISCUSSION









SUPPLEMENTARY DATA


ACKNOWLEDGEMENTS


FUNDING



REFERENCES










































































DISCUSSION









SUPPLEMENTARY DATA


ACKNOWLEDGEMENTS


FUNDING



REFERENCES











































































SUPPLEMENTARY DATA


ACKNOWLEDGEMENTS


FUNDING



REFERENCES









































































prmt5-mediated histone h4 arginine-3 symmetrical ... · or suv420h1/h2 (kmt5b/c) affected h3k9me3...

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