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Histone arginine methylation regulates pluripotency in the early mouse embryo
Author: Maria-Elena Torres-Padilla, David-Emlyn Parfitt, Tony Kouzarides & Magdalena Zernicka-Goetz
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"LETTERS Histone arginine methylation regulates pluripotency in the early mouse embryo Maria-Elena Torres-Padilla 1 , David-Emlyn Parfitt 1 , Tony Kouzarides 1 & Magdalena Zernicka-Goetz 1 It has been generally accepted that the mammalian embryo starts its development with all cells identical, and only when inside and outside cells form do differences between cells first emerge. However, recent findings show that cells in the mouse embryo can differ in their developmental fate and potency as early as the four-cell stage 1?4 .These differences dependon theorientation and order of the cleavage divisions that generated them 2,5 . Because epigenetic marks are suggested to be involved in sustaining plur- ipotency 6,7 , we considered that such developmental properties might be achieved through epigenetic mechanisms. Here we show that modification of histone H3, through the methylation of spe- cific arginine residues, is correlated with cell fate and potency. LevelsofH3methylationatspecificarginineresiduesaremaximal in four-cell blastomeres that will contribute to the inner cell mass (ICM) and polar trophectoderm and undertake full development when combined together in chimaeras. Arginine methylation of H3 is minimal in cells whose progeny contributes more to the mural trophectoderm and that show compromised development when combined in chimaeras. This suggests that higher levels of H3 arginine methylation predispose blastomeres to contribute to the pluripotent cells of the ICM. We confirm this prediction by overexpressing the H3-specific arginine methyltransferase CARM1 in individual blastomeres and show that this directs their progeny to the ICM and results in a dramatic upregulation of Nanog and Sox2. Thus, our results identify specific histone mod- ifications as the earliest known epigenetic marker contributing to development of ICM and show that manipulation of epigenetic information influences cell fate determination. To address whether epigenetic differences exist between blasto- meresatthefour-cellstage,wefocusedonhistonemethylationmarks relatedtotranscriptional activation 8?10 .Because thedivisionsoftwo- cell-stage blastomeres differ in orientation in relation to the animal? vegetal axis of the egg 5,11 , the shapes of four-cell embryos vary: blas- tomeresfilltheapicesofatetrahedronwhentheyundergooneequat- orial (E) and one meridional (M) division, or they lie on a similar plane when they undergo either two E or two M divisions (Fig. 1a). Although any combination of the temporal sequence of such divi- sions is possible, sequential M and E divisions are most common (about 80%), but they can occur in either order. We found that whereas embryos that underwent either two E or two M divisions did not show significant variations in the levels of H3 methylation at Arg26 (H3R26me) between four-cell-stage blastomeres (Fig. 1a, EEandMM embryos; blue bars), tetrahedral embryos showed marked differences in H3R26me levels between their blastomeres (Fig. 1a, EM and ME embryos; red bars). In the latter, the weakest level of H3R26me was generally less than 40% of the cell giving the strongest signal (P5 0.0002). Measuring the intensity of DNA stain- ing indicated that the variation of H3R26me levels was not related to differences in the content of DNA resulting from replication (not shown).We confirmed thatthis variation in H3R26melevels didnot result from the confocal scanning of embryos of differing shapes by scanningindividualcellsofdisaggregatedembryos,wherewefounda similar outcome (Supplementary Fig. S1). In addressing whether the methylation of other arginine residues also showed differences between four-cell blastomeres, we found that levels of H3R2me andH3R17me(SupplementaryFigsS2andS3a)variedbetweenblas- tomeres correlating with embryo morphology in a similar way to H3R26me. This is consistent with Arg2 and Arg17 being targets for the same methyltransferase, CARM1, as Arg26 (refs 12, 13). When we analysed CARM1 distribution in four-cell blastomeres, we found that CARM1 levels varied with the same tendency as those ofH3R26me(SupplementaryFig.S4b,c).Incontrast,methylationof H4R3, which is the target of a different methyltransferase, protein arginine methyltransferase 1 (PRMT1; refs 14, 15), seemed equival- ent betweenblastomeres regardless ofembryo morphology (Fig. 1b). Thus, the differences in the levels of histone arginine methylation in four-cell-stage blastomeres are specific. We also observed that levels of BrUTP incorporation in late four-cell-stage embryos were highest in the blastomeres that were enriched for H3R26me, indicative of elevated levels of global transcription in these cells (Fig. 1c?e). Because H3R26me showed the biggest difference in its distribution between blastomeres (Supplementary Fig. S3b), we concentrated on analysing this modification further. Becausedifferentdevelopmentalfateandpotentialcanbeascribed to blastomeres depending on the orientation and the order of divi- sionfromthetwo-celltothefour-cellstageinrelationtotheanimal? vegetal axis, we wished to determine whether differences in H3R26merelatedtopatternsofdivision.Tothisendwefirstgrouped embryos according to their cleavage patterns to the four-cell stage. When the earlier of the second cleavages is M and the later E (ME embryos), the earlier dividing two-cell blastomere contributes most of its progeny to the embryonic (ICM and adjacent polar trophecto- derm) and the later one to the abembryonic part (ICM and mural trophectoderm)oftheblastocyst.Whentheearliersecondcleavageis EandthelaterisM(EMembryos),theearlierblastomerecangiverise to either the embryonic or abembryonic part of the blastocyst. By contrast, when second cleavage divisions are of similar orientation (MM or EE), the allocation of blastomere progeny is random. The ME group of embryos thus allowed the identification of individual four-cell blastomeres that have a predictable fate within the blasto- cyst 5 . Moreover, blastomeres resulting from the E division that inherit the ?vegetal? cytoplasm in ME embryos tend to contribute moretothemuraltrophectodermanddonotcompletedevelopment when combined with the same type of ?vegetal? cells in chimaeric embryos 2 . In contrast, chimaeras of blastomeres that arise from early Mdivisions(with?animal?vegetal?cytoplasm)couldcompletedevel- opment with full success, and chimaeras constructed only from ?ani- mal? blastomeres could also develop although with reduced success. 1 The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK. Vol 445|11 January 2007|doi:10.1038/nature05458 214 Nature �2007 Publishing Group Todistinguishtheprogenyoftwo-cell-stageblastomeres,weinjected one two-cell blastomere at random with rhodamine-dextran and then monitored the second cleavage divisions (Supplementary Fig. S5a). Immunostaining then revealed similar levels of H3R26me in the progeny of both E and M divisions of EM embryos (compare M cells with E cells; P5 0.112; Supplementary Fig. S5b, c). In ME embryos, however, the M sister cells had significantly higher levels of H3R26 methylation than both E sisters, with one of the E-derived blastomeres showing significantly less methylation than either of the M-derived blastomeres (P,0.0001; Supplementary Fig. S5b, c). In confirmation of our earlier observations (Fig. 1), EE and MM embryos did not show differences in their relative H3R26me levels. Thus, in ME embryos in which prediction of developmental prop- erties is possible, higher levels of H3R26me are seen in blastomeres expected to contribute more cells to the embryonic part of the blas- tocyst and lower levels in blastomeres expected to contribute to the abembryonic part. Given the reported differences in developmental potential of ?animal? and ?vegetal? blastomeres arising from E divisions in ME embryos 2 , we next examined whether these cells differed in their H3R26me levels. To this end we randomly injected a two-cell-stage blastomerewithrhodamine-dextranasbeforeandthenappliedgreen fluorescentbeadstothevegetalmembranesofthetwoblastomeresas a second label (Fig. 2a and ref. 2). The rhodamine label allowed us to scoretheorderandtheplaneofdivisiontothefour-cell-stageandthe beads served as ?vegetal? markers. We found that in ME embryos, the ?vegetal?blastomerealwayshadsignificantlylowerlevelsofH3R26me than the ?animal? or ?animal/vegetal? blastomeres (P,0.0001; ab Random injection at the late two- cell stage Place beads Score first 2nd division Score second 2nd division 100 ME embryos EM embryos 75 50 25 0 A/V A/V A V 100 75 50 25 0 A/V A/V A V Relative H3R26me fluor escence intensity Figure 2 | The?vegetal?blastomerein MEembryoshasthelowestlevelsof H3R26me. a,Designtodeterminetheidentityoffour-cell-stageblastomeres accordingtodivisionorientation,order,andblastomerepositioning.Atwo- cell-stage blastomere was microinjected with rhodamine-dextran. We then placed a green fluorescent bead in the ?vegetal? membrane of the two blastomeres. Divisions were scoredand embryoswere stainedfor H3R26me at the late four-cell stage. The position of the bead and the presence of rhodamine allowed the identification of the blastomeres as animal/vegetal (A/V,derivedfromMdivisions),oranimalorvegetal(AorV,derivedfromE divisions)inEM(n 510)orME(n 59)embryos.b,Thevegetalblastomere of ME embryos shows the lowest levels of H3R26me, whereas the vegetal blastomere of EM embryos shows similar levels to the animal or animal/ vegetal blastomeres. H3R26me levels were quantified as in Fig. 1. Because there are two A/V blastomeres after one M division there are two columns labelled A/V. Error bars indicate s.d. ab DNA H3R26me DNA DNA BrUTP H3R26me H4R3me MM EE EM and ME MM EE EM and ME 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 1234 0 20 40 60 80 100 1234 Relative fluor escence intensity Relative fluor escence intensity c e d Figure 1 | Levels of H3R26me are different in blastomeres of four-cell- stage embryos and correlate with their spatial arrangement. a, Four-cell- stage embryos were stained for H3R26me and grouped according to their shape:tetrahedral(EMandME),EE(flattened,polarbodyononeside)orMM (flattened, polar body in the middle). Shown are projections, including all sections, of representative embryos. Fluorescence levels were quantified and normalizedagainstthe blastomere showing thehighest level,whichwasset at 100%. Decreasing values of fluorescence were normalized and averaged accordingly(n518).Eachbarrepresentstherelativefluorescencelevelofeach of the four blastomeres: red bars, EM and ME embryos; blue bars, MM or EE embryos. Scale bar, 50mm. b, Differences in histone arginine methylation levelsinfour-cell-stageblastomeresarespecific:onlyresiduesthatareCARM1 targets, and not PRMT1, show differential distribution. The histogram gives resultsforembryosofallshapes.c,Four-cell-stageblastomeresshowdifferent global transcriptional activities. BrUTP incorporation was measured in sections from nuclei of four-cell-stage embryos captured every 0.6mm (n512).ProjectionswereusedaftercroppingoffthenucleiwiththeVolocity softwaretoquantifyactive(nuclear)transcription.Valueswerenormalizedas in a. d, e, Global transcription levels correlate with global H3R26me levels. d, Quantification of BrUTP incorporation (green) and H3R26me (red) of nucleiofeachfour-cellblastomereofarepresentativeembryo.e,Nucleishown at the same scale, numbers at the bottom correspond to the blastomere numbers of the graph in d. Error bars indicate s.d. NATURE|Vol 445|11 January 2007 LETTERS 215 Nature �2007 Publishing Group Fig. 2b). In contrast, in EM embryos the ?animal? and ?vegetal? blas- tomeres from the E division had equivalent levels of H3R26me (Fig. 2b). Taken together, our results indicate that more extensive H3 arginine methylation in M blastomeres of ME embryos is corre- lated with their greater contribution to the embryonic part of the blastocyst. In contrast, blastomeres having the lowest levels of H3 arginine methylation (that is, ?vegetal? blastomeres) are those pre- dicted to contribute mainly to the abembryonic part. Because levels of methylation at Arg2, Arg17 and Arg26 varied similarly and because these three residues are the specific targets of CARM1/PRMT4, which is expressed maternally in mouse embryos (Supplementary Figs S4 and S6), we wondered whether CARM1 WB anti-tub Non-injected CARM1.HA kDa 87 42 Total no. of cells No. of inner (% all cells) Red that are inner (%) Inner positive (% of inner) No. of red cells (% of all cells) No. of outer (% of all cells) Outer positive (% of outer) Red that are outer (%) No. of embryos analysed iv i ix ii iii viv vii viii CARM1(E267Q). HA/DsRed DsRed CARM1.HA/DsRed CARM1.HA/DsRed CARM1.HA/DsRed Top DsRed DsRed DsRed Phase Mer ge Middle Bottom WB anti-HA P<0.0001 P<0.0001 a dc e b Figure 3 | CARM1 overexpression in a two-cell blastomere results in the contribution of that cell predominantly to the ICM. A late two-cell-stage blastomere was injected with mRNA for DsRed alone (control) or in combination with mRNA for CARM1.HA (HA stands for haemagglutinin). This resulted in CARM1 overexpression from the mid-four-cell stage because CARM1.HA/DsRed expression starts 6?8h after injection (not shown). Embryos were cultured until the blastocyst stage and observed under fluorescencemicroscopy.DsRedwasusedasalineagetracer.a,Representative embryos derived from DsRed only (n517) or DsRed/CARM1.HA- overexpression experiments (n535). b, Blastocysts were stained with phalloidin?Texas red and TOTO-3 (to reveal cell membranes and DNA, respectively) and analysed under confocal microscopy. Representative top, middleandbottomsectionsareshown.DNAisinblue;phalloidin(red)canbe distinguished from DsRed because the latter is exclusively cytoplasmic. The red channel is shown as a greyscale. The progeny of the CARM1- overexpressing blastomere is predominantly within the inner cells of the blastocyst. c, Overexpression of CARM1 was verified by western blot (WB) analysis in zygotes injected with mRNA for CARM1.HA/DsRed. d, Representative three-dimensional reconstructions of blastocysts in which mRNA for CARM1.HA/DsRed (i), Ds/Red only (iv) or CARM1(E267Q).HA/ DsRed(vii)wasmicroinjectedatthetwo-cellstage.Blastocystswerestainedas inb.Confocalz-stacksweretakenat1-mmintervals.IMARISsoftwarewasused tooutlinecellmembranestocreatethree-dimensionalmodelsofallcellsofthe embryo. Cells were then scored according to their position: cells completely surrounded by others are denoted as inner, those with an outer surface as outer. Cells were scored as either positive or negative for DsRed. Progeny of injected blastomere is shown in red. A middle slice is shown in ii, v and viii, wherethecavityisdepictedwithaline.Iniii,viandix,onlytheprogenyofthe injected blastomere is shown; the contour of the embryo is indicated by a dashed line and the position of the cavity by a solid line. Scale bar, 10mm. e, Analysis of the distribution of the progeny of CARM1- and DsRed- overexpressing blastomere at the blastocyst stage. Results are means6s.d. LETTERS NATURE|Vol 445|11 January 2007 216 Nature �2007 Publishing Group might have a function in directing developmental fate and potency. Totestthis,weinjectedCARM1mRNAintosinglelatetwo-cell-stage blastomeres aiming to elevate levels of arginine methylation in H3 in the progeny of these cells from the mid-four-cell stage. We followed the effect on cell fate by co-injecting mRNA for DsRed as a lineage tracer (Fig. 3a, b). Strikingly, the labelled clone was located in the embryonic part of the blastocyst in 31 out of 35 embryos (89%), and in no embryos were labelled cells found exclusively in the abembryo- nic part. We randomly selected ten of these embryos for reconstruc- tion in three dimensions to locate every cell and determine to which lineage (ICM or trophectoderm) labelled cells had contributed. This showedthattheICMcomprised37%ofallcellsatthisstageandthat, on average, 88.5% of ICM cells were derived from the blastomere in which CARM1 had been overexpressed (Fig. 3d, e, and Sup- plementary Table S1). Interestingly, in half of the embryos, all of the ICM comprised the exclusive progeny of the CARM1-over- expressing blastomere. By contrast, even though most (63%) of the blastocyst cells are outer cells, the proportion of outer cells derived from the CARM1-overexpressing blastomere was only 12% (Fig. 3e, and Supplementary Table S1). This was in contrast with control embryos, injected with mRNA for DsRed only, in which we found that 41% and 59% of cells labelled with the lineage tracer were inner and outer cells, respectively (P,0.0001; Fig. 3e, and Supplementary Table S2). We therefore conclude that forced overexpression of CARM1 in a two-cell blastomere leads that cell to contribute pre- dominantly (if not exclusively) to the ICM. To establish whether the methyltransferase activity of CARM1 was required for this effect, we injected mRNA for CARM1 containing a point mutation (E267Q) and devoid of catalytic activity 16 into single two-cell blastomeres as above. We found that the progeny of the labelled cells could contrib- ute to both embryonic and abembryonic regions of the blastocyst. The distribution of the progeny of the blastomere injected with CARM1(E267Q) was similar to that of the DsRed control (Fig. 3d, e, and Supplementary Table S3). The progeny of the CARM1-over- expressing blastomereshowedincreasedlevelsofH3R26me,whereas blastomeres overexpressing CARM1(E267Q) did not (Fig. 4a, b). Hence, the ability of CARM1 to direct the progeny of a blastomere towardstheICMisstrictlydependentonitsmethyltransferaseactivity. We next assessed expression levels of transcription factors known to influence the development of ICM cells. We found that over- expression of CARM1 led to an early and marked upregulation of Nanog in the injected blastomeres, suggesting that the Nanog pro- moter is regulated by arginine methylation (Fig. 4c). In contrast, Cdx2, a trophectoderm marker, showed no induction after over- expressionofCARM1(notshown).Wealsodetectedincreasedlevels of Sox2 in the progeny of the CARM1-injected blastomere (Fig. 4c). However, Oct4/Pou5f1 showed variable levels of expression in non- injected blastomeres as well as in cells overexpressing CARM1 (Supplementary Fig. S7). Note that, in contrast with Nanog, both Oct4/Pou5f1 and Sox2 were also present in the non-injected blasto- meres, reflecting an earlier expression and/or their maternal inher- itance 17,18 . The co-expression by the blastocyst stage of the ICM markers Oct4/Pou5f1 and Nanog in the progeny of the CARM1- overexpressing blastomere is consistent with the observed change in cell fate (Fig. 4d). Hence, by manipulating epigenetic information through the overexpression of a histone modifier it is possible to direct cells towards the ICM. Our findings, in control experiments, that either of the two-cell blastomeresnormallycontributesitsprogenytobothinnerandouter cells of the blastocyst are in accordance with our earlier findings 1,2,4,5 . However, they contrast with a report that shows that as a con- sequence of differential Cdx2 levels between two-cell blastomeres, oneblastomerecontributesexclusivelytooutercells,whichnormally express Cdx2 (ref. 19). Unlike those authors we do not observe expression of Cdx2 at the two-cell stage. Ourstudydemonstratesthatepigeneticdifferencesdevelopbetween blastomeres by the four-cell stage. We cannot exclude the possibility that CARM1 mediates some of its effects through targets other than histoneH3.However,thedifferentialcellularlevelsofH3methylation within four-cell blastomeres can account for their different cell fates and potencies 1,4,5 . Cells with more extensive H3 arginine methylation aredestinedtocontributepluripotentprogenytotheblastocyst.These cellsshowincreasedlevelsoftranscription,includingtheexpressionof a select set of genes responsible for maintaining pluripotency such as Nanog and Sox2 (refs 18, 20, 21). An enrichment in modifications characteristicofeuchromatinmayhaveadditionaleffectsonthechro- matin to generate an ?open? configuration that could sustain pluripo- tency in the embryo, as has been suggested for ES cells 22 . Indeed, the relative importance of potential changes in chromatin structure in relation to cell plasticity demands further study. a cd b Non -injecte d Non -inje cte d CARM1.HA C ARM1. (E267Q).H A H3R26me fluor escence r e lative to non-injected cells Nanog Nanog/DsRed Oct4/DsRed Figure 4 | Overexpression of CARM1 results in elevated levels of arginine methylation and upregulation of Nanog and Sox2. a, A two-cell-stage blastomere was injected with mRNA for CARM1.HA/DsRed. Embryos were cultured to the eight-cell stage and stained for H3R26me. Shown are three nuclei of cells from a representative embryo. The progeny of the injected blastomere is indicated by arrows (note the presence of DsRed). For overexpressionoftheCARM1(E267Q).HAmutant,fivecellsfromthesame embryo are shown (n 56). PB, polar body. b, Quantification of H3R26me levelsincellsoverexpressingCARM1werenormalizedagainstthoseofnon- injectedcellswithinthesameembryo(asterisk,P 5 0.0006;n 5 14).Bottom: data derived from overexpression of CARM1(E267Q).HA. Error bars indicates.d.c,Embryoswereinjectedasinaandstainedwithananti-Nanog (n 5 5)orananti-Sox2(n 5 11)antibodybetweenthesix-cellandtheeight- cell stage. For Nanog, four nuclei from the same embryo, two of them deriving from the CARM1-overexpressing blastomere, are shown (white arrows; note the presence of DsRed). Nanog is detectable only in the blastomeres deriving from the two-cell-stage blastomere injected with CARM1mRNA.ForSox2,arepresentativeembryoisshown.NotethatSox2 is mainly cytoplasmic at this stage 18 . We were unable to address CARM1 functionbytheconverseexperimentbyRNA-mediatedinterferencebecause the protein is provided maternally and its mRNA is rapidly downregulated after fertilization. However, treatment of zygotes with specific arginine methyltransferase inhibitors 25 showed that decreasing the level of histone argininemethylationimpaireddevelopment(SupplementaryFig.S8).d,The progeny of a CARM1-overexpressing blastomere expresses ICM markers in the blastocyst. Blastocysts were stained for Oct4/Pou5f1 (n 5 7) or Nanog (n 5 3; green). The presence of DsRed indicates the progeny of the injected blastomere. DNA is shown in blue. NATURE|Vol 445|11 January 2007 LETTERS 217 Nature �2007 Publishing Group METHODS Embryos were collected from F 1 crosses (C57BL/6XCBA/H). To monitor the divi- sion from the two-cell stage, one blastomere was microinjected randomly with dextran-tetramethylrhodamine (3kDa; Molecular Probes). Green fluorescent beads were placed in the membrane of blastomeres with the use of a Piezo driller. Embryoswereobservedevery20mintodeterminetheplaneandorderofdivision 5 . Immunostaining and BrUTP labelling were performed as described 23 .Anti-H3 asymmetric-dimethyl-Arg2, anti-H3 dimethyl-Arg17, anti-H3 dimethyl-Arg26 and anti-H4 symmetric-dimethyl-Arg3 antibodies were from Abcam (Sup- plementary Fig. S9); anti-Oct4 was from R&D Systems; and anti-Nanog and anti-Sox2werefromSantaCruz.Foranalysisoffour-cellembryos,confocalsections weretakenevery0.8mmthroughthewholeembryoandthefluorescencesignalwas measured in projections with the Volocity software (Improvision). For three- dimensional reconstructions, blastocysts were stained with phalloidin?Texas red and TOTO-3. Confocal sections were captured every 1mm and processed with IMARIS (Bitplane) and 3DVirtual Embryo software 24 . Received 9 August; accepted 17 November 2006. 1. Piotrowska, K., Wianny, F., Pedersen, R. A. & Zernicka-Goetz, M. Blastomeres arisingfromthefirstcleavagedivisionhavedistinguishablefatesinnormalmouse development. Development 128, 3739?3748 (2001). 2. Piotrowska-Nitsche, K., Perea-Gomez, A., Haraguchi, S. & Zernicka-Goetz, M. Four-cell stage mouse blastomeres have different developmental properties. Development 132, 479?490 (2005). 3. Gardner, R. L. Specification of embryonic axes begins before cleavage in normal mouse development. Development 128, 839?847 (2001). 4. Fujimori, T., Kurotaki, Y., Miyazaki, J. & Nabeshima, Y. Analysis of cell lineage in two- and four-cell mouse embryos. Development 130, 5113?5122 (2003). 5. Piotrowska-Nitsche, K. & Zernicka-Goetz, M. Spatial arrangement of individual 4-cellstageblastomeresandtheorderinwhichtheyaregeneratedcorrelatewith blastocyst pattern in the mouse embryo. Mech. Dev. 122, 487?500 (2005). 6. Li, E. Chromatin modification and epigenetic reprogramming in mammalian development. Nature Rev. Genet. 3, 662?673 (2002). 7. Morgan, H. D., Santos, F., Green, K., Dean, W. & Reik, W. Epigenetic reprogramminginmammals.Hum.Mol. Genet.14(Spec.Iss.1),R47?R58(2005). 8. Chen, D. et al. Regulation of transcription by a protein methyltransferase. Science 284, 2174?2177 (1999). 9. Ma,H.etal.Hormone-dependent,CARM1-directed,arginine-specificmethylationof histone H3 on a steroid-regulated promoter. Curr. Biol. 11, 1981?1985 (2001). 10. Bauer,U.M.,Daujat,S.,Nielsen,S.J.,Nightingale,K.&Kouzarides,T.Methylation at arginine 17 of histone H3 is linked to gene activation. EMBO Rep. 3, 39?44 (2002). 11. Gardner, R. L. Experimental analysis of second cleavage in the mouse. Hum. Reprod. 17, 3178?3189 (2002). 12. Bannister, A. J. & Kouzarides, T. Reversing histone methylation. Nature 436, 1103?1106 (2005). 13. Schurter,B.T. et al. MethylationofhistoneH3bycoactivator-associatedarginine methyltransferase 1. Biochemistry 40, 5747?5756 (2001). 14. Wang, H. et al. Methylation of histone H4 at arginine 3 facilitating transcriptional activation by nuclear hormone receptor. Science 293, 853?857 (2001). 15. Strahl, B. D. et al. Methylation of histone H4 at arginine 3 occurs in vivo and is mediated by the nuclear receptor coactivator PRMT1. Curr. Biol. 11, 996?1000 (2001). 16. Lee, Y. H., Koh, S. S., Zhang, X., Cheng, X. & Stallcup, M. R. Synergy among nuclear receptor coactivators: selective requirement for protein methyltransferase and acetyltransferase activities. Mol. Cell. Biol. 22, 3621?3632 (2002). 17. Scholer, H. R., Hatzopoulos, A. K., Balling, R., Suzuki, N. & Gruss, P. A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor. EMBO J. 8, 2543?2550 (1989). 18. Avilion, A. A. et al. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17, 126?140 (2003). 19. Deb, K., Sivaguru, M., Yong, H. Y. & Roberts, R. M. Cdx2 gene expression and trophectoderm lineage specification in mouse embryos. Science 311, 992?996 (2006). 20. Mitsui, K. et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631?642 (2003). 21. Chambers, I. et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643?655 (2003). 22. Meshorer, E. et al. Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells. Dev. Cell 10, 105?116 (2006). 23. Torres-Padilla, M. E. & Zernicka-Goetz, M. Role of TIF1a as a modulator of embryonic transcription in the mouse zygote. J. Cell Biol. 174, 329?338 (2006). 24. Tassy, O., Daian, F., Hudson, C., Bertrand, V. & Lemaire, P. A quantitative approach to the study of cell shapes and interactions during early chordate embryogenesis. Curr. Biol. 16, 345?358 (2006). 25. Cheng, D. et al. Small moleculeregulators ofprotein arginine methyltransferases. J. Biol. Chem. 279, 23892?23899 (2004). Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements We thank P. Greda for bead labelling, C. Lee for assistance, D. Glover for comments on the manuscript, M. Stallcup for the CARM1 expression vectors, and M. Bedford for providing the Carm1 2/2 MEFs and the PRMT inhibitor. M.-E.T.-P. is an EMBO long-term fellow. We are grateful to the Wellcome Trust Senior Fellowship to M.Z.-G., which supported this work. Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to M.Z.-G. (mzg@mole.bio.cam.ac.uk). LETTERS NATURE|Vol 445|11 January 2007 218 Nature �2007 Publishing Group "
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