A mono-allelic bivalent chromatin domain controls tissue-specific imprinting at Grb10
2008; Springer Nature; Volume: 27; Issue: 19 Linguagem: Inglês
10.1038/emboj.2008.142
ISSN1460-2075
AutoresLionel A. Sanz, Stormy J. Chamberlain, Jean-Charles Sabourin, Amandine Henckel, Terry Magnuson, Jean-Philippe Hugnot, Robert Feil, Philippe Arnaúd,
Tópico(s)Prenatal Screening and Diagnostics
ResumoArticle24 July 2008free access A mono-allelic bivalent chromatin domain controls tissue-specific imprinting at Grb10 Lionel A Sanz Lionel A Sanz Institute of Molecular Genetics, CNRS UMR-5535 and University of Montpellier II, Montpellier, France Search for more papers by this author Stormy Chamberlain Stormy Chamberlain Department of Genetics, Carolina Center for the Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Jean-Charles Sabourin Jean-Charles Sabourin INSERM U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital St Eloi, Montpellier, France Search for more papers by this author Amandine Henckel Amandine Henckel Institute of Molecular Genetics, CNRS UMR-5535 and University of Montpellier II, Montpellier, France Search for more papers by this author Terry Magnuson Terry Magnuson Department of Genetics, Carolina Center for the Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Jean-Philippe Hugnot Jean-Philippe Hugnot INSERM U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital St Eloi, Montpellier, France Search for more papers by this author Robert Feil Corresponding Author Robert Feil Institute of Molecular Genetics, CNRS UMR-5535 and University of Montpellier II, Montpellier, France Search for more papers by this author Philippe Arnaud Corresponding Author Philippe Arnaud Institute of Molecular Genetics, CNRS UMR-5535 and University of Montpellier II, Montpellier, France Search for more papers by this author Lionel A Sanz Lionel A Sanz Institute of Molecular Genetics, CNRS UMR-5535 and University of Montpellier II, Montpellier, France Search for more papers by this author Stormy Chamberlain Stormy Chamberlain Department of Genetics, Carolina Center for the Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Jean-Charles Sabourin Jean-Charles Sabourin INSERM U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital St Eloi, Montpellier, France Search for more papers by this author Amandine Henckel Amandine Henckel Institute of Molecular Genetics, CNRS UMR-5535 and University of Montpellier II, Montpellier, France Search for more papers by this author Terry Magnuson Terry Magnuson Department of Genetics, Carolina Center for the Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Jean-Philippe Hugnot Jean-Philippe Hugnot INSERM U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital St Eloi, Montpellier, France Search for more papers by this author Robert Feil Corresponding Author Robert Feil Institute of Molecular Genetics, CNRS UMR-5535 and University of Montpellier II, Montpellier, France Search for more papers by this author Philippe Arnaud Corresponding Author Philippe Arnaud Institute of Molecular Genetics, CNRS UMR-5535 and University of Montpellier II, Montpellier, France Search for more papers by this author Author Information Lionel A Sanz1, Stormy Chamberlain2,‡, Jean-Charles Sabourin3,‡, Amandine Henckel1, Terry Magnuson2, Jean-Philippe Hugnot3, Robert Feil 1 and Philippe Arnaud 1 1Institute of Molecular Genetics, CNRS UMR-5535 and University of Montpellier II, Montpellier, France 2Department of Genetics, Carolina Center for the Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 3INSERM U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital St Eloi, Montpellier, France ‡These authors contributed equally to this work *Corresponding authors: Institute of Molecular Genetics, CNRS UMR-5535 and University of Montpellier II, 1919 route de Mende, Montpellier 34293, France. Tel.: +33 4 6761 3661; Fax: +33 4 6704 0231; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2008)27:2523-2532https://doi.org/10.1038/emboj.2008.142 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Genomic imprinting is a developmental mechanism that mediates parent-of-origin-specific expression in a subset of genes. How the tissue specificity of imprinted gene expression is controlled remains poorly understood. As a model to address this question, we studied Grb10, a gene that displays brain-specific expression from the paternal chromosome. Here, we show in the mouse that the paternal promoter region is marked by allelic bivalent chromatin enriched in both H3K4me2 and H3K27me3, from early embryonic stages onwards. This is maintained in all somatic tissues, but brain. The bivalent domain is resolved upon neural commitment, during the developmental window in which paternal expression is activated. Our data indicate that bivalent chromatin, in combination with neuronal factors, controls the paternal expression of Grb10 in brain. This finding highlights a novel mechanism to control tissue-specific imprinting. Introduction Genomic imprinting is a form of non-Mendelian inheritance in mammals, whereby some genes are expressed from a single allele, depending on whether it is inherited from the mother or the father. Many of the 80 imprinted genes discovered so far in humans and mice are involved in the regulation of cellular proliferation and growth, whereas others have key functions in neurological processes and behaviour (Smith et al, 2006; Wilkinson et al, 2007). Not surprisingly, therefore, deregulation of imprinting gives rise to abnormal development and is causally involved in a number of growth and behavioural syndromes in humans (Arnaud and Feil, 2005). To achieve allele-specific expression, imprinted loci are regulated by epigenetic modifications that differentially mark the parental alleles as either active or repressed. This is manifest at key control elements known as imprinting control regions (ICRs), which mediate imprinted gene expression. All known ICRs are marked by DNA methylation on either their maternal or their paternal allele. This allelic methylation is established in the female or male germ line and is subsequently maintained throughout development (Delaval and Feil, 2004). In addition to DNA methylation, ICRs are also marked by differential histone modifications in somatic cells. The allele harbouring DNA methylation is associated with 'repressive' histone marks such as trimethylation of lysine 9 of histone H3 (H3K9me3), whereas the unmethylated allele is marked by 'permissive' marks such as H3K4me2 (Fournier et al, 2002; Yang et al, 2003; Delaval et al, 2007). DNA methylation at ICRs is essential for orchestrating mono-allelic gene expression. However, the precise role of histone tail modifications in this process remains poorly understood. One possibility is that histone modifications could be important for the ICR-mediated repression of imprinted domains. It is well documented that these modifications can influence gene expression (Margueron et al, 2005; Li et al, 2007). Thus, their allelic distribution at imprinted loci could be involved in regulating mono-allelic expression. Recent studies of tissue-specific imprinting at the Igf2r, NDN and Gnas loci (Yang et al, 2003; Lau et al, 2004; Li et al, 2004) have correlated imprinted expression with tissue-specific differences in histone modifications. A more direct relationship between histone modifications and tissue-specific imprinting exists at the imprinted Kcnq1 domain in the mice, where a maternally methylated ICR controls a number of paternally repressed genes, some of which are imprinted in the placenta only. Across the Kcnq1 domain, the repressed paternal allele is enriched in repressive marks (H3K9me2 and H3K27me3), especially in the placenta (Umlauf et al, 2004). A functional role for these marks is suggested by studies showing that placenta-specific imprinting at Kcnq1 domain is perturbed in mouse conceptuses deficient in G9A, a H3K9me2 methyltransferase (Wagschal et al, 2008), or in EED (embryonic ectoderm development), a component of the PRC2 complex that mediates H3K27 methylation (Mager et al, 2003). The growth factor receptor-binding protein 10, GRB10, is a potent growth inhibitor (Charalambous et al, 2003). We and others have shown that the mouse Grb10 gene displays a tissue-specific reciprocal imprinting pattern based on the use of tissue-specific promoters (Arnaud et al, 2003; Hikichi et al, 2003). In most tissues, Grb10 is expressed from the maternal allele, whereas in brain, its expression is mainly from the paternal allele. Strikingly, the maternal and paternal expressions initiate from different promoter regions. DNA methylation has been shown to be important in the imprinting of Grb10 (Arnaud et al, 2006). This agrees with the current model to explain the maternal Grb10 expression that proposes the existence of a methylation-sensitive boundary mechanism that prevents promoter–enhancer interactions (Arnaud et al, 2003; Hikichi et al, 2003). However, this model does not account for the brain-specific paternal expression where other epigenetic modifications are likely to be involved. To investigate whether histone modifications could be involved, we compared their distribution across key chromosomal regions of Grb10 in brain and several other tissues. We focused our analysis on the CpG island (CGI) region that corresponds to the putative ICR and comprises brain-specific paternal promoters. We identified a mono-allelic bivalent chromatin domain that carries both active (H3K4me2) and repressive (H3K27me3) histone marks and that controls paternal expression of Grb10. The loss of this bivalent chromatin, together with neuronal-specific factors, is important to de-repress Grb10 expression in brain. Bi-allelic bivalent chromatin has been observed at the promoters of many genes in embryonic stem (ES) cells as well as in differentiated cells (Azuara et al, 2006; Bernstein et al, 2006; Roh et al, 2006; Pan et al, 2007; Mikkelsen et al, 2007; Zhao et al, 2007), and has been considered to be involved in maintaining key developmental genes in a 'permissive' chromatin configuration, while being repressed by H3K27me3 (Azuara et al, 2006; Bernstein et al, 2006). Our work shows that bivalent chromatin is also important in the control of imprinted gene expression. Furthermore, our data provide one of the first examples of the implication of bivalent chromatin in the control of gene expression in a differentiated tissue. Results Paternal enrichment for H3K27me3 on the brain-specific paternal promoters is lost in brain We previously established that in neonatal tissues, the three CGIs located in the 5′ untranslated region (UTR) of Grb10 display reciprocally imprinted and tissue-specific promoter activities (Arnaud et al, 2003). Here, we extended and confirmed this analysis by using the material obtained from reciprocal crosses between C57BL/6J (B6) and Mus musculus molossinus JF1 mice. Maternal expression was detected in all tissues and developmental stages analysed, except in brain, and initiates from exon 1A located in CGI1. By contrast, the brain-specific paternal expression arises from three independent regions: exon 1C in CGI3 and exons 1B1 and 1B2, immediately upstream and at the 3′ end of CGI2 (Figure 1A; Supplementary Figures 1 and 2A). Further analysis of RNA from glial cells and a neuron-enriched fraction of cells derived from embryonic cerebral cortices showed that the paternal expression from exons 1B2 (Yamasaki-Ishizaki et al, 2007) and 1B1 (this study) is restricted to neuronal cells (Supplementary Figure 1D). Figure 1.Paternal enrichment of H3K27me3 at CGI2 is lost in brain. (A) Overview of tissue-specific imprinted expression of Grb10. The upper panel gives a schematic representation of the Grb10 gene. Untranslated and coding exons are shown in grey and black, respectively. The 5′ UTR region encompassing the three CpG islands (CGI, CGI3 and CGI2) is drawn to scale. CGI1 is located roughly 70 kb upstream of the first coding exon; CGI2 10 kb downstream of CGI1 and CGI3 8.5 kb downstream of CGI1. Maternal expression arises from exon 1A within CGI1, whereas brain-specific paternal expression arises from exons 1C, 1B1 and 1B2 located in CGI3 and CGI2. (B) Representative results of allele-specific analysis of histone marks at CGI2. Native ChIP followed by SSCP-mediated allelic discrimination in the antibody-bound (B) and unbound (U) fractions was performed on the material from (B6 × JF1)F1 mice (indicated on the left). Maternal (mat.) and paternal (pat.) alleles are indicated. The relative position of the SSCP polymorphism at CGI2 is shown (black star). (C) Relative enrichment of the paternal versus maternal allele in chromatin precipitated with anti-H3K27me3 and anti-H3K9ac antisera, respectively. For each tissue, the value is reported as the mean of the results obtained from at least two independent ChIP experiments (n) normalized to the allelic results obtained in the corresponding input chromatin. Brain (n=3); neonatal brain (n=3); 12.5 d.p.c. embryo (n=4); 12.5 d.p.c. placenta (n=2); liver (n=4); kidney (n=5). Download figure Download PowerPoint We previously observed brain-specific hypomethylation of CGI3 on the paternal allele (Arnaud et al, 2003), suggesting that DNA methylation is involved in controlling the expression from promoter 1C. However, the regulation of Grb10 paternal expression cannot be explained solely by DNA methylation. Indeed, DNA methylation pattern does not vary at the brain-specific paternal promoters 1B1 and 1B2 between brain and other tissues. The putative ICR CGI2, which comprises 1B1 and 1B2, maintains its maternal methylation in all tissues and developmental stages analysed (Arnaud et al, 2003; Hikichi et al, 2003; Yamasaki-Ishizaki et al, 2007). Therefore, we focused our analysis on histone tail modifications at CGI2. Particularly, we wished to determine whether allelic histone modification patterns could vary between brain and other tissues. To this purpose, we performed chromatin immunoprecipitation (ChIP), followed by electrophoretic detection of single strand conformational polymorphism (SSCP) to evaluate for each antibody-bound and unbound fractions the relative abundance of the maternal and paternal alleles. ChIP was performed with antisera against permissive (H3K9ac and H3K4me2) and repressive (H3K27me3, H3K9me3 and H4K20me3) marks. In all tissues and developmental stages analysed, we observed constitutive allelic enrichment of specific histone modifications. H3K4me2 was always associated with the unmethylated paternal allele of CGI2, whereas H3K9me3 and H4K20me3 were consistently associated with the DNA-methylated maternal allele (Figure 1B, not shown). By contrast H3K9ac, which marks active chromatin (Jenuwein and Allis, 2001), was found to be specifically associated with the paternal allele in brain and, to a lesser extent, in 12.5 days post-coitum (d.p.c.) embryos in which paternal expression arising from the developing brain is detected (not shown) (Figure 1B and C). A similar distribution was observed for H3K27ac, with no allelic enrichment in adult liver and kidney and a clear paternal enrichment in brain (Supplementary Figure 3A). Unexpectedly, we observed that the repressive mark H3K27me3 was enriched on the unmethylated paternal allele of CGI2 (Figure 1B). This observation suggested that both the repressive H3K27me3 and the permissive H3K4me2 coexisted on the paternal allele of CGI2. Moreover, paternal H3K27me3 enrichment was specifically lost in brain (Figure 1B and C, not shown). Indeed, relative H3K27me3 paternal enrichment in placenta and kidney (3.29±0.56 and 3.20±0.35, respectively) was much higher than in adult (1.03±0.05) and neonatal brain (1.26±0.04) (Figure 1C). These observations indicated that at CGI2, the paternal enrichment in H3K27me3 correlates with repression of this promoter region, suggesting a role for this modification in the regulation of the paternal Grb10 expression. Allele-specific bivalent 'H3K4me2/H3K27me3' chromatin marks CGI2 The finding that the permissive H3K4me2 and the repressive H3K27me3 marks are both enriched on the paternal allele of CGI2 is reminiscent of the bivalent chromatin domains recently described in ES cells (Azuara et al, 2006; Bernstein et al, 2006). Accordingly, we established that the allelic chromatin pattern across CGI2 in undifferentiated ES cells (line SF1-G) was also characterized by paternal co-enrichment of H3K4me2 and H3K27me3 (Figure 2A). This chromatin pattern was also observed in undifferentiated trophoblast stem (TS) cells (Supplementary Figure 3B). To firmly establish that CGI2 is mono-allelically marked by bivalent chromatin in differentiated cells, we performed sequential ChIP on mouse embryonic fibroblasts (MEFs). In this approach, a first round of ChIP precipitated chromatin fragments enriched in H3K4me2. These fragments were subsequently precipitated with antibodies against H3K27me3. This sequential round of immunoprecipitation retains only chromatin fragments that carry both the histone modifications. To validate this approach, we included the Irx2 and Tcf4 genes as controls. The promoter region of Irx2 has a H3K4me3/H3K27me3 bivalent chromatin in ES cells (Bernstein et al, 2006; Azuara et al, 2006) that is maintained in MEFs (Mikkelsen et al, 2007; http://www.broad.mit.edu/seq_platform/chip/). The detection of the Irx2 promoter in the final bound fractions was consistent with these observations (Figure 2B). Conversely, the Tcf4 promoter is known to be enriched in H3K4me3 only in MEFs (Mikkelsen et al, 2007; http://www.broad.mit.edu/seq_platform/chip/), and could be PCR amplified only after the first round of ChIP with the anti-H3K4me2 antibody (Figure 2B). Analysis performed on the CGI2 region demonstrated that both the modifications were present on the same chromatin fragments (Figure 2B), indicating the presence of a bivalent chromatin in this region in somatic cells. Figure 2.CGI2 is specifically marked by an H3K4me2/H3K27me3 bivalent domain. (A) H3K4me2 and H3K27me3 are enriched on the paternal allele of CGI2 in undifferentiated ES cells. Analysis was performed on (C57BL/6J × M. spretus) F1 undifferentiated ES cells. (B) H3K4me2 and H3K27me3 are enriched on the same chromatin fragments at the CGI2 region. Sequential ChIP was performed on MEFs to detect chromatin fragments enriched for both H3K4me2 and H3K27me3. Irx2 was used as a positive and Tcf4 as a negative control. (C) The H3K4me2/H3K27me3 bivalent domain is restricted to CGI2. Allelic analysis of histone modifications in kidney and brain at CGI1, CGI3 and a non-CpG-rich region located upstream of the transcriptional unit (region 'UR'). Kidney data are shown as an example of representative results obtained with 'non-brain tissues'. At the three regions, H3K27me3, but not H3K4me2, was enriched on the paternal allele. This paternal enrichment is lost in brain tissue. Relative positions of polymorphisms used for the SSCP-based allelic discrimination are shown (black stars). TSS: transcriptional start site. Download figure Download PowerPoint To evaluate the extent of this bivalent chromatin domain, we analysed chromatin at CGI1, CGI3 and at a third region upstream of the transcriptional unit, which we called UR, for upstream region (Figure 2C). At all three regions, in 'non-brain-tissues' H3K27me3 was associated with the paternal allele. However, H3K4me2 was enriched only on the maternal CGI1 allele and displayed no allelic enrichment at CGI3. This finding shows that the bivalent domain observed in non-brain tissues does not extend beyond the CGI2 region. We completed these observations by consulting an online ChIP database (Mikkelsen et al, 2007; http://www.broad.mit.edu/seq_platform/chip/). Chromatin patterns obtained from ES and MEF cells indicate that the entire Grb10 5′ UTR region is enriched in H3K27me3, whereas H3K4me3 is more specifically localized in CGI2 and CGI1 (Supplementary Figure 4). This information together with our allelic analysis establish that the paternal allele of the Grb10 5′ UTR region is enriched in H3K27me3 and that at CGI2, this mark is specifically associated with H3K4me2/me3, thus forming a bivalent chromatin domain. During neural precursor cell differentiation the CGI2 bivalent chromatin domain is resolved and Grb10 paternal expression is induced To evaluate how the CGI2 bivalent chromatin evolves during nervous system development and to explore its effects on Grb10 imprinted expression, we used cultures of highly enriched embryonic neural precursor cells called neurospheres (Reynolds and Weiss, 1992). Neurospheres are generated in suspension by clonal expansion of neural stem cells and are composed of a mixture of neural stem–progenitor cells with little or no differentiated cells (Deleyrolle et al, 2006; Supplementary Figure 5A). Upon growth factor withdrawal in adherent culture conditions, neurospheres generated about 70% astrocytic cells (GFAP+), 10–20% neurons (β-3-tubulin+) and approximately 1% oligodendrocytes (O4+) (Supplementary Figure 5B; Deleyrolle et al, 2006). By exon-specific RT–PCR amplification coupled with sequencing, we revealed that in undifferentiated embryonic neurospheres, Grb10 was maternally expressed from exon 1A (Figure 3A; Supplementary Figure 2B). This expression pattern correlated with the presence of bivalent H3K4me2/H3K27me3 chromatin on the paternal CGI2 allele (Figure 3B, upper panel). Upon differentiation, we detected both maternal expression from exon 1A and paternal expression from exon 1B1 (Figure 3A; Supplementary Figure 2B). The bivalent chromatin was almost resolved, as the H3K27me3 enrichment on the paternal allele was largely reduced, whereas H3K4me2 remained unchanged (Figure 3B). To evaluate whether this relative decrease in paternal H3K27me3 was due to the loss of H3K27me3, we determined its abundance by real-time PCR amplification after ChIP. A decrease of nearly 75% was detected at CGI2 upon neurosphere differentiation (Figure 3C). A similar decrease was also observed at CGI1 (Supplementary Figure 5D). Paternal enrichment of H3K9ac was detected in both undifferentiated and differentiated neuronal cells. However, the amount of this modification at CGI2 clearly increased upon differentiation (Figure 3C), suggesting that the allelic enrichment observed in undifferentiated neurospheres arises from a limited subset of cells. We observed a similar pattern for H3K27ac (Supplementary Figure 5C). Thus, during neural stem/progenitor cell differentiation, the CGI2 bivalent domain is almost completely resolved through a decrease of H3K27me3 whereas acetylation increases. This chromatin change is concomitant with the induction of Grb10 paternal expression from exon 1B1 (Figure 3A). Figure 3.Resolution of the bivalent domain upon neural differentiation correlates with paternal Grb10 expression from CGI2. (A) The promoter usage in undifferentiated (undiff.) and differentiated (diff.) embryonic neurospheres was determined by exon-specific RT–PCR amplification. Relative positions of the polymorphism (black star) and primers used are indicated. Paternal expression from the CGI2 region was detected only after differentiation. De-repression appeared to be limited to exon1B1, as we failed to detect transcripts from exon1B2. (B) Representative allele-specific analysis of histone modifications at CGI2 in undifferentiated (undiff.) and differentiated (diff.) neurospheres. The upper panel shows the results of the SSCP analysis. The lower panel shows the relative enrichment of the paternal versus the maternal allele in H3K27me3 precipitations. (C) The level of H3K27me3 decreases at CGI2 during neurosphere differentiation, whereas H3K9ac increases. Following ChIP, the abundance of H3K27me3 and H3K9ac was determined by real-time PCR quantification of bound fractions and reported as a percentage of the value obtained with input chromatin. Download figure Download PowerPoint Ectopic Grb10 paternal expression in Eed−/− ES cells and embryos In ES cells, bivalent chromatin is thought to maintain key developmental genes in a 'permissive' chromatin configuration through H3K4me2, while repressing them by means of H3K27me3 (Azuara et al, 2006; Bernstein et al, 2006; [35]Mikkelsen et al, 2007). This idea is supported by the finding that some of these genes are inappropriately upregulated in ES cells deficient for EED (Azuara et al, 2006), a component of the PRC2 complex that mediates H3K27 tri-methylation. Therefore, to investigate the functional significance of the developmental loss of H3K27me3, we analysed Grb10 imprinted expression in two Eed−/− ES cell lines (i.e. G8.1 and B1.3) (Azuara et al, 2006). As controls, undifferentiated wild-type ES cell lines were analysed. Expression from the 1B1 promoter was detected in both mutant lines, but not in the wild-type cells (Figure 4A). However, the absence of allelic polymorphisms in G8.1 and B1.3 cells did not allow us to draw formal conclusions about the parental origin of this transcript. Nevertheless, bisulphite treatment of DNA followed by sequencing suggested that the maternal DNA methylation at CGI2 was unaltered in the two mutant cell lines (data not shown). The detected transcript was thus likely to originate from the unmethylated paternal allele. These results indicate that, in ES cells, EED deficiency and the resulting loss of H3K27me3 leads to a partial de-repression of brain-specific promoters at CGI2. Figure 4.Effects of EED deficiency on Grb10 imprinted expression. (A) EED is required for repressing the GCI2 promoter 1B1 in ES cells. Eed-deficient and wild-type ES cells were studied by RT–PCR amplification; Gapdh is used as a semiquantitative reference. We failed to detect transcripts initiating from exons 1C and1B2 in both wild-type and Eed-deficient ES cell lines. (B) EED deficiency has a limited impact on Grb10 paternal expression in 6.5 d.p.c. Grb10XC302 conceptuses. Embryos from a cross between an Eed−/+ female and an Eed−/+; Grb10XC302 male were collected at 6.5 d.p.c., genotyped and stained with X-Gal. Download figure Download PowerPoint To extend our analysis, we studied mice bearing a novel gene-trap insertion in intron 7 of Grb10, referred to as Grb10XC302. In these mice, LacZ staining can be used to visualize Grb10 expression. RNA in situ hybridization confirmed that LacZ staining faithfully recapitulated endogenous Grb10 expression (data not shown). Through paternal and maternal transmission of Grb10XC302, Grb10 imprinted expression was monitored in wild-type and Eed−/− embryos. As expected, Grb10 was widely expressed from the maternal allele in both cases, as indicated by the ubiquitous blue staining (Supplementary Figure 6). Conversely, following paternal transmission, Grb10 expression was restricted to specific cell populations in wild-type 6.5 d.p.c. embryos (Figure 4B). Particularly, Grb10 was expressed in the anterior visceral endoderm (AVE), an extra-embryonic tissue required for specifying early anterior patterning in the mouse embryo. In Grb10XC302/Eed−/− conceptuses, ectopic paternal expression of Grb10 was not found to be ubiquitous, but rather, was limited to one part of the extra-embryonic ectoderm (Figure 4B). This ectopic expression is likely to be a direct consequence of the absence of EED rather than an expansion of the AVE region in the mutant embryo. Previous lineage analysis showed that the AVE does not expand into the extra-embryonic region (Faust et al, 1998; Rivera-Pérez et al, 2003). Together these observations suggest that absence of EED (and the associated H3K27me3 mark), although involved in, is not sufficient on its own to promote the paternal expression of Grb10. Discussion In this study, we unravelled how the paternal expression of Grb10 in brain is controlled. Our main finding is that this process is regulated at least in part by a mono-allelic bivalent chromatin domain that marks the brain-specific paternal promoters of Grb10. We confirmed and extended previous Grb10 imprinted expression studies (Miyoshi et al, 1998; Arnaud et al, 2003; Hikichi et al, 2003). Grb10 is likely to be imprinted at pre-implantation stages as maternal expression is detected in both undifferentiated ES and trophoblast cells. Maternal expression is subsequently maintained in the developing embryo, placenta and in all adult tissues, except brain. The reciprocally imprinted, paternal expression observed in brain relies on three different promoters and is restricted to neuronal cells (this study; Yamasaki-Ishizaki et al, 2007). This is consistent with the expression pattern we observed in neurospheres, where paternal expression from the promoter in exon 1B1 is initiated upon differentiation. Hence, the paternal expression is activated in the neural lineage, when the neural stem/progenitor cells are committed towards the neuronal fate. Nonetheless, the three promoters are not activated simultaneously, and paternal expression from exon 1C is detected only after birth (data not shown). Therefore, the discrete paternal expression we observed in 6.5 d.p.c. conceptuses is intriguing. This paternal expression could result from a 'leakage' of imprinting at this stage, or could somehow pre-empt the forthcoming brain-specific paternal expression. The CGI2 region corresponds to the putati
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