H19 lncRNA controls gene expression of the Imprinted Gene Network by recruiting MBD1 Paul Monniera,b, Clémence Martineta, Julien Pontisc, Irina Stanchevad, Slimane Ait-Si-Alic, and Luisa Dandoloa,1 aGenetics and Development Department, Institut National de la Santé et de la Recherche Médicale U1016, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 8104, University Paris Descartes, Institut Cochin, Paris 75014, France; bUniversity Paris Pierre and Marie Curie, Paris 75005, France; cUniversity Paris Diderot, Sorbonne Paris Cité, Laboratoire Epigénétique et Destin Cellulaire, CNRS UMR 7216, Paris 75013, France; and dWellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom Edited by Marisa Bartolomei, University of Pennsylvania, Philadelphia, PA, and accepted by the Editorial Board November 11, 2013 (received for review May 30, 2013) The H19 gene controls the expression of several genes within the this control is transcriptional or posttranscriptional and whether Imprinted Gene Network (IGN), involved in growth control of the these nine targets are direct or indirect targets remain elusive. embryo. However, the underlying mechanisms of this control re- Several lncRNAs interact with chromatin-modifying com- main elusive. Here, we identified the methyl-CpG–binding domain plexes and appear to exert a transcriptional control by targeting fi protein 1 MBD1 as a physical and functional partner of the H19 local chromatin modi cations at discrete genomic regions (8, 9). long noncoding RNA (lncRNA). The H19 lncRNA–MBD1 complex is In the case of imprinted clusters, the DMRs controlling the ex- fi pression of imprinted genes exhibit parent-of-origin epigenetic required for the control of ve genes of the IGN. For three of these fi fi genes—Igf2 (insulin-like growth factor 2), Slc38a4 (solute carrier modi cations (DNA methylation and histone modi cations) that family 38 member 4), and Peg1 (paternally expressed gene 1)— govern the imprinting of the locus. In some of these clusters, lncRNAs control in cis the transcription of adjacent genes. For both MBD1 and H3K9me3 binding were detected on their differ- – example, the Kcnq1ot1 lncRNA associates with the lysine entially methylated regions. The H19 lncRNA MBD1 complex, methyltransferase G9a and the Polycomb Repressive Complex through its interaction with histone lysine methyltransferases, (PRC2) to regulate the expression of other genes of the locus in therefore acts by bringing repressive histone marks on the differ- the placenta (10, 11). Similarly, in the context of X-inactivation, entially methylated regions of these three direct targets of the the Xist lncRNA associates with PRC2 and creates domains of H19 gene. Our data suggest that, besides the differential DNA repressive control on the inactive X chromosome (8). Alterna- methylation found on the differentially methylated regions of tively, some lncRNAs, termed macroRNAs, such as Airn and imprinted genes, an additional fine tuning of the expressed allele Nespas, act by silencing promoters and enhancers by transcrip- is achieved by a modulation of the H3K9me3 marks, mediated by tional overlap (12, 13). Finally, in other nonimprinted regions, the association of the H19 lncRNA with chromatin-modifying com- several lncRNAs, such as HOTAIR, seem to exert their func- plexes, such as MBD1. This results in a precise control of the level tional role by recruiting chromatin-modifying complexes for of expression of growth factors in the embryo. transregulation (14, 15). To elucidate the mechanism of action of the H19 lncRNA genomic imprinting | embryonic growth | long noncoding RNA partner | on the genes of the IGN, we performed RNA immunoprecipi- Dlk1 | Cdkn1c tation (RNA-IP) with specific proteins and discovered that H19 RNA binds the methyl-CpG-binding domain protein 1 (MBD1). MBD1 belongs to the family of the methyl-CpG–binding domain he imprinted H19 locus belongs to a conserved gene cluster proteins, such as MBD2, MBD3, MBD4, and MECP2 (methyl- Ton chromosome 7 in the mouse and 11p15.5 in human, and it CpG-binding protein 2) (16). MeCP2, MBD1, and MBD2 pro- plays an important role in embryonic development and growth teins bind to methylated DNA and recruit different histone control. The cluster contains the insulin-like growth factor 2 GENETICS (Igf2) gene, located 90 kb away from the H19 gene, and both Significance genes are coordinately regulated by an intergenic differentially methylated region (DMR) also called imprinting control region (ICR) and by downstream enhancers, with H19 being expressed The H19 imprinted gene produces a long noncoding RNA from the maternal and Igf2 from the paternal allele (1, 2). The (lncRNA) exclusively expressed from the maternal allele. It is Igf2 gene is under the additional control of somatic DMRs 1 and involved in the control of embryonic growth and regulates 2 in the embryo. Both genes are strongly expressed during em- nine genes of an Imprinted Gene Network (IGN). Our goal was bryogenesis and down-regulated after birth, with H19 remaining to decipher the molecular mechanisms that drive this control of expressed in adult skeletal muscle and heart. the IGN. We show that this lncRNA represses several target The H19 gene produces a 2.3 kb spliced, capped, and poly- genes through interaction with the methyl-CpG–binding do- adenylated long noncoding RNA (lncRNA) (3). The H19 locus main protein 1 MBD1. This protein is involved in the mainte- also produces a microRNA (miR) from a highly conserved re- nance of repressive H3K9me3 histone marks. The H19 RNA is gion in the first exon. This miR-675 plays a role in controlling required for the recruitment of MBD1 to some of its targets, placental growth at the end of gestation by regulating the ex- including the adjacent insulin-like growth factor 2 gene, and pression of the Igf1r gene (4). acts by a fine-tuned regulation on the expression levels of Δ The targeted deletion of the gene (H19 3) induces an over- these growth-controlling genes of the IGN. growth phenotype (+ 8% compared with WT mice), which can be rescued by transgenic reexpression of H19 (5, 6). Expression Author contributions: P.M. and L.D. designed research; P.M., C.M., and J.P. performed of the Igf2 gene is affected by the deletion of the H19 gene, and it research; P.M. and L.D. analyzed data; J.P., I.S., and S.A.-S.-A. contributed new reagents/ becomes biallelically expressed, with a 35% level of expression analytic tools; and P.M., I.S., S.A.-S.-A., and L.D. wrote the paper. from the usually silent maternal allele. Similarly, eight other The authors declare no conflict of interest. genes belonging to an Imprinted Gene Network (IGN) (7) also This article is a PNAS Direct Submission. M.B. is a guest editor invited by the Editorial show an increased expression level in the absence of H19, which Board. is restored to a normal level by transgenic reexpression. These 1To whom correspondence should be addressed. E-mail: [email protected]. data suggest that H19 acts in trans to regulate the expression of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. these genes and to control growth of the embryo (6). Whether 1073/pnas.1310201110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1310201110 PNAS | December 17, 2013 | vol. 110 | no. 51 | 20693–20698 Downloaded by guest on September 23, 2021 A H19 RNA represses the maternal expression of the nearby Igf2 gene in trans and thus regulates Igf2 gene levels in embryonic limb muscle. We then focused on the other imprinted genes whose ex- pression was increased in absence of the H19 gene. In contrast to the Igf2 gene, RT-PCR followed by sequencing indicated that the other H19 targets of the IGN (such as Slc38a4, Peg1, Dlk1, − + B Cdkn1c, and Gtl2) remained monoallelicaly expressed in H19 / embryonic limb muscle (Fig. 1B). Dcn is biallelically expressed, however sequencing in WT embryonic limb muscle showed that this gene is actually not imprinted in this tissue (Fig. S1). The H19 lncRNA Represses the IGN in MEFs. To study the molecular mechanisms that drive the H19-mediated repression of the IGN in the embryo, we chose to use primary mouse embryo fibroblasts (MEFs) as a model system. We measured IGN expression levels in − + − + WT, H19 / ,andH19 / ;Tg MEFs. We observed, as previously shown in limb muscles, that Igf2, Slc38a4, Dcn, Dlk1, Peg1, Gtl2, Cdkn1c, and Igf2r are overexpressed in cells lacking H19 RNA expression (Fig. 2A and Table S1). In MEFs that ectopically ex- − + − + press the H19 RNA in an H19 / background (H19 / ;Tg), the expression of these genes is restored to WT level. Thus, both in MEFs and in embryonic limb muscle, the H19 gene negatively Fig. 1. Allele-specific expression of the IGN. (A) Allele-specific expression regulates several genes of the IGN. The level of expression of analysis of the Igf2 gene detected by RT-PCR followed by MspI digestion in these target genes was often higher in MEFs compared with that E14.5 limb muscle samples. Molossinus (mol, paternal) allele presents an in embryonic muscle samples. This suggests that a strict control of MspI restriction site absent in the domesticus (dom, maternal) allele. the expression of growth-controlling genes is exerted in vivo, Nondigested (ND) and digested (D) RT-PCR products are presented. On the whereas this control is more flexible in an in vitro culture system. left, results obtained with a H19−/+dom;Tg × JF1 mating. On the right, −/+;YAC × We also evaluated the levels of expression of the primary results obtained with a H19 JF1 mating. Arrows show the ma- transcripts using primers in introns of target genes (Fig. 2B). For ternal Igf2 allele. (B) Allele-specific expression analysis of other imprinted −/+ Igf2, Slc38a4, and Peg1 genes, an increase in the level of primary genes of the network detected by RT-PCR and sequencing in H19 E14.5 −/+ limb muscle samples.