PMC

H19 represses its targets via the MBD1 protein. (A) Expression levels of the IGN in Mbd1^−/− samples were detected by RT-qPCR. The expression level in WT MEFs was set at 1, and histograms show modifications relative to this level (n = 4). (B) siRNA-mediated knockdown experiments of MBD1 in WT MEFs. The expression level in MEFs treated with a nonsilencing control was set at 1, and histograms show modifications relative to this level (n = 6). (C) siRNA-mediated knockdown experiments of H19 in Mbd1^−/− MEFs. The expression level in MEFs treated with a nonsilencing control was set at 1, and histograms show modifications relative to this level (n = 6).

H19 is necessary for recruitment of MBD1 and H3K9me3 to the Igf2 DMR1 and Slc38a4 and Peg1 DMRs. (A) Chromatin immunoprecipitation with an antibody to MBD1 in WT and H19^−/+ MEFs (n = 4). Histograms represent the ratio of immunoprecipitated DNA relative to the input sample. Igf2 DMR1, Slc38a4, Peg1, and Cdkn1c DMR regions were analyzed. Actin B was included as a negative control. (B) Chromatin immunoprecipitation with an antibody to H3K9me3 in WT and H19^−/+ MEFs (n = 3). (C) Sequencing of immunoprecipitated Igf2 DMR1 in WT MEFs. Maternal (dom, domesticus) and paternal (mol, molossinus) sequences are indicated. Star shows polymorphism between the two alleles.

Allele-specific expression of the IGN. (A) Allele-specific expression analysis of the Igf2 gene detected by RT-PCR followed by MspI digestion in E14.5 limb muscle samples. Molossinus (mol, paternal) allele presents an MspI restriction site absent in the domesticus (dom, maternal) allele. Nondigested (ND) and digested (D) RT-PCR products are presented. On the left, results obtained with a H19^−/+dom;Tg × JF1 mating. On the right, results obtained with a H19^−/+;YAC × JF1 mating. Arrows show the maternal Igf2 allele. (B) Allele-specific expression analysis of other imprinted genes of the network detected by RT-PCR and sequencing in H19^−/+ E14.5 limb muscle samples. Maternal (dom, domesticus) and paternal (mol, molossinus) sequences are indicated. Stars indicate polymorphisms between the two alleles.

H19 modulates the IGN in MEFs and interacts with the MBD1 protein. (A) Expression levels in E14.5 primary MEF samples were detected by RT-qPCR. The expression level of WT MEFs was set at 1, and histograms show modifications relative to this level (n = 4 for each genotype). (B) Primary transcript levels were detected using primers located in introns of the genes. (C) RIP with an antibody to MBD1 indicates binding to H19 in WT MEFs. The enrichment of RNA over a random IgG is shown. Igf2 mRNA was used as a negative control. The specificity of the antibody was tested by performing the experiment in Mbd1^−/− MEFs.

Model of H19-mediated regulation of Igf2, Slc38a4, and Peg1 genes. In WT cells, the H19 lncRNA interacts with the MBD1 protein and recruits it to the Igf2 DMR1, both on the maternal and paternal allele (Upper Left). This recruitment induces H3K9me3 on both alleles, probably via interaction with an H3K9 KMT. In H19^−/+ cells, MBD1 cannot be recruited to the Igf2 DMR1, leading to a loss of H3K9me3 (Upper Right). This results in an increase of Igf2 transcription, concomitant with a loss of Igf2 imprinting. On the Slc38a4 and Peg1 paternal DMRs, the H19 lncRNA recruits MBD1 and induces H3K9me3 (Lower Left). In absence of H19, the lack of binding of MBD1 results in a loss of H3K9me3 and in overexpression of the paternal allele (Lower Right). Therefore, H19 exerts a fine-tuned regulation of these genes, by modulating the presence of the repressive H3K9me3 histone mark on the active alleles.