H2A.B regulates the transcription of the imprinted Igf2r locus. (A) Schematic sketch of the Igf2r locus. (B) Retinoic acid (RA) induces the enrichment of H2A.B at the Igf2r locus. ChIP analysis on the Igf2r locus is performed in mouse ES cells with or without RA treatment. Data are presented as mean ± SEM (n = 3). (C,D) H2A.B is deposited at the maternally methylated DMR of Igf2r following RA treatment. Allele-specific incorporation of H2A.B was determined by DNA sequencing (C) or PCR-SSCP (D) (see Methods). (E) Knockdown of H2A.B suppresses the incorporation of H2A.B at the DMR of Igf2r. ChIP analysis is performed in shRNA-treated ES cells with or without RA induction. Data are presented as mean ± SEM (n = 3). (F) Knockdown of H2A.B suppresses the RA-induced transcription of Igf2r. The relative mRNA level of Igf2r is examined by qPCR. Data are presented as mean ± SEM (n = 4). (G) Knockdown of H2A.B suppresses the transcription of Igf2r from the maternal allele following RA treatment. Left panel shows that maternal and paternal alleles are digested by TaqI into different patterns because of SNP. Right panel shows that the transcription of Igf2r is dependent on the maternally methylated allele following RA treatment, and that depletion of H2A.B suppresses the RA-induced transcription of Igf2r from the maternal allele. The reduction of the transcription of Igf2r from each allele following RA treatment is summarized in the histogram.
