Fig. 3

Characterization of MSH2 and MSH6 binding at the ICRs. a Screen shots from the UCSC Genome Browser showing the Bio-ChIP-seq peaks detected for Biotin-tagged MSH2 in BirA-expressing E14 ESCs along 8 ICRs compared with previously determined ZFP57 and KAP1 binding regions. The ICRs are shown as horizontal boxes coloured according to the mean DNA methylation level determined by RRBS. Enrichment of Bio-ChIP-seq reads in Msh2-AviTag-transfected ESCs (2 replicates) and mock-transfected BirA-expressing ESCs (MSH2 control) are shown in light blue and green, respectively; enrichment of ZFP57 and KAP1 ChIP-seq reads are in pink and purple, respectively. b Bar-plots showing the relative enrichment of Biotin-tagged MSH2 (blue) and MSH6 (green) at ICRs as determined by Bio-ChIP and ChIP, respectively, in WT E14 ESCs. The Smg7 and Utp3 promoter CpGI were used as positive controls, while a region of the Hoxa3 gene that is not overlapping a CpGI and showed lower enrichment of MSH2 by Bio-ChIP-seq was used as negative control. ChIP values are expressed as % input. Error bars represent the SD of three biological duplicates. Significant enrichment of MSH2-AviTag was demonstrated by Bio-ChIP at ICRs in Msh2-AviTag-transfected with respect to mock-transfected BirA-expressing ESCs (Mock), and significant enrichment of MSH6 was demonstrated at ICRs with anti-MSH6 antibodies with respect to IgG. c Bar-plots showing the relative enrichment of MSH2 and MSH6 at the Plagl1 and Inpp5f ICRs and flanking regions determined as in b. Screen-shots from the UCSC Genome Browser showing features of the analysed regions are reported above the bar-plots. The ICRs are indicated in dark grey, CpGI in green, ZFP57 peaks in pink. Orange bars represent the ICRs overlapping the ChIP-seq peaks of ZFP57. Blue, orange and light grey bars indicate the location of the primers used for Q-PCR. ChIP values are expressed as % input. Error bars represent the SD of two biological replicates each averaged over two technical replicates. *p < 0.05, **p < 0.01, ***p < 0.001