Figure 3.

Chromatin interactions at the ICR2 domain determined by 3C experiments using control cell lines. The Figures a-b are to scale and have a reverse orientation with respect to Fig. 1 because these loci are classically reported with a reverse orientation since the genes are transcribed on the negative strand. (a) Schemes of the interactome for the ICR2 domain, showing the main ROIs observed in the control cell lines. Red triangles, interactions between different elements in the region; the intensity of the red colour is directly proportional to the number of interactions present in that sub-region. Black circles, mean association frequencies of the controls (CTRL1 and 2). A linear representation of the ICR2 imprinted domain is depicted below the interactome. Black triangles and bold characters indicate the anchors used for 3C analysis. The data of each control derive from two independent 3C experiments. The overall results represent the sum of the chromatin conformations of the paternal and maternal alleles. (b) ICR2 domain looping profiles for the different anchors in control cell lines CTRL1 (purple) and CTRL2 (blue). BglII restriction sites are indicated above. Each point in the profiles is the mean ± standard deviation of the two replicates for each one of the controls and indicates the association frequency between the anchor and the fragment on the left of the corresponding BglII restriction site. (c) Allele specificity of the ICR2 chromatin associations, in the heterozygous CTRL2 cell line, by 3C-SNP analysis. Analysis of the two SNPs rs2283197 G/C and rs2283196 T/G (indicated by asterisks) was performed by Sanger sequencing and showed that all interactions were biallelic, with the exception of CTCF4-ICR2, which was monoallelic (C and G alleles). The 3C ligation product, indicating the orientation of primers (black triangles), SNPs, and BglII restriction sites, is depicted below the electropherograms.