Cohesin Mediates Chromatin Conformation Figure 3. Chromatin conformation of the IGF2-H19 locus is conserved between G1 and G2 phase of the cell cycle. (A) Schematic representation of a 350 Kb genomic region including INS and the IGF2-H19 locus. Blue boxes represent the position of genes and the green oval depicts the localisation of the enhancer (Enh) (see also Figure 1). Similarly, red boxes in the second line represent the CTCF/cohesin binding regions CTCF AD, CCD, ICR, and CTCF DS. Vertical blue lines indicate the position of BamHI restriction sites within the locus and letters point out the restriction sites analysed by 3C (3C RS) in the graphs and histograms shown in (B, D, F, G). The blue vertical lines in (B, D, F, G) link the positions of the restriction sites used as anchors in the different panels with the overview of the locus in (A). Thicker vertical lines indicate the position of the anchor in the respective graph. The anchor restriction sites fragments are: b1 in the CTCF AD/DMR0 region, k in the ICR, m in the enhancer and q in the CTCF DS region. The X-axis is labelled according to genomic position and position 0 was arbitrarily fixed 42 Kb upstream of INS. (B) Associations detected with the anchor site in the ICR (primer k). An association is present between the ICR and the CTCF AD/DMR0 region in both G1 and G2 phase and with the CTCF DS preferentially in G2. (C) The peak of association frequencies between the ICR and CTCF AD/DMR0 compared to random ligation (f–k) is shown in the histogram. Restriction site a is adjacent to the CTCF AD region (Figure 1) and the high association frequency between a and k might reflect directly the CTCF/cohesin binding to the CTCF AD. (D) Associations detected with an anchor in the enhancer (primer m). Peaks indicating interactions between the enhancer and the CTCF AD/DMR0 region can be seen in G1 and G2 phase cells. Positions of primers P2 and P3 are indicated. (E) The peak of association frequencies between the enhancer and CTCF AD/DMR0 region is shown in the histogram. Association values at restriction sites a–m report on the CTCF AD/DMR0 interaction with the enhancer, while b2-m, c1-m and d–m report on the IGF2 promoters P2 and P3. (F) Associations with the CTCF AD/DMR0 region (restriction site b1 as anchor site). Very high association frequencies are visible for the interactions with the CCD and the distant CTCF DS in G1 and G2 phase cells, presumably due to the strong and biallelic binding of CTCF and cohesin at these sites. (G) Associations of the CTCF DS region (q anchor) throughout the locus during G2 phase. Reciprocal association peaks with the CTCF AD/DMR0 and the CCD are visible. doi:10.1371/journal.pgen.1000739.g003 PLoS Genetics | www.plosgenetics.org 5 November 2009 | Volume 5 | Issue 11 | e1000739