of a 350 Kb genomic region including INS and the IGF2/H19 locus. Blue boxes represent the position of genes, the green oval depicts the localisation of the enhancer (Enh) (see also Figure 1) and red boxes in the second line represent the CTCF/cohesin binding regions CTCF AD, CCD, ICR, and CTCF DS. Vertical blue lines in a row 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 the other panels. The blue vertical line across each graph indicates the position of the anchor restriction site (j) in the ICR. (B,C) Association frequencies of the ICR (anchor site j) throughout the locus are analysed in unsynchronised cells as well as in cells synchronised in G1 and G2 phases. Association frequencies are displayed on a log scale to include all data points (B) but also on a linear scale (C) to better visualise differences. For all three cell populations, associations of the ICR can be detected with the CTCF AD/DMR0 region as well as with the CTCF DS. The CCD is too close to the ICR to distinguish an interaction. ICR association frequencies with intervening regions between the CCD and the IGF2 gene (restriction sites e, f and g) vary between G1 and G2 phase. Since these are random ligation interactions we don’t know what this means. We do not see this with an anchor near the k restriction site (Figure 3). The association frequencies between ICR and the CTCF AD/DMR0 were similar; however we do see a higher association frequency in G2 compared to G1 at the CTCF DS region. We also see this difference when we use the k primer as an anchor (Figure 3). The high interaction with the d restriction site is not reproducible. (D) Associations of the ICR with individual restriction sites close to the CTCF AD/DMR0 are depicted as histograms for higher resolution. (E,F) Association frequencies of the ICR (anchor site j) throughout the locus are analysed in cohesin depleted cells (SCC1 RNAi) or cells treated with control RNAi. Association frequencies are first displayed on a log scale to include all data points (E) but also on a linear scale in (F) to better visualise differences. All associations detected within the locus are reduced after SCC1 depletion. Statistical evaluation for all interactions with the j primers are in Table S1. (G) For a detailed view of ICR interactions with the CTCF AD/DMR0 region, effects of cohesin depletion are displayed as a histogram. # Denotes significant differences (P,0.05) between control and SCC1 RNAi. Found at: doi:10.1371/journal.pgen.1000739.s003 (1.13 MB TIF) Figure S4 Effect of cohesin depletion on chromatin conforma- tion in the IGF2/H19 locus in G1 phase cells. (A) Schematic representation of a 350 Kb genomic region including INS and the IGF2-H19 locus. Blue boxes represent the position of genes, the green oval depicts the localisation of the enhancer (Enh) (see also Figure 1) and red boxes in the second line represent the CTCF/ cohesin binding regions CTCF AD, CCD, ICR, and CTCF DS. Vertical blue lines in a row indicate the position of BamHI restriction sites within the locus and letters point out the restriction sites analysed by 3C (3C RS). Anchor primers are shown as black arrows below the restriction site map. (B) Enlarged view of the region comprising the CTCF AD/DMR0 and the IGF2 gene showing exons as boxes (blue for coding and empty for non-coding exons). Promoters are shown as arrows above the exons and the DMR0 region is shown as a yellow bar. Reciprocal 3C primers are shown as grey arrowheads. (C) Association frequencies of the ICR (j-primer as anchor) with restriction sites in the CTCF AD/DMR0 (a and b1) and IGF2 gene promoter (c1) in cohesin depleted (SCC1 RNAi) and control (G1 control) cells synchronised in G1 phase. Association frequencies with restriction sites in IGF2 upstream Cohesin Mediates Chromatin Conformation region drop significantly in cohesin depleted cells compared to a random ligation with a restriction site in the intervening region (restriction site e) that is not reduced. (D) The effect of cohesin depletion on associations between the enhancer (m primer) and restriction sites close to promoters P2 (b1 restriction site), and P3 (d restriction site), was analysed. Upon cohesin depletion no significant change in the association frequencies was observed, confirming that these interactions occur independently of CTCF/ cohesin binding (Table S1). # Denotes significant differences (P,0.05) between control and SCC1 RNAi. Found at: doi:10.1371/journal.pgen.1000739.s004 (0.74 MB TIF) Figure S5 Effects of normalisation using circularisation of a restriction fragment as an internal control. BamHI restriction of i and j sites followed by random ligation results in circularisation of this fragment (i-fragment). Circularisation of i-fragment can be detected by using primers amplifying 180 bp spanning the re- ligated i and j sites. (Primer sequences are located in Table S3.) Inefficient digestion or ligation will give very low yields of this PCR product and therefore it is a good ligation and digestion control. (A) We found that the variation of i-fragment circularisation relative to the copy number of template between 3 biological replicates was low. There was also no significant difference in the relative circularisation frequency of i-fragment between control and cohesin depleted samples. (B) All interactions in our 3C samples were normalised using the circularisation frequency of the i-fragment. There was no significant difference between control and cohesin depleted samples at an interaction between the enhancer anchor (primer m) and a restriction site between the enhancer and the CTCF DS (site p). The interaction between m and p is not dependent on CTCF/cohesin and is therefore not expected to change after cohesin depletion. Found at: doi:10.1371/journal.pgen.1000739.s005 (0.29 MB TIF) Table S1 Statistical analysis of difference in locus-wide associ- ation frequencies with ICR, enhancer, CTCF AD/DMR0, and CTCF DS anchors. Found at: doi:10.1371/journal.pgen.1000739.s006 (0.31 MB DOC) Table S2 ChIP primers. Found at: doi:10.1371/journal.pgen.1000739.s007 (0.04 DOC) Table S3 3C primers for BamH1 template. Found at: doi:10.1371/journal.pgen.1000739.s008 (0.12 DOC) Table S4 3C primers for BglII template. Found at: doi:10.1371/journal.pgen.1000739.s009 (0.06 DOC) MB MB MB Acknowledgments We thank Thierry Forne and members in his laboratory in Montpellier for advice and discussions on the quantitative 3C technique, Maja Jagodic for tuition on real-time PCR, Sarah Vowler for statistical advice, Christian Bourne for his assistance with DNA sequencing, and Mariette Kemner-van de Corput and Iris Jonkers for advice on the RNA/DNA FISH. Author Contributions Conceived and designed the experiments: RN KSW WR JMP AM. Performed the experiments: RN KSW YI JEH SUL KW. Analyzed the data: RN KSW YI JEH SUL KW CK AM. Contributed reagents/ materials/analysis tools: RN KSW YI SUL KW CK AM. Wrote the paper: JMP AM. PLoS Genetics | www.plosgenetics.org 14 November 2009 | Volume 5 | Issue 11 | e1000739