Figure 1

5′-3′ intragenic chromatin loops in CASP9 gene induced by retinoic acid (RA). (A) CASP9 gene structure and regulatory elements are shown as colored boxes in a blue line with the arrow indicating the direction of transcription; promoter (red), enhancer (yellow), exons (green), polyA addition sites (black). The black vertical lines indicate the restriction sites used to digest formaldehyde-fixed chromatin from MCF7 breast cancer cells induced for 15 min with 10 nM RA. Numbered horizontal arrows show the primers used to detect specific ligated fragments. The black curved lines show the interactions found in CASP9 chromatin derived from cells exposed to RA, using the promoter (upper panel) or the 5′of the RARE (lower panel) as baits. The bar graphs show the quantification of the interactions measured by 3C analysis (qPCR) between the baits and the primers (arrows and numbers in the lower panel) in the chromatin from unstimulated (basal), RA-stimulated cells for 15 min (RA 15 min) and treated with RNAse H1 for 1 h. Wilcoxon sign-rank test for matched pairs * or **p ≤ 0.001 compared to the basal or to the sample not treated with RNAse H1, respectively. (B) 3C analysis of CASP9 intragenic loops. CASP9 ligated fragments were separated by agarose gel electrophoresis (left panel) or identified by qPCR (right panel). The ligated fragments were sequenced to confirm the results obtained by qPCR and electrophoresis. Enhancer regions were detectable both with primers located at 5′ and 3′ end of the Nco I fragment containing the RARE. Where indicated, digestion with RNase H1 (100 U/ml) of formaldehyde-fixed chromatin for 60 min was carried out prior to DNA ligation. The 3C protocol used is described in Materials and Methods and in refs^5,8. (C) DNA-RNA immunoprecipitation (DRIP) analysis of promoter-RARE and polyA sites in CASP9 gene upon RA induction. Cells were induced with RA for different periods of time, DNA was extracted and DRIP analysis was carried as described in Materials and Methods. DNA was treated in parallel samples with RNAse H1 for 1 h before DRIP. Exon 9 of the TSH receptor gene was used as control. The data set corresponding to (C) are shown in Supplemental Fig. S1A, t test: *p ≤ 0.001 as compared to the basal, Wilcoxon sign-rank test for matched pairs: **p ≤ 0.001 as compared to the sample not treated with RNAse H1. (D) Chromatin immunoprecipitation of the RAD21cohesin subunit, at the promoter-RARE and polyA sites in CASP9 gene upon RA induction. Cells were induced with RA for different periods of time, fixed, and the nuclei prepared and subjected to ChIP analysis with antibodies to RAD21. In parallel samples, fixed chromatin was treated with RNAse H1 before ChIP. Exon 9 of the TSH receptor gene was used as control. The data set corresponding to (D) are shown in Supplemental Fig. S1B, t test: *p ≤ 0.001 as compared to the basal.