Figure 2
From: RNA Stabilizes Transcription-Dependent Chromatin Loops Induced By Nuclear Hormones
5′-3′ intragenic chromatin loops in BCL2 and CAV1 gene induced by estrogen. (A) BCL2 gene structure and regulatory elements. The black vertical lines indicate the restriction sites used to digest formaldehyde-fixed chromatin after induction with 10 nM E2 for various periods of time (min). Horizontal arrows indicate the primers used to detect specific ligated fragments. All primer combinations were tested using the promoter (oligonucleotide 1 or 5), the ERE (oligonucleotides 6 or 2) and polyA site (oligonucleotide 8 and 4) as bait sequences. The red and the blue curved lines show interactions in BCL2 chromatin derived from cells exposed to E2 digested with Bam HI (blue) or Bgl II (red). The blue lines show interacting regions identified by paired-end tag sequencing (ChIA-PET), which detects global chromatin interactions of estrogen receptor α (ERα)8. E2-dependent interactions were selected for further analysis with RNAse H1. The histograms show the quantification by qPCR of the promoter-polyA loop (oligonucleotides 5 and 8) under various conditions. Non-parametric 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) BCL2 ligated fragments separated by agarose gel electrophoresis (left panel) or identified by qPCR (right panel). The primers used to detect the ligated fragment are indicated on the left side of the gel. INPUT shows the input DNA in the 3C experiment; a PCR fragment amplified with primers 9 and 10 containing the ERE. Sequencing the ligated fragments confirmed the qPCR and electrophoresis results. Where indicated, digestion with RNase H1 (100 U/ml) of formaldehyde-fixed chromatin for 60 min was carried out before DNA ligation. The right panel shows the quantification by qPCR of the promoter-polyA loop shown in the left hand side gel. Non-parametric 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. (C) DNA-RNA immunoprecipitation (DRIP) analysis of promoter and polyA sites in BCL2 gene upon E2 induction. Cells were induced with E2 for 45 min, DNA was extracted and DRIP analysis was carried as described in Materials and Methods. DNA was also treated with RNAse H1 for 1 h before immunoprecipitation. Exon 9 of the TSH receptor gene was used as control. The data set corresponding to Fig. 2C are shown in Supplemental Fig. S2A, t test: *p ≤ 0.001 ascompared to the basal, Wilcoxon sign-rank test for matched pairs: **p ≤ 0.01 as compared to the sample not treated with RNAse H1. (D) CAV1 gene structure and regulatory elements. The black vertical lines indicate the Ava II restriction sites used to digest formaldehyde-fixed chromatin of cells exposed to 10 nM E2 for various periods of time (min). Horizontal arrows indicate the primers used to detect specific ligated fragments. All combinations of the primers were tested using the promoter (oligonucleotide 1 and 2), the ERE (oligonucleotides 9, 3, 4 and 5) and the polyA site (oligonucleotide 7 and 8) as bait sequences. The black curved lines show the interactions found in CAV1 chromatin derived from cells treated with E2 and digested with Ava II. E2-dependent interactions were selected for testing with RNAse H1. The bar graphs at the left and right of CAV1 gene show the quantitative analysis of the promoter-ERE (primers 1–9) and ERE-polyA (primers 3–7) loops respectively. The ANOVA test on the time course is shown in Supplemental Fig. S2B. (E) CAV1 ligated fragments separated by agarose gel electrophoresis (left panel) or identified by qPCR (right panel). The primers used to detect the ligated fragment are indicated on the left side of the gel. INPUT indicates the PCR of the fragment amplified by primers 14 and 15 containing the ERE. The ligated fragments were sequenced to confirm the qPCR and electrophoresis results. Where indicated, digestion with RNase H1 (100 U/ml) of formaldehyde-fixed chromatin for 60 min was carried out before DNA ligation. The right panel shows the quantification by qPCR of the ERE-polyA loop shown on the gel on the left side. Non-parametric 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. The ANOVA test on the time course is shown in Supplemental Fig. S4A. We found no interaction between the following CAV1 pairs of primers on restricted chromatin derived from E2-stimulated cells: 1 + 2; 1 + 4; 1 + 5; 1 + 6; 1 + 7; 1 + 8; 2 + 3; 2 + 4; 2 + 5; 2 + 6; 2 + 7; 2 + 8; 3 + 4; 3 + 5; 3 + 6; 7 + 7; 3 + 8; 4 + 6; 4 + 7; 4 + 8; 5 + 6; 5 + 7; 5 + 8; 6 + 8 (Supplemental Table 1). (F) DNA-RNA immunoprecipitation analysis of promoter and polyA sites in CAV1 gene upon E2 induction. Cells were induced with E2 for 45 min, DNA was extracted and DRIP analysis was carried as described in Materials and Methods. Parallel DNA samples were treated with RNAse H1 for 1 h before immunoprecipitation. Exon 9 of the TSH receptor gene was used as control. The data set corresponding to Fig. 2F are shown in Supplemental Fig. S4B, t test: *p ≤ 0.001 ascompared to the basal, Wilcoxon sign-rank test for matched pairs: **p ≤ 0.01 as compared to the sample not treated with RNAse H1.
