Fig. 4

4C-seq analysis of two female-biased and two male-biased genes. a–c, e Shown are WashU Epigenome Browser screenshots for four genomic regions investigated by 4C-seq. The upper track presents 4C-seq data for four viewpoints, marked by a vertical highlight in each panel. The 4C-seq track is based on merged data from three biological replicates for each sex, calculated from the median value for a sliding window of 11 restriction fragments, in reads per million normalized 4C-seq signal per sex. These values are overlaid for visualization using the Matplot functionality built into the WashU genome browser with default parameters. The next four tracks show normalized DNase-seq or ChIP-seq signal for the indicated factors, and correspond to those described in Fig. 3. Sex-biased lncRNAs are shown below the Refseq gene track in pink (female-specific lncRNAs) or blue (male-specific lncRNAs). c, e also show locations of intra-TAD loops (pink arcs) below the Refseq gene track. 4C-seq data for individual biological replicates is presented in Additional file 1: Figure S6. a Distal enhancer viewpoint near A1bg and female-biased lncRNAs. The region shown includes 12 female-biased and nuclear-enriched mono-exonic lncRNAs, which fall into three clusters. The lncRNAs in each cluster are all transcribed from the same strand, as indicated by the arrow marking the TSS and direction of transcription of the most upstream lncRNA in each cluster. LncRNAs 12590–12593 show the strongest 4C-seq interactions with the viewpoint and also the most consistent female bias (Additional file 1: Figure S5C). Interactions marked by red arrows were significantly sex-biased by FourCSeq analysis at FDR < 0.1; from left to right, FDR = 0.0015, FDR = 0.0058, and FDR = 0.067. The region shown spans chr15:60733512–60954051. b Enhancer viewpoint 8 kb upstream of Gm4794 interacts primarily with the proximal promoter (left red arrow) and a strong female-biased enhancer (right red arrow). Viewpoint enhancer and the interacting enhancer both contain female-biased CNC peaks (marked below cohesin track in dark pink). Sult3a1 shows a weak and broad pattern of interaction with this viewpoint (red bracket). Interactions marked by red arrows were significantly sex-biased by FourCSeq: left region FDR = 0.038, right region FDR = 0.036. The region shown spans chr10:33418446–33680888. c Viewpoint at the promoter of C9 interacts with a distal male-biased enhancer, bypassing the gene Dab2. This viewpoint shows a strong male-biased interaction with distal male-biased enhancer indicated by a red arrow. The nested intra-TAD loop structure (loops at bottom) may facilitate the high and male-biased expression of C9 by looping out the intervening gene Dab2. There is a weaker, but apparent male-biased interaction with the TSS of the short isoform of Dab2. Based on CAGE data and Refseq annotation, the shorter isoform of Dab2 is predominantly expressed in liver (data not shown). Interaction marked by red arrows was significantly sex-biased by FourCSeq, FDR = 0.0009. The region shown spans chr15:6147917–6461799. Thick blue arrows at bottom, CTCF anchor orientation. d Cohesin depletion significantly reduces expression of C9 and its antisense lncRNA (lnc12340): 66% reduction, p = 0.0056; and 82% reduction, p = 0.0049, respectively. Bars represent mean expression compared to wild-type liver (WT, set equal to 1), with error bars showing standard deviation for n = 4 per group. eNudt7 lacks male-biased DHS or H3K27ac at its promoter, but interacts with a distal male-biased enhancer within the same intra-TAD loop. This viewpoint is anchored at a male-biased DHS (red highlighting). Prominent interactions with this viewpoint (red arrows) are with a neighboring male-biased enhancer and the promoter of Nudt7. While the TSS of lnc7430 and Nudt7 are close, the viewpoint enhancer interacts specifically with the TSS of Nudt7. Interactions marked by red arrows were significantly sex-biased by FourCSeq: left region, FDR = 0.0187; right region, FDR = 0.0041. The region shown spans chr8:116592444–116707613. f Upon loss of chromatin-bound cohesin, expression of both Nudt7 and its bi-directionally transcribed lncRNA (lnc7430) are significantly reduced (69% decrease, p = 0.0116; and 93% decrease, p = 0.0113, respectively). Expression of lnc7423 was not significantly impacted. Data presentation is as described in d