Fig. 5

Nox4 results with 4C-seq for two viewpoints and confirming nested intra-TAD loop structure. a Two viewpoints for Nox4 gene region: VP1 is anchored ~ 11 kb upstream of the Nox4 TSS (light red highlight), and VP2 is anchored ~ 125 kb downstream of the TSS (green highlight). VP1 has the strongest male-biased H3K27ac mark. This region shown spans chr7:94248242–94726358. In addition to the ChIP-seq (H3K27ac, Cohesin, and CTCF) and DNase-seq tracks shown, the tracks at the top show the chromatin state of this genomic region in male liver (top) and female liver (bottom). Chromatin states are colored: green indicates an enhancer-like state, blue indicates a promoter-like state, and purple a transcribed-like state. Red indicates an inactive chromatin state (see Additional file 1: Figure S7C for further details). Regions Y1 and Y2 are in chromatin state E13 in male liver but in inactive state E2 in female liver. Y1 and Y2 both include a short enhancer state region in male liver (E11 within region Y1; E10 in region Y2). The absence of focal interactions between VP1 and VP2 supports the model of two nested and insulated intra-TAD loops shown at the bottom. All tracks were normalized and are presented as described in Fig. 4. Interactions between VP1 and the Y1 region were significantly sex-biased by FourCSeq (FDR = 0.020), but interactions between VP1 and the Y2 region were not significant in the same analysis (FDR = 0.137). Within the shown window, no interactions originating from enh2 were found to be significantly sex-biased. Thick blue arrows, CTCF anchor orientation. b Expression of Nox4, but not the neighboring gene Tyr, is cohesin-dependent. Although Nox4 is primarily regulated by proximal enhancers within the shorter intra-TAD loop, its full expression is nevertheless dependent on cohesin. This may be due to the need for the intra-TAD loop structure; however, loss of this insulation did not increase expression of Tyr. Expression of Nox4 was reduced by 62% (p < 0.0001). Data presentation are as described in Fig. 4d