Fig. 3

Proximal and distal sex-biased regulatory elements and impact of cohesin depletion. Shown are WashU Epigenome Browser screenshots of sex-biased cohesin and CTCF binding events proximal (≤ 20 kb from a sex-biased gene TSS) and distal (> 20 kb) to sex-biased genes in mouse liver. Tracks shown are (from top to bottom): TAD and TAD boundary location [indicated by a vertical shift in the TAD track, as on the left and on the right sides of panel C], H3K27ac ChIP-seq, DNase-seq, cohesin (Rad21) ChIP-seq, CTCF ChIP-seq, and Ref-seq genes. ChIP-seq and DNase-seq data in each track is shown superimposed for male (blue) and female (red) liver after normalization to total reads per million in the union of peaks for that factor (see “Materials and methods”). Below each ChIP-seq track, a horizontal bar identifies genomic regions that show significant male-bias (blue bar) or female-bias (pink bar), with darker and lighter shades indicating strict and lenient cutoffs for sex-bias, respectively (see “Materials and methods”). Green arrows indicate CTCF sex-differential CAC, and red arrows indicate CTCF sex-differential CNC and Lone peaks. Male/Female stranded polyA + RNA-seq gene expression ratios [9] are indicated above each panel. aSlc22a29 is a female-biased gene with a distal female-biased non-anchor CAC peak overlapping a robust female-biased DHS with female-biased H3K27ac histone mark accumulation ~ 34 kb upstream (green arrow). The region shown spans chr19:8290981–8333632. b Two male-biased genes, Cml5 and Nat8, with proximal male-biased cohesin and CTCF peaks overlapping male-biased DHS (red, green arrows). Cml5 has a ~ 3 kb upstream male-biased CNC peak overlapping a strongly male-biased DHS. The Cml5 promoter shows strong male-biased H3K27ac marks and a weaker male-biased DHS. Nat8 has a weakly male-biased DHS at its promoter and a stronger male-biased DHS ~ 12 kb upstream that overlaps a male-biased CAC peak. The region shown spans chr6:85766132–85794443. c Male-biased genes C8a and C8b have distal male-biased CAC and CNC peaks overlapping male-biased DHS and H3K27ac marks (green and red arrows, respectively). The linear distance between the upstream male-biased CAC peaks and downstream male-biased genes is > 1.5 Mb. C8a and C8b reside on opposite ends of the same TAD. Oma1 is close in linear distance to these sex-biased regulatory elements, but shows no sex differences in expression; based on TAD structure it is not be predicted to interact with the highlighted male-biased enhancers. See Additional file 1: Figure S4A for the full length of the TAD and a model of spatial positions. The left portion of this figure spans chr4:103027454–103167067, and the right portion spans chr4:104433514–104583344. d Loss of cohesin binding decreases expression of C8a and C8b significantly (p ≤ 0.0001 and p = 0.0020; M–W t-test), while expression of Oma1 increases (p = 0.0102; M–W t-test). Bars represent the mean expression of the group for a given gene relative to the mean of the WT group (equal to 1), and error bars show the standard deviation based on n = 4 per group. e Loss of cohesin binding has a tenfold greater suppressive effect on male-biased genes with distal sex-biased enhancers than those with proximal sex-biased enhancers. Shown is the mean expression for cohesin-depleted versus wild-type liver, such that a value of 0.1 represents a tenfold reduction in expression after cohesin loss. The median relative expression for DHS/H3K27ac-proximal genes is 0.69 (representing a modest suppressive effect of cohesin loss) and the corresponding median for DHS/H3K27ac-distal genes is 0.07, indicating a > 10-fold greater reduction in gene expression (p = 0.0087; M–W). Similar results were obtained when the definition of proximally regulated genes was relaxed to include genes with a TSS < 20 kb from either a male-biased DHS or a male-biased H3K27ac peak (median relative expression of 0.45 versus 0.042 for distal genes). Also see Additional file 5: Table S2