Features and predictors of cohesin-dependent gene expression. (A) The T cell receptor locus Tcra and 3′ flanking region of chr14 are shown alongside Hi-C heat maps at 140-kb resolution for control (top) and cohesin-deficient thymocytes (bottom), epigenomic features, and genes that are up- (red) or down-regulated (blue, no examples in this region). Differentially interacting (DI) regions as determined by HOMER analysis of Hi-C data are shown in red (up-regulated) or blue (down-regulated). Dashed gray lines demarcate increased interactions with the region 3′ of Tcra. (B) HOMER-identified increased and decreased interactions around the Cd3 gene cluster on chr9. (Top) increased interactions marked as (i) chr9:44300000–44400000 and (ii) chr9:44800000–44900000 contain the Bcl9 and Mpzl2 genes that are up-regulated in cohesin-deficient thymocytes. (Bottom) Gene expression and 3C analysis of region (ii). Hind III fragments containing CTCF and RAD21 binding sites are shaded gray. (Left inset) Primary 3C data (mean ± SD, n = 3). (Right inset) RT-PCR validation of Mpzl2 expression (mean ± SD, n = 3). (C) Multinomial logistic regression model integrating gene expression, Hi-C, and ChIP-seq data to predict up-regulated, down-regulated, and unchanged genes. We tested each gene for the following features: (1) the presence or absence of ChIP-seq peaks near the promoter (TSS ± 2.5 kb); (2) location within 100-kb differentially interacting regions (divided into interactions that were stronger, DI region [Up], or weaker, DI region [Down], in cohesin-deficient thymocytes); (3) the presence of the H3K4me3 histone modification; (4) the binding of RAD21, CTCF, NIPBL and Mediator (MED1), RNA Pol II (RNAP2), paused RNA Pol II at the promoter (Hendrix et al. 2008); (5) the presence of a promoter CpG island (CGI); and (6) gene length. Error bars represent 95% confidence intervals. Variables are ranked by coefficient significance from left to right. Of 17,849 genes assayed by RNA-seq, 1461 resided in DI regions. Of 450 down-regulated genes, 83 resided in DI regions, which represents a strong enrichment (P-value < 10−11, odds ratio 2.63). Up-regulated genes were only slightly enriched in DI regions (P < 0.05, odds ratio 1.27).
