Promoter-enhancer communication occurs primarily within Gene Unit Sub-TADs (GUSTs)

The mammalian genome is sequentially partitioned from chromosomes into looped chromatin regions termed Topologically Associating Domains (TADs), and then into numerous smaller sub-TAD loops, each bounded and insulated by CTCF and Cohesin. The sub-TADs encompass enhancers, promoters and often multiple genes but whether promoter-enhancer interactions and gene regulation are broadly restricted to within the sub-TAD loops has not been functionally determined. Here we identify “Gene Unit Sub-TADs” or GUSTs as a class of sub-TADs that confine promoter-enhancer interactions and demarcate functional gene regulatory regions. We depleted Estrogen-related receptor β (Esrrb), which binds to the Mediator co-activator complex, to impair the activity of enhancers controlling 245 mouse embryonic stem cell genes. We find that most Esrrb-responsive enhancers lack significant Cohesin binding but instead correlate with Mediator binding. Esrrb depletion causes reduced Mediator binding, decreased nascent RNA expression and diminished promoter-enhancer looping. In 88% of the cases examined, the effects of Esrrb depletion are restricted to enhancers and target genes within GUSTs. In some GUSTs, active genes lay alongside inactive ones but are distinguished by their proximal promoter chromatin accessibility, explaining further the specificity of enhancer-promoter interactions within GUSTs. Our data indicate that GUSTs represent functional gene regulons in mammalian genomes. Systematic analysis on how enhancer inactivation by Esrrb knockdown affects gene expression in a GUST. All treatments contain 2 biological replicates except for Smc1 ChIP-seq and Nascent RNA-seq. Please note that the processed data generated from merged replicates are available as Series supplementary files.