Accessibility at a primed distal Fshb-Kcna4 super-enhancer is facilitated by Foxl2 during gonadotrope differentiation

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Abstract

A large "gene desert" located far upstream from Fshb and Kcna4 contains several gonadotrope-specific accessible chromatin sites which were seen in chromatin conformation capture (3C) to make distinct contacts with both genes. Expression of Fshb and Kcna4 was strongly inhibited by JQ-1, which represses super-enhancer activity, and the region displays super-enhancer characteristics. The sites of open chromatin were seen, in chromatin immunoprecipitation (ChIP), to bind Brd4 and Med1, most notably at a site -67 kb from the Fshb gene, as well as binding Ctcf further upstream (-123 kb), all of which were increased following activin exposure. The locus is transcribed to chromatin-associated lncRNAs whose levels correlate with Fshb and Kcna4 mRNA levels in vivo and in cultured gonadotrope cells, indicating coordinated regulation. CRISPR interference (CRISPRi) confirmed distinct functions for each element and, together with the 3C data, indicate that the -67 kb locus mediates basal and activin-stimulated Fshb expression, while the site at -59 kb contributes to activin-stimulation of both genes. Single-cell multiomics revealed that the -67 kb locus is accessible in pituitary stem cells and throughout gonadotrope differentiation, preceding opening of the Fshb promoter, although it is closed in other differentiated cell types, suggesting a gonadotrope-specific factor that keeps it open at this stage. Foxl2 was found to bind this element, contributes to maintaining its chromatin accessibility, and recruits Supt16h, a component of the Facilitates Active Chromatin Transcription (FACT) histone chaperone complex. These findings define a distal, Foxl2-bound super-enhancer that regulates Fshb transcription and shapes the gonadotrope regulatory landscape.