GRHL and PGR control WNT4 expression via 3D looping of conserved and species-specific enhancers

Developmental gene expression is tightly regulated by tissue-specific enhancers. Understanding this process in the context of the three-dimensional (3D) genome requires dedicated interrogation on a per gene and per tissue basis. Here, we functionally dissect the mechanisms that regulate Wnt4/WNT4 gene expression. We focus our efforts on the mouse mammary gland and human breast, where Wnt4/WNT4 expression is restricted to hormone-responsive luminal epithelial cells and generally assumed to lie downstream of progesterone. Using freshly isolated cells and cell lines, we map the Wnt4/WNT4 enhancer network, reveal Grainyhead-like (GRHL) proteins as novel co-regulators of Wnt4/WNT4 expression, and identify a conserved distal enhancer that physically loops to Wnt4/WNT4. We integrate our findings to propose a working model in which a conserved distal chromatin hub, bound by CTCF and GRHL, recruits other species-specific enhancers, to facilitate PGR-dependent and -independent Wnt4/WNT4 gene regulation via 3D chromatin looping.