Unexpected nuclear hormone receptor and chromatin dynamics regulate estrous cycle dependent gene expression [RNA-seq]

Hormone dependent uterine gene expression changes that occur during the estrous cycle suggest hormone receptor binding to chromatin may also be dynamic. Therefore, we employed a multi-faceted approach to examine in vivo dynamics of hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq for estrogen receptor alpha (ERa) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ERa and PGR binding as well as chromatin accessibility. There were 4,159 differentially expressed genes between estrus and diestrus. At transcription start sites, accessibility generally correlated with the directionality of gene expression and there was reduced PGR in estrus compared to diestrus but little change in ERa. There were 2,727 enhancers with dynamic accessibility near these genes and 77% of those correlated with directionality of gene expression changes. However, most enhancers were constitutively open (8,694). In both dynamic and constitutively open enhancers, ERa and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ERa binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ERa binding occurred at transcription factor deserts in relatively closed chromatin while estrus specific accessible regions were highly enriched for ATF, ELF and ELK motifs. Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) revealed many estrous cycle dependent partners of ERa (diestrus, 60; estrus, 24). PGR was found in complex with ERa during diestrus but not estrus supporting coordinated binding of both receptors during diestrus. Two of the cohesin complex proteins, SMC1A and SMC3, were found in complex with ERa during diestrus but not estrus; overlap of SMC1A with ERa confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ERa interactions have functional consequences on chromatin structure. Taken together, a complex series of interactions between hormone receptors, chromatin structure and accessibility orchestrate estrous cycle dependent changes in gene expression. Overall design: Uteri from intact, untreated CD-1 mice were collected at 2 months of age at diestrus (CoD) or estrus (CoE). RNA-seq, ATAC-seq, ER alpha ChIP-seq, PgR ChIP-seq, Hif2a ChIP-seq and HiC-seq were performed as well as RIME assay using ER alpha as the immunoprecipitating antibody.