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 (ERα) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ERα 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 ERα. 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, ERα and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ERα binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ERα 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 ERα (diestrus, 60; estrus, 24). PGR was found in complex with ERα 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 ERα during diestrus but not estrus; overlap of SMC1A with ERα confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ERα 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. 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.

动情周期中发生的激素依赖性子宫基因表达变化提示，激素受体与染色质的结合或许同样具有动态性。为此，我们采用多维度方法，通过结合雌激素受体α（ERα）与孕酮受体（PGR）的RNA测序（RNA-seq）、转座酶可及性测序（ATAC-seq）、HiC测序（HiC-seq）及染色质免疫共沉淀测序（ChIP-seq），探究激素受体占据、染色质可及性及染色质结构的体内动态变化。
全基因组范围内，ERα与PGR的结合以及染色质可及性均存在广泛的动情周期依赖性变化。动情期与间情期之间存在4159个差异表达基因。在转录起始位点处，染色质可及性通常与基因表达的方向性相关；相较于间情期，动情期的PGR结合水平有所降低，而ERα结合水平则几乎无变化。
这些基因附近存在2727个具有动态可及性的增强子，其中77%与基因表达变化的方向性相关。不过，大多数增强子（8694个）处于组成型开放状态。在动态开放与组成型开放的增强子中，ERα与PGR的结合均从间情期到动情期发生协同性丢失。
间情期特异性的ERα结合区域与可及区域，富集了PGR、FOX、GATA及SOX的结合基序。与之相反，动情期特异性的ERα结合发生在相对闭合染色质中的转录因子荒漠区域，而动情期特异性的可及区域则高度富集ATF、ELF及ELK基序。
内源蛋白快速免疫沉淀质谱（RIME）实验揭示了多个动情周期依赖性的ERα结合伴侣（间情期60个，动情期24个）。研究发现，PGR在间情期与ERα形成复合物，但在动情期并未形成，这支持两种受体在间情期存在协同结合。
黏连蛋白复合体（cohesin complex）的两种蛋白SMC1A与SMC3，同样在间情期与ERα形成复合物，而动情期未检测到该相互作用；SMC1A与ERα的重叠峰分析证实了这一间情期偏好性的相互作用。此外，HiC分析显示，间情期特异性的染色质相互作用数量多于动情期（476对vs263对），提示SMC1A/ERα相互作用对染色质结构具有功能性影响。
综上，激素受体、染色质结构与染色质可及性之间一系列复杂的相互作用，共同调控了动情周期依赖性的基因表达变化。
我们从2月龄未处理的完整CD-1小鼠中，分别于间情期（CoD）与动情期（CoE）采集子宫组织。随后开展了RNA-seq、ATAC-seq、ERα ChIP-seq、PGR ChIP-seq、低氧诱导因子2α（Hif2a）ChIP-seq及HiC-seq实验，并以ERα作为免疫沉淀抗体进行了RIME实验。