Unexpected nuclear hormone receptor and chromatin dynamics regulate estrous cycle dependent gene expression [ChIP-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.

发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同样具有动态性。因此，我们采用多维度方法，结合RNA测序（RNA-seq）、转座酶可及性测序（ATAC-seq）、染色质构象捕获测序（HiC-seq）以及针对雌激素受体α（ERα）和孕酮受体（PGR）的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大量发情周期依赖性变化。发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调控方向相关；与间情期相比，发情期的PGR结合水平降低，但ERα结合几乎无变化。上述差异表达基因附近存在2727个具有动态可及性的增强子，其中77%与基因表达变化的调控方向相关。不过，大多数增强子（8694个）为组成型开放状态。
无论是动态开放还是组成型开放的增强子，从间情期到发情期，ERα与PGR的结合均发生协同性丢失。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家族的结合基序。与之相反，发情期特异性的ERα结合发生在染色质相对闭合的转录因子荒漠区域；而发情期特异性的可及区域则高度富集ATF、ELF及ELK家族的结合基序。
内源蛋白快速免疫沉淀质谱（Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins, RIME）实验揭示了多个随发情周期变化的ERα互作蛋白：间情期有60种，发情期有24种。研究发现，间情期时PGR与ERα形成复合物，而发情期则无此现象，这支持两种受体在间情期存在协同结合。黏连蛋白复合物中的两种蛋白SMC1A与SMC3，在间情期可与ERα形成复合物，发情期则无此相互作用；SMC1A与ERα的共定位分析验证了这种在间情期的偏好性互作。此外，HiC-seq分析显示，间情期特异性染色质相互作用数量（476个）多于发情期（263个），这提示SMC1A与ERα的互作对染色质结构具有功能性影响。
综上，激素受体、染色质结构与可及性之间一系列复杂的相互作用，共同调控了发情周期依赖性的基因表达变化。
实验整体设计：选取2月龄、未接受任何处理的完整CD-1小鼠，分别在间情期（CoD）与发情期（CoE）采集其子宫组织。实验涵盖RNA-seq、ATAC-seq、ERα ChIP-seq、PGR ChIP-seq、Hif2a ChIP-seq及HiC-seq，同时以ERα为免疫沉淀抗体开展RIME实验。