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 (ER) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ER 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 ER. 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, ER and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ER binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ER 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 ER (diestrus, 60; estrus, 24). PGR was found in complex with ER 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 ER during diestrus but not estrus; overlap of SMC1A with ER confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ER 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.动情周期中依赖激素的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，我们采用多维度研究策略，结合RNA测序（RNA-seq）、转座酶可及性测序（ATAC-seq）、Hi-C测序（HiC-seq）以及针对雌激素受体α（ER）和孕酮受体（PGR）的染色质免疫共沉淀测序（ChIP-seq），对激素受体占据、染色质可及性与染色质结构的体内动态变化展开了系统性分析。全基因组水平上，ER与PGR的结合模式以及染色质可及性均存在广泛的动情周期依赖性改变。动情期与间情期的子宫组织间共鉴定出4159个差异表达基因。在转录起始位点区域，染色质可及性通常与基因表达的变化方向呈正相关；与间情期相比，动情期的PGR结合水平显著降低，但ER的结合水平无明显变化。上述差异表达基因附近共存在2727个可及性动态变化的增强子，其中77%的增强子可及性变化与对应基因的表达变化方向高度相关。不过，绝大多数增强子（共8694个）始终处于开放状态。无论是动态开放还是持续开放的增强子，ER与PGR的结合均从间情期到动情期发生协同性减弱。间情期特异性的ER结合区域与染色质可及区域，显著富集了PGR、FOX、GATA以及SOX家族转录因子的结合基序。与之形成对比的是，动情期特异性的ER结合区域位于转录因子荒漠（transcription factor deserts）内，所处染色质相对闭合；而动情期特异性的染色质可及区域则高度富集ATF、ELF与ELK家族转录因子的结合基序。内源蛋白快速免疫沉淀质谱（RIME）实验结果显示，ER存在多个随动情周期变化的互作蛋白：间情期中可鉴定到60种互作蛋白，动情期中仅鉴定到24种。研究发现，在间情期，PGR可与ER形成蛋白复合物，但动情期无该复合物形成，这一结果支持两种受体在间情期协同结合的结论。黏连蛋白复合体的两个核心亚基SMC1A与SMC3，同样在间情期与ER形成蛋白复合物，而动情期未检测到该相互作用；SMC1A与ER的共定位实验进一步验证了间情期的这种优先互作关系。此外，Hi-C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vs 263对），提示SMC1A与ER的互作对染色质结构具有功能性调控作用。综上，激素受体、染色质结构与染色质可及性之间的一系列复杂相互作用，共同调控了动情周期依赖性的基因表达变化。本研究采集了2月龄未经过任何处理的CD-1小鼠在间情期（CoD）与动情期（CoE）的子宫组织。随后开展了RNA-seq、ATAC-seq、ERα ChIP-seq、PGR ChIP-seq、Hif2a ChIP-seq及HiC-seq实验，并以ERα作为免疫沉淀抗体完成了RIME检测。