Estrogen induces dynamic ERa and RING1B recruitment to control gene and enhancer activities in luminal breast cancer. Estrogen induces dynamic ERa and RING1B recruitment to control gene and enhancer activities in luminal breast cancer

We have previously shown that RING1B, a core Polycomb Repressive Complex 1 subunit, is amplified in 22% of breast cancer patients. In ER+ breast cancer, RING1B functionally interacts and co-localizes with ERa at enhancers and actively transcibed genes to modulate oncogenic transcriptional programs. However, the molecular mechanisms underlying this interaction is unclear. Here we demonstrate that in ER+ breast cancer cells, prolonged estrogen (E2) administration induces fluctuations in transcriptional output and chromatin landscape. RING1B loss impairs full E2-mediated gene expression and chromatin accessibility at genomic loci where key breast cancer transcription factors bind. RING1B mediates E2-induced gene expression and chromatin architectural changes both dependently and independently of its enzymatic activity and nucleosomal binding ability. We found that RING1B is recruited to ER, FOXA1, and GRHL2 co-bound sites in a cyclic manner during E2 administration and regulates E2-induced enhancers and ER recruitment. Finally, we characterized the spacial organization pattern between ER, FOXA1, GRHL2, and RING1B on the chromatin at single-nucleotide resolution genome-wide. Overall design: ChIP-seq of H3K27Ac, RING1B, ERa, and FOXA1 in T47D shCtrl and shRING1B cells in HD and after 45', 8h, and 24h of E2 treatment. Pair-end ATAC-seq of T47D shCtrl and shRING1B cells in HD and after 4h, 8h, 12h, and 24h of E2 in duplicates. RNA-seq of T47D shCtrl and shRING1B cells (same cells as the ATAC-seq) in HD and after 4h, 8h, 12h, and 24h of E2 in duplicates. RNA-seq of T47D cells expressing RING1B wild type, RING1B R98A, and RING1B I53A in HD and after 24h of E2 in duplicates. ChIP-exo of RING1B, ERa, FOXA1, and GRHL2 in T47D parental cells in HD and after 45' of E2 treatment. RNA-seq of T47D shRING1B cells rescued with exogenous RING1B wild type in HD and after 24h of E2 in duplicates.

我们此前已证实，多梳抑制复合体1（Polycomb Repressive Complex 1, PRC1）的核心亚基RING1B在22%的乳腺癌患者中存在扩增。在雌激素受体阳性（ER+）乳腺癌中，RING1B可与雌激素受体α（ERα）在增强子区域及活跃转录基因位点发生功能互作并共定位，以调控致癌性转录程序。然而，该互作背后的分子机制尚不明确。
本研究证实，在ER+乳腺癌细胞中，长期雌激素（E2）处理会诱导转录输出与染色质景观发生动态波动。RING1B缺失会损害E2介导的完整基因表达程序，以及关键乳腺癌转录因子结合基因组位点处的染色质可及性。RING1B所介导的E2诱导基因表达与染色质构象变化，既依赖于其酶促活性与核小体结合能力，也可不依赖于该二者发挥功能。
我们发现，在E2处理过程中，RING1B会以循环招募的方式结合至ER、FOXA1与GRHL2的共结合位点，并调控E2诱导的增强子区域与ER的招募过程。最后，我们在全基因组单核苷酸分辨率水平上，解析了ER、FOXA1、GRHL2与RING1B在染色质上的空间组织模式。

实验整体设计：
1. 针对T47D细胞中转入对照短发卡RNA（shCtrl）与RING1B靶向短发卡RNA（shRING1B）的细胞，在激素剥夺（hormone-deprived, HD）培养条件下，以及E2处理45分钟、8小时、24小时后，进行H3K27乙酰化（H3K27Ac）、RING1B、ERα以及FOXA1的染色质免疫共沉淀测序（ChIP-seq）。
2. 对上述T47D shCtrl与shRING1B细胞，在HD条件下以及E2处理4小时、8小时、12小时、24小时后，进行双生物学重复的双端转座酶可及性测序（ATAC-seq）。
3. 采用与ATAC-seq相同来源的T47D shCtrl与shRING1B细胞，在HD条件下以及E2处理4小时、8小时、12小时、24小时后，进行双生物学重复的转录组测序（RNA-seq）。
4. 对分别过表达野生型RING1B、RING1B R98A突变体与RING1B I53A突变体的T47D细胞，在HD条件下以及E2处理24小时后，进行双生物学重复的RNA-seq。
5. 在T47D亲本细胞中，于HD条件下以及E2处理45分钟后，对RING1B、ERα、FOXA1以及GRHL2进行染色质免疫共沉淀-外切酶测序（ChIP-exo）。
6. 对通过外源野生型RING1B挽救RING1B敲低的T47D细胞，在HD条件下以及E2处理24小时后，进行双生物学重复的RNA-seq。