RNA fine-tunes estrogen receptor-alpha binding on low-affinity DNA motifs for transcriptional regulation

Transcription factors (TFs) regulate gene expression by binding, with varying strength, to DNA via their DNA-binding domain. Additionally, some TFs also interact with RNA, which modulates transcription factor binding to chromatin. However, whether RNA-mediated TF binding results in differential transcriptional outcomes remains unknown. In this study, we demonstrate that estrogen receptor α (ERα), a ligand-activated TF, interacts with RNA in a ligand-dependent manner. Defects in RNA binding lead to genome-wide loss of ERα recruitment, particularly at weaker ERα-motifs. Furthermore, ERα mobility in the nucleus increases in the absence of its RNA-binding capacity. Unexpectedly, this increased mobility coincides with robust polymerase loading and transcription of ERα-regulated genes that harbor low-strength motifs. However, highly stable binding of ERα on chromatin negatively impacts ligand-dependent transcription. Collectively, our results suggest that RNA interactions spatially confine ERα on low-affinity sites to fine-tune gene transcription. Chromatin Immunoprecipitation (ChIP-seq) for the FLAG tagged WT and RBM Estrogen receptor-alpha upon 72 hours of hormone deprivation and 1 hour of E2 signaling. RNAseA ChIP-seq of Estrogen receptor- alpha with and without pre-extraction after 72 hours of hormone deprivation and 1 hour of E2 siganling . ChIP-seq of total PolII upon 1 hour of E2 siganling with expression of either FLAG tagged WT or RBM Estrogen receptor alpha. Paired factor ChIP with antiFLAG and anti-CTCF upon expression of either FLAG tagged WT or RBM Estrogen receptor alpha.

转录因子（Transcription factors, TFs）通过其DNA结合结构域与DNA以不同强度特异性结合，进而调控基因表达。此外，部分转录因子还可与RNA发生相互作用，该过程会调节转录因子与染色质的结合能力。然而，RNA介导的转录因子结合是否会引发差异化的转录结局，目前仍未明确。
本研究证实，配体激活型转录因子雌激素受体α（estrogen receptor α, ERα）能够以配体依赖性的方式与RNA结合。RNA结合功能缺陷会导致全基因组范围内ERα招募的丢失，尤其富集于较弱的ERα结合基序区域。进一步研究发现，当ERα丧失RNA结合能力时，其在细胞核内的移动性会显著升高。
出乎意料的是，这种移动性增强与携带低强度结合基序的ERα调控基因的聚合酶大量加载及转录激活同步发生。然而，ERα在染色质上的高度稳定结合则会对配体依赖性的转录过程产生负面影响。综上，本研究结果表明，RNA相互作用可将ERα空间限制在低亲和力结合位点，以此精准微调基因的转录过程。
本研究生成的测序数据集如下：
1. 经72小时激素剥夺、1小时雌二醇（E2）信号刺激后，对FLAG标记的野生型（WT）与RBM突变型ERα进行染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）；
2. 经72小时激素剥夺、1小时E2信号刺激后，分别在预抽提与未预抽提条件下，对ERα进行核糖核酸酶A（RNAseA）辅助的ChIP-seq；
3. 分别表达FLAG标记的野生型或RBM突变型ERα，经1小时E2信号刺激后，对总RNA聚合酶II（PolII）进行ChIP-seq；
4. 分别表达FLAG标记的野生型或RBM突变型ERα，利用抗FLAG与抗CTCF抗体进行配对因子染色质免疫共沉淀测序。