Pioneering new enhancers by GATA3: role of facilitating transcription factors and chromatin remodeling [CUTnRUN]

The mechanistic details by which pioneer transcription factors interact with partner transcription factors and chromatin modification/remodeling enzymes to activate enhancers remains an emerging area of study. Here, we utilize inducible expression of the pioneer factor GATA3 and study cofactor association and structural alterations in local chromatin. ATAC-seq analysis and footprinting revealed co-occurrence of GATA3 with other transcription factors, including AP-1, in newly accessible chromatin. GATA3 and AP-1 co-localize at primed enhancers with p300 and BRG1 where nucleosome positioning is responsive to GATA3 location, not to AP-1. Genetic inhibition of AP-1 binding alters chromatin opening at some, but not most, GATA3-bound loci. Pharmacologic inhibition of SWI/SNF ATPase function results in loss of GATA3-dependent chromatin accessibility, binding, and alterations in local chromatin architecture. We conclude that GATA3-dependent gains in chromatin accessibility require chromatin remodeling and that accessibility at some loci is facilitated by a partner transcription factor, in this case AP-1. Overall design: Doxycycline (dox) inducible wildtype (wt; 1-444aa) and TA1del (ta1; 126-444aa) GATA3 cell lines were generated in SUM159PT basal breast cancer cell line. CUT&RUN was performed for histone modifications, transcription factor binding and chromatin remodeler binding in +/- DOX conditions and +/- inhibitiors of transcription factor binding (AFOS) or chromatin remodeling (BRM014).

先锋转录因子（pioneer transcription factor）与伴侣转录因子、染色质修饰/重塑酶相互作用并激活增强子的具体机制，仍是当前研究的新兴前沿方向。
本研究通过诱导表达先锋转录因子GATA3，探究其辅助因子结合模式与局部染色质的结构变化。
转座酶可及性测序（ATAC-seq）与足迹分析（footprinting）结果显示，GATA3与包括AP-1在内的其他转录因子共同出现在新开放的染色质区域中。
GATA3与AP-1可与p300、BRG1共同定位于预激活增强子区域，该区域的核小体定位仅受GATA3的位置调控，而非AP-1。
对AP-1结合的遗传抑制会改变部分（而非全部）GATA3结合位点处的染色质开放状态。
对SWI/SNF ATP酶功能的药物抑制会导致GATA3依赖的染色质可及性、结合能力丧失，并改变局部染色质结构。
本研究结论表明，GATA3介导的染色质可及性提升依赖于染色质重塑，且部分位点的可及性可通过伴侣转录因子（本研究中为AP-1）得以增强。
实验整体设计：在SUM159PT基底型乳腺癌细胞系中构建了多西环素（doxycycline，简称dox）诱导的野生型GATA3细胞系（wt；氨基酸序列1-444）与TA1缺失型GATA3细胞系（TA1del；氨基酸序列126-444）。
分别在添加/不添加多西环素，以及添加/不添加转录因子结合抑制剂（AFOS）或染色质重塑抑制剂（BRM014）的条件下，针对组蛋白修饰、转录因子结合与染色质重塑因子结合开展CUT&RUN实验。