Phase Separation of FOXA1 Unpacks the Condensed Chromatin to Function as a Pioneer Factor [ATAC-seq]

Eukaryotic DNA is packaged into chromatin in the nucleus, which restricts the binding of transcription factors (TFs) to the target DNA sites. FOXA1 functions as a pioneer TF to bind condensed chromatin and initiates the opening of local chromatin for gene expression. However, the principles of how FOXA1 recruits to and unpacks the condensed chromatin remain elusive. Here, we revealed that FOXA1 intrinsically undergoes phase separation through its N-PrLD and C-IDR regions, identified as phase separation region (PSR). Notably, the phase separation property confers FOXA1 the ability to dissolve the chromatin condensates. In addition, the DNA-binding capacity of FOXA1 contributes to its phase separation property. Further genome-wide investigation showed that FOXA1 is recruited to and unpacks the condensed chromatin by its PSR to regulate the function of breast cancer cells. This work provides a principal example of how pioneer TFs function to initiate competent chromatin states by phase separation. ChIP-seq,ATAC-seq and RNA-seq for vector,overexpress FH and FOXA1 in MDA-MB-231 cells

真核生物的DNA在细胞核内被包装为染色质，这一结构会限制转录因子（transcription factors, TFs）与靶DNA位点的结合。FOXA1作为一类先锋转录因子（pioneer TF），能够结合浓缩状态的染色质，并启动局部染色质的开放以调控基因表达。然而，FOXA1被招募至浓缩染色质并介导其解聚的分子机制仍有待阐明。本研究发现，FOXA1可通过其N端朊蛋白样结构域（prion-like domain, PrLD）与C端内在无序区域（intrinsically disordered region, IDR）发生内在相分离，上述区域被鉴定为相分离区域（phase separation region, PSR）。值得注意的是，相分离特性赋予FOXA1溶解染色质凝聚物的能力。此外，FOXA1的DNA结合能力对其相分离特性具有促进作用。进一步的全基因组分析显示，FOXA1可通过其相分离区域被招募至浓缩染色质并介导其解聚，进而调控乳腺癌细胞的生物学功能。本研究为先锋转录因子如何通过相分离启动具备转录活性的染色质状态提供了典型范例。本研究针对在MDA-MB-231细胞中转染空载体、过表达FH与FOXA1的样本，开展了ChIP-seq、ATAC-seq及RNA-seq实验。