Phase Separation of FOXA1 Unpacks the Condensed Chromatin to Function as a Pioneer Factor (NIH-3T3 cells). Phase Separation of FOXA1 Unpacks the Condensed Chromatin to Function as a Pioneer Factor (NIH-3T3 cells)

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. Overall design: ChIP-seq and ATAC-seq for vector,overexpress FH and FOXA1 in NIH-3T3 cells.

真核生物DNA在细胞核内被包装为染色质，该过程会限制转录因子（TFs）与靶DNA位点的结合。FOXA1作为先驱转录因子，可结合致密染色质并启动局部染色质的开放以调控基因表达。然而，FOXA1被招募至致密染色质并对其进行解聚的分子机制仍不明确。本研究发现，FOXA1可通过其N端朊病毒样低复杂度结构域（N-PrLD）与C端内在无序区域（C-IDR）发生固有相分离，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结合能力亦有助于其相分离特性的发挥。进一步的全基因组分析表明，FOXA1可通过其相分离区域被招募至致密染色质并对其解聚，从而调控乳腺癌细胞的功能。本研究为先驱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。整体实验设计：在NIH-3T3细胞中分别转染空载体、过表达FH与过表达FOXA1，随后开展染色质免疫共沉淀测序（ChIP-seq）与转座酶可及性测序（ATAC-seq）。