Distinct Chromatin Scanning Modes Lead to Targeting of Compacted Chromatin by Pioneer Factors FOXA1 and SOX2 [Cut&Run]

Pioneer transcription factors, by interacting with nucleosomes, scan silent, compact chromatin to target regulatory sequences, enabling cooperative binding events that modulate local chromatin structure and gene activity. However, not all cognate motifs are targeted by pioneer factors and dynamic scanning parameters required to scan compact chromatin are unknown. A combined genomics and single-molecule tracking approach shows that to target DNase-resistant, low-histone turnover sites, pioneer factors FOXA1 and SOX2 display opposite dynamics of chromatin scanning: slow, with low nucleoplasmic diffusion and stable interactions, versus fast, with high nucleoplasmic diffusion and transient interactions, respectively. Despite such differences, the ability of FOXA1 and SOX2 to scan low-mobility chromatin, mediated by protein domains outside of the respective DNA binding domains, leads to targeting silent chromatin. By contrast, non-pioneer HNF4A predominantly targets DNase-sensitive, nucleosome-depleted regions. We conclude that the targeting of compact chromatin sites by pioneer factors can be performed through diverse dynamic processes. Cleavage under Targets and Release Using Nuclease (CUT&RUN) of SOX2 and SOX2-DBD after ectopic expression in BJ fibroblasts. Overall design: Andrew,Katznelson

先驱转录因子（Pioneer transcription factors）通过与核小体相互作用，扫描沉默且致密的染色质以靶向调控序列，进而介导协同结合事件，调节局部染色质结构与基因活性。然而，并非所有同源基序均可被先驱转录因子靶向，且扫描致密染色质所需的动态扫描参数仍未被阐明。结合基因组学与单分子追踪（single-molecule tracking）的研究方法表明，为靶向抗DNase、低组蛋白更新的染色质位点，先驱转录因子FOXA1与SOX2展现出截然相反的染色质扫描动力学特征：前者表现为缓慢的核质扩散与稳定的相互作用，后者则表现为快速的核质扩散与瞬时的相互作用。尽管存在上述差异，FOXA1与SOX2凭借其DNA结合结构域之外的蛋白质结构域（protein domains）所介导的扫描低迁移率染色质的能力，仍可实现对沉默染色质的靶向。相较而言，非先驱型转录因子HNF4A主要靶向DNase敏感、核小体缺失区域（nucleosome-depleted regions）。本研究得出结论：先驱转录因子可通过多样化的动态过程实现对致密染色质位点的靶向。本研究在BJ成纤维细胞（BJ fibroblasts）中外源异位表达SOX2及SOX2-DBD后，开展了核酸酶靶标切割与释放（Cleavage under Targets and Release Using Nuclease, CUT&RUN）实验。整体实验设计：Andrew、Katznelson