Influences of DNA sequence and histone features on transcription factor binding to nucleosomes

摘要 基因表达程序是动态调节的染色质的可及性， 转录因子（TF）结合，但TF如何识别特定的调控区域闭塞 核小体仍不清楚。某些TF，称为先驱因子，可以识别它们的目标 核小体内的位点，导致染色质的开放。通过引发顺式调节 随后的转录调控活动的元件，先驱者作为看门人， 细胞分化。虽然先驱者可以结合核小体位点，但它们仅结合核小体的一个子集。 它们在基因组中的潜在识别位点通常因细胞类型而异，因此 表明它们与序列、表观遗传或其他细胞特征的相互作用。 尽管先锋因素的重要性，什么限制先锋绑定是知之甚少。 关于它们在核小体中的位点的序列背景， 组蛋白变体或组蛋白的翻译后修饰（PTM），或与 识别这些PTM的辅因子或染色质阅读器可能会影响先锋结合， 核小体尚未开发高通量技术来调查 这些参数对TF先锋绑定的影响。 在这个项目中，我们将开发新的，高通量的生化分析，以研究如何 核小体序列背景、组蛋白变体或组蛋白PTM影响 将人TF转化为核小体。我们还将研究先驱者，辅因子和 pioneer binding中的染色质阅读器。这些生化试验的结果将在 体外和用于分析人体细胞中的体内基因组数据，以了解这些 各种特征有助于TF先锋在细胞中的结合。 由于先锋因素在监管层级的最高层发挥着至关重要的作用，这些结果将有助于 了解细胞状态的基因调控如何在基因组中编码， 它被读出的机制。

Abstract Gene expression programs are dynamically regulated by the accessibility of chromatin for transcription factor (TF) binding, but how TFs recognize specific regulatory regions occluded by nucleosomes remains unclear. Certain TFs, termed pioneer factors, can recognize their target sites within nucleosomes, leading to the opening of chromatin. By priming cis-regulatory elements for subsequent transcriptional regulatory activity, pioneers serve as gatekeepers to cellular differentiation. Although pioneers can bind nucleosomal sites, they bind only a subset of their potential recognition sites in the genome that typically varies across cell types, thus indicating their interplay with sequence, epigenetic or other cellular features. Despite the importance of pioneer factors, what restricts pioneer binding is poorly understood. Little is known about how the sequence context of their sites in nucleosomes, the presence of histone variants or post-translational modifications (PTMs) of histones, or interactions with cofactors or chromatin readers that recognize those PTMs might influence pioneer binding to nucleosomes. No high-throughput technologies have been developed to survey the impact of these many parameters on TF pioneer binding. In this project, we will develop novel, high-throughput biochemical assays to investigate how nucleosomal sequence context, histone variants or histone PTMs influence pioneer binding of human TFs to nucleosomes. We will also investigate the interplay of pioneers, cofactors, and chromatin readers in pioneer binding. Results from these biochemical assays will be validated in vitro and used in analysis of in vivo genomic data in human cells to understand how these various features contribute to TF pioneer binding in cells. As pioneer factors play crucial roles at the top of regulatory hierarchies, these results will aid in understanding how gene regulation of cell states is encoded in the genome and the mechanisms by which it is read out.