Analyzing pioneer factor dynamics and function during differentiation and reprogramming

项目总结/摘要 这项建议的长远目标包括： 确定先驱因子-核小体相互作用的频率和必要性 在细胞分化期间全基因组，和2）表征个体 和合作先锋因子-核小体相互作用， 诱导多能性。细胞命运的决定需要精确的时空 调节转录因子（TF）与增强子的结合以介导基因 调节，这在各种疾病中经常被选择。公 确定了活性增强子局部耗尽核小体，并且 TF结合与局部染色质一致，并且通常是局部染色质所需的 可访问性。然而，目前尚不清楚TF如何在以下方面发挥积极作用： 在增强子处置换核小体。一种假设是，“先驱”TF， 因为它们在细胞分化的早期表达，并与它们的 增强子靶点在它们被赋予强可及性之前，直接结合并 取代核小体以便于接近。然而，这种机制 从未在体内单个模板上进行验证，主要是由于 在测试推定的中间TF-核小体颗粒的存在。 由于缺乏对体内先锋活性的直接测试， 先驱TF的核小体驱逐仍然未经测试，并且 先驱活动在很大程度上是未知的。此外， 自然分化系统或TF过表达中的TF先锋活性 细胞重编程过程中的环境仍然没有特征。直接测试 先驱TF模型，我将利用原位，微球菌核酸酶（MNase） 基于消化的ChIP-seq替代品，称为CUT&RUN，其中DNA 片段大小是最小蛋白质保护序列的直接信息， 因此可以区分直接TF结合和通过 核小体中间体我要用这些信息来确定基因组- 胚胎干细胞中先驱因子-核小体相互作用的广阔前景 分化和诱导多能性过程中，并测试这样的必要性， 利用功能性遗传学实验进行相互作用。这些见解将帮助我们 了解先驱因子如何激活致病基因表达 在癌症等疾病中。我觉得我提出的项目符合使命， NIGMS资助基础生物学研究，揭示新的见解， 对人类健康有积极影响。 !

Project Summary/Abstract The long-term objectives of this proposal include the following: 1) to determine the frequency and necessity of pioneer factor-nucleosome interactions genome wide during cell differentiation, and 2) to characterize the individual and cooperative pioneer factor-nucleosome interactions that occur during induced pluripotency. Cell fate decisions require exquisite spatiotemporal regulation of transcription factor (TF) binding to enhancers to mediate gene regulation, which is frequently co-opted in a variety of diseases. It is well established that active enhancers are locally depleted of nucleosomes, and that TF binding is coincident with, and often required for, local chromatin accessibility. However, it remains unclear how TFs might play an active role in displacing nucleosomes at enhancers. One hypothesis is that “pioneer” TFs, so called because they are expressed early in cell differentiation and bind to their enhancer targets before they are rendered strongly accessible, directly bind and displace nucleosomes to facilitate accessibility. However, such a mechanism has never been validated on individual templates in vivo, chiefly due to the difficulty in testing for the presence of the putative intermediate TF-nucleosome particle. Lacking a direct test for in vivo pioneer activity, putative mechanisms for nucleosome eviction by pioneer TFs remain untested, and the locus specificity of pioneer activity is largely unknown. Moreover, the frequency and importance of TF pioneer activity in either natural differentiation systems or TF overexpression contexts during cellular reprogramming remain uncharacterized. To directly test the pioneer TF model, I will leverage an in situ, micrococcal nuclease (MNase) digestion-based alternative to ChIP-seq known as CUT&RUN, in which DNA fragment size is directly informative of the minimal protein-protected sequence, and therefore can distinguish direct TF binding from binding through a nucleosomal intermediate. I will use this information to determine the genome- wide landscape of pioneer factor-nucleosome interactions in embryonic stem cell differentiation and during induced pluripotency, and test the necessity of such interactions using functional genetic experiments. These insights will help us to understand how pioneer factors function to activate pathogenic gene expression in diseases such as cancer. I feel that my proposed project fits the mission of NIGMS to fund research in fundamental biology that uncovers new insights that could positively impact human health. !