Role of nucleosome architecture in cellular reprogramming

项目摘要 通过转录因子在精确的染色质位点形成增强子使得适当的基因激活成为可能 生物需求。这一基本过程受到数百万个转录匹配的挑战 因子结合基序存在于人类基因组中。染色质结构也被认为是作为一个 许多转录因子的物理屏障。转录因子如何克服染色质屏障 并选择性地激活适当的基因组位点，同时它们与染色质屏障竞争？ 这个基本问题的答案在很大程度上仍然是未知的。最近，转录的一个子集 这些因子，即所谓的先锋因子，被认为是修饰染色质所必需的蛋白质 在细胞重编程过程中的可访问性。与其他转录因子不同，先锋因子能够 在封闭的（非活性的）染色质位点与核小体结合并诱导染色质开放。以来 核小体的形成被认为是一个物理屏障，确定先锋因子的作用， 染色质是理解细胞重编程过程的基础。的误调节 先锋因子与包括癌症在内的多种人类疾病有关。因此，有一个关键的 需要确定潜在的先锋因子诱导细胞重编程的分子机制。 我们的长期目标是阐明转录因子诱导的细胞凋亡的分子机制。 重新编程我们以前证明GATA 3作为一个先锋因子，可以直接结合 关闭染色质并在间质向上皮转化期间激活上皮标记基因 (MET)在三阴性乳腺癌细胞中。使用该细胞重编程模型系统，我们进一步 鉴定了在GATA 3靶基因座富集的独特核小体定位。我们的Cryo-EM结构 对GATA 3-核小体复合物的分析表明，GATA 3对核小体上的基序识别， 核小体不同于无组蛋白的DNA。MET期间的染色质结构分析还确定， GATA3的染色质开放和从头增强子形成是位点特异性的，并且仅在一个细胞中观察到。 GATA3结合位点的子集。基于这些证据，我们假设核小体定位和 先锋因子GATA3结合位点的结构决定了读数（染色质开放和基因表达）。 激活）GATA3结合，并直接参与MET。此应用程序的主要目标是： (1)确定特定核小体定位和构象是成功GATA 3- 诱导细胞重编程;（2）确定染色质重塑酶在GATA 3- 诱导MET，和（3）发现GATA 3诱导增强子的最小染色质组分 阵我们将使用几种新的方法，包括高分辨率映射方法， 核小体定位和转录因子的足迹。这些结果将为 理解转录因子诱导的细胞重编程过程。

PROJECT SUMMARY Enhancer formation at precise chromatin loci by transcription factors enables appropriate gene activation to biological demand. This essential process is challenged by the millions of matches to transcription factor binding motifs present in the human genome. Chromatin structure is also known to act as a physical barrier for many transcription factors. How do transcription factors overcome chromatin barriers and selectively activate appropriate genomic loci while they are competing against chromatin barriers? The answer to this fundamental question is still largely unknown. Recently, a subset of transcription factors, so-called pioneer factors, have been identified as essential proteins required to modify chromatin accessibility during cellular reprogramming. Unlike other transcription factors, pioneer factors are capable of binding to nucleosomes at closed (inactive) chromatin sites and inducing chromatin opening. Since nucleosome formation was thought to act as a physical barrier, identification of pioneer factors’ action on chromatin is fundamental to understanding the cellular reprogramming processes. Misregulation of pioneer factors is associated with various human diseases including cancer. Therefore, there is a critical need to identify the molecular mechanisms underlying pioneer factor-induced cellular reprogramming. Our long-term goal is to elucidate the molecular mechanisms of transcription factor-induced cellular reprogramming. We previously demonstrated that GATA3 acts as a pioneer factor that can directly bind closed chromatin and activate epithelial marker genes during the mesenchymal-to-epithelial transition (MET) in triple-negative breast cancer cells. Using this cellular reprogramming model system, we further identified the unique nucleosome positioning enriched at GATA3 target loci. Our Cryo-EM structure analysis of the GATA3-nucleosome complex revealed that the motif recognition by GATA3 on the nucleosome differs from histone-free DNAs. Chromatin structural analysis during MET also identified that chromatin opening and de novo enhancer formation by GATA3 is site-specific and only observed at a subset of GATA3 binding sites. Based on this evidence, we hypothesize that nucleosome positioning and structure at the pioneer factor GATA3 binding site dictate the readout (chromatin opening and gene activation) of GATA3 binding and is directly involved in MET. The primary goals of this application are to: (1) identify specific nucleosome positioning and conformation that are essential for successful GATA3- induced cellular reprogramming; (2) identify the roles of chromatin remodeling enzymes during GATA3- induced MET, and; (3) discover minimum chromatin components for GATA3-induced enhancer formation. We will use several novel approaches including high-resolution mapping methods for nucleosome positioning and transcription factors’ footprint. These results will provide a basis for understanding cellular reprogramming processes induced by transcription factors.