Role of nucleosome architecture in cellular reprogramming

项目总结 转录因子在染色质精确位点形成增强子，从而实现适当的基因激活 以满足生物需求。这一基本的过程受到了数百万匹配转录的挑战 人类基因组中存在的因子结合基序。染色质结构也被认为是一种 许多转录因子的物理屏障。转录因子如何克服染色质障碍 并在与染色质障碍竞争时选择性地激活适当的基因组基因座？ 这个根本问题的答案在很大程度上仍是个未知数。最近，转录的一个子集 因子，即所谓的先驱因子，已被确定为修饰染色质所需的基本蛋白质。 在蜂窝重新编程期间的可访问性。与其他转录因子不同，先锋因子能够 在封闭的(不活跃的)染色质部位与核小体结合，并诱导染色质开放。自.以来 核小体的形成被认为是一种物理屏障，识别先锋因子在 染色质是理解细胞重编程过程的基础。监管不善 先锋因子与包括癌症在内的各种人类疾病有关。因此，有一个关键的 需要确定先锋因子诱导的细胞重编程背后的分子机制。 我们的长期目标是阐明转录因子诱导的细胞的分子机制。 重新编程。我们以前证明了GATA3是一个先驱因子，可以直接结合 间充质向上皮间充质转化过程中封闭染色质和激活的上皮标记基因 (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GATA3靶点丰富的独特的核小体定位。我们的低温电磁结构 对GATA3-核小体复合体的分析表明，GATA3对 核小体不同于不含组蛋白的DNA。MET期间的染色质结构分析也证实了 GATA3的染色质开放和从头形成增强子是位点特异性的，仅在 GATA3结合位点的子集。基于这一证据，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出(染色质开放和基因 GATA3结合的激活)，并直接参与MET。此应用程序的主要目标是： (1)确定对成功的GATA3-至关重要的特定核小体定位和构象 诱导细胞重编程；(2)确定染色质重塑酶在GATA3- (3)发现GATA3诱导增强子的最低染色质组分 队形。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。这些结果将为今后的研究提供依据 了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.