Mechanism of Chromatin Accessibility Mediated by Pioneer Transcription Factors

 描述(由申请人提供)：我研究生工作的目标是解决细胞类型特定基因在细胞转分化过程中如何被激活的问题。为了提高转分化细胞用于再生治疗的效率；我们的知识 在控制细胞转分化和细胞类型基因激活的机制中，需要极大地扩展。在真核细胞中，染色质的DNA紧致作用是通过调节转录因子对DNA的可及性来调控基因的机制。然而，转录因子最初参与基因组并对其进行组织以允许特定细胞类型决定的机制仍有待阐明。我们的实验室和其他实验室已经表明，FOXA是一种肝脏特异性因子，在内胚层形成的早期阶段与白蛋白增强剂中的核小体结合。因此，FOXA扮演着“先锋因子”的角色，与核小体结合，打开局部染色质，并允许与随后的转录因子结合。因此，识别具有类似FOXA的先锋活性的转分化因子，为理解细胞转分化的初始步骤提供了重要的工具。基于我们实验室以前的发现和专业知识，我建议：(1)通过比较不同的转分化因子在体外结合核小体的能力来鉴定作为细胞转分化先驱启动者的转录因子。将使用生物物理技术，包括质谱氢交换和荧光共振能量转移(FRET)来比较由核小体结合能力确定的TF。这将使我们深入了解TF结合引起的核小体结构的变化。2)确定转录因子转分化能力是否需要核小体结合能力。所提出的目标的实现将提供对细胞转分化的体内基因组机制的洞察，生化分析将提供结构细节 功能域和先锋因子的活性，这是我培训计划的一个显著特点。

 DESCRIPTION (provided by applicant): The goal of my graduate work is to address the question of how cell type specific genes become activated during cellular transdifferentiation. In order to increase efficiency of transdifferentiated cells for regenerative therapies; our knowledge of the mechanisms that control cell transdifferentiation and cell type gene activation needs to be greatly expanded. In eukaryotic cells, DNA compaction of chromatin serves as a mechanism for gene regulation by modulating the accessibility of transcription factors (TFs) to DNA. However, the mechanism by which TFs initially engage in the genome and organize it to allow specific cell type decisions remains to be elucidated. Our lab and others have shown that Foxa, a liver-specific factor, binds in early stages of endoderm formation to a nucleosome in the albumin enhancer. Thereby Foxa acts as a "pioneer factor", binding nucleosomes, opening local chromatin and allowing binding of subsequent transcription factors. Identification of transdifferentiation factors with pioneer activity similar to Foxa, thus provides a crucial tool inthe understanding of the initial steps in cell transdifferentiation. Based on our lab's previous discoveries and expertise I propose to: (1) identity TFs that act as pioneer initiators of cell transdifferentiation by comparing diverse transdifferentiation factors ability to bind nucleosomes in vitro. TFs identified by nucleosome binding ability will be compared using biophysical techniques including hydrogen exchange by mass spectrometry and fluorescence resonance energy transfer (FRET). This will give insight into changes in nucleosome structure caused by TF binding. 2) Determine if nucleosome binding capacity is required for TFs transdifferentiation ability. Achievement of the proposed goal will provide insights into the in vivo genomic mechanisms of cellular transdifferentiation and biochemical analysis will provide structural detail of functional domains and activity of pioneer factors, which is a distinguishing feature of my training program.