Mechanism of Chromatin Accessibility Mediated by Pioneer Transcription Factors

先锋转录因子介导的染色质可及性机制

• 批准号: 9307877
• 负责人: Meilin M. Fernandez Garcia
• 金额: $ 4.4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9307877
• 关键词: Achievement; Address; Affect; Affinity; Agreement; Albumins; Amaze; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Cell Therapy; Cells; Characteristics; Chromatin; Chromatin Structure; Chromosomes; Competence; Coupled; DNA; DNA Binding; Data; Dissociation; Ectopic Expression; Elements; Endoderm; Enhancers; Eukaryotic Cell; Event; Fibroblasts; Fluorescence Resonance Energy Transfer; GATA4 gene; Gene Activation; Gene Expression Regulation; Generations; Genes; Genome; Genomics; Goals; Head; Hepatic; Hepatocyte; Hydrogen; In Vitro; Knowledge; Literature; Liver; Maps; Mass Spectrum Analysis; Measures; Mediating; Modeling; Molecular; Mutagenesis; Neurons; Nucleosomes; Parents; Pennsylvania; Process; Protocols documentation; Public Health; Recruitment Activity; Regenerative Medicine; Regulator Genes; Research; Seminal; Structure; Students; Testing; Therapeutic; Training Programs; Translations; United States National Institutes of Health; Universities; Validation; Work; base; biophysical techniques; cell injury; cell type; improved; in vivo; insight; public health relevance; reconstitution; regenerative therapy; tool; transcription factor; transdifferentiation

 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.

 描述（由申请人提供）：我的研究生工作的目标是解决细胞类型特异性基因如何在细胞转分化过程中被激活的问题。为了提高转分化细胞用于再生疗法的效率;我们的知识 控制细胞转分化和细胞类型基因激活的机制需要大大扩展。在真核细胞中，染色质的DNA致密化通过调节转录因子（TF）对DNA的可接近性来作为基因调控的机制。然而，TF最初参与基因组并组织它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人已经表明，Foxa，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子中的核小体结合。因此，Foxa作为“先锋因子”，结合核小体，打开局部染色质并允许随后的转录因子结合。因此，鉴定具有类似于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工具。基于本实验室以往的发现和经验，我提出：（1）通过比较不同的转分化因子在体外与核小体结合的能力，鉴定作为细胞转分化先驱引发剂的转分化因子。通过核小体结合能力鉴定的TF将使用生物物理技术进行比较，包括通过质谱法和荧光共振能量转移（FRET）的氢交换。这将使深入了解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。该目标的实现将为深入了解细胞转分化的体内基因组机制提供帮助，生化分析将提供结构细节 功能领域和先锋因素的活动，这是我的培训计划的一个显着特点。