Dissecting principles of transcription factor binding

解析转录因子结合的原理

• 批准号: 9907905
• 负责人: Amy Fan Chen
• 金额: $ 6.53万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907905
• 关键词: ATAC-seq; Address; Affect; Automobile Driving; Bar Codes; Binding; Binding Sites; Bone Marrow Cells; CRISPR/Cas technology; Cell Line; Cell physiology; Cell surface; Cells; ChIP-seq; Characteristics; Chromatin; Collaborations; DNA; Data; Defect; Development; Disease; Ectopic Expression; Environment; Epigenetic Process; GATA1 gene; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome; Genomic Segment; Genomics; Goals; Histones; Human; Human Development; Individual; K-562; Learning; Link; Malignant Neoplasms; Measures; Metabolic Pathway; Methods; Modeling; Molecular; Mutation; Myelogenous; Myeloid Progenitor Cells; Normal Cell; Nucleosomes; Oligonucleotides; Organism; Phenotype; Play; Population; Proteins; Protocols documentation; Regulation; Regulator Genes; Research; Resources; Role; Signal Pathway; Site; Somatic Cell; Specificity; Stains; Techniques; Technology; Testing; Time; Training; Variant; antibody conjugate; base; cell type; chromatin modification; combinatorial; developmental disease; differential expression; dosage; effective therapy; epigenetic variation; improved; induced pluripotent stem cell; next generation sequencing; overexpression; pluripotency; predictive modeling; progenitor; spatiotemporal; transcription factor; transcriptome sequencing

Project Summary Transcription factors (TFs) are the master regulators of cell identity, coordinating precise control of specific gene networks to drive all the cellular processes necessary for a particular cell fate. The ability of TFs to regulate cell identity is highlighted by the discovery that ectopic expression of a cocktail of pluripotency TFs can reprogram somatic cells into induced pluripotent stem cells. Mutations or improper dosage of certain TFs can also result in various diseases such as cancer. While proper TF regulation is clearly important for normal cell function, our understanding of how TFs achieve such precise control of over a gene network is lacking. Specifically, the principles that govern how TFs bind to the correct set of target sites in a cell remain unclear. For example, TFs generally do not bind all sites containing its target sequence motif within the genome, and many TF binding sites do not contain the canonical sequence motif. Furthermore, a TF expressed in multiple cell types can bind to distinct sites in each cell type. This suggests that the cellular context plays a critical role in determining a TF’s binding sites. To improve our ability to predict and model TF binding and activity, and therefore drive cell fate, I propose to dissect how the molecular environment within a cell influences where a TF binds and what the subsequent effects are on chromatin accessibility and gene expression. I hypothesize that while the binding sequence motif is most important in directing TF binding, the presence of specific chromatin modifications and cooperative binding partners is important to facilitate TF binding especially at lower TF expression levels, even for pioneer factors that can access nucleosome-occluded DNA. I will test this hypothesis through three specific aims. In aim 1, I will examine how TF expression levels affect binding, chromatin accessibility, and gene expression using new methods I have optimized to measure intracellular protein levels and chromatin accessibility profiles in the same bulk cell population and in single cells. In aim 2, I will determine how the presence of specific chromatin modifications can influence where a TF binds in different cell types. In aim 3, I will directly connect the variability in expression of a TF and its cooperative binding partners with differential chromatin accessibility profiles observed in the highly heterogeneous common myeloid progenitor population and determine how this affects cell fate. These studies will provide a detailed analysis of the role that various factors play in regulating TF binding and enable us to develop more accurate predictions of where specific TFs are bound in a cell, how they will affect the gene regulatory network to drive a particular cell state, and how disrupting TF activity can result in disease states. These studies will be carried out in the Stanford Genetics department, where I will have access to the latest next-generation sequencing technology as well as other world-class resources. By carrying out this project, I will further my graduate training in investigating the mechanisms underlying gene regulation by learning and developing cutting edge genomic techniques to answer questions that have previously been difficult to address using existing methods.

项目摘要 转录因子（TF）是细胞身份的主要调节因子，协调特异性转录因子的精确控制。 基因网络来驱动特定细胞命运所必需的所有细胞过程。TF的能力， 多能性TF混合物的异位表达， 可以将体细胞重编程为诱导多能干细胞。某些TF的突变或剂量不当 也可能导致各种疾病，如癌症。虽然适当的TF调节对于正常的 细胞功能，我们对TF如何实现对基因网络的精确控制的理解是缺乏的。 具体来说，控制TF如何与细胞中正确的靶位点结合的原理仍然不清楚。 例如，TF通常不结合基因组内含有其靶序列基序的所有位点，并且 许多TF结合位点不含典型序列基序。此外，以多个表达的TF 细胞类型可以结合到每种细胞类型中的不同位点。这表明细胞环境起着关键作用 来确定TF的结合位点。为了提高我们预测和建模TF结合和活性的能力， 因此，驱动细胞命运，我建议解剖细胞内的分子环境如何影响， TF结合以及随后对染色质可及性和基因表达的影响。我假设 虽然结合序列基序在指导TF结合中是最重要的，但特异性结合序列基序的存在是重要的。 染色质修饰和协同结合配偶体对于促进TF结合是重要的，特别是在 较低的TF表达水平，甚至对于可以进入核小体封闭的DNA的先锋因子。我来测试一下 通过三个具体目标的假设。在目标1中，我将研究TF表达水平如何影响结合， 染色质可及性和基因表达，使用我优化的新方法来测量细胞内 蛋白水平和染色质可及性概况。在目标2中，我 将确定特定染色质修饰的存在如何影响TF在不同细胞中的结合位置， 细胞类型。在目标3中，我将直接将TF表达的变异性与其协同结合联系起来 在高度异质性的常见染色体中观察到具有差异染色质可及性特征的伴侣， 骨髓祖细胞群体，并确定这如何影响细胞命运。这些研究将提供详细的 分析各种因素在调节TF结合中的作用，使我们能够更准确地 预测特定的转录因子在细胞中的结合位置，它们将如何影响基因调控网络，以驱动一个新的转录因子。 特定的细胞状态，以及破坏TF活性如何导致疾病状态。这些研究将在 在斯坦福大学的遗传学系，我可以接触到最新的下一代测序技术 技术以及其他世界级的资源。通过实施这个项目，我将进一步我的毕业 通过学习和发展尖端技术研究基因调控机制的培训 基因组技术来回答以前难以使用现有方法解决的问题。