Dissecting principles of transcription factor binding

项目摘要 转录因子是细胞特性的主要调节者，协调对特定基因的精确控制。 基因网络驱动特定细胞命运所必需的所有细胞过程。信托基金的能力 多能因子鸡尾酒的异位表达的发现突显了调节细胞身份 可以将体细胞重新编程为诱导的多能干细胞。某些转铁蛋白的突变或不适当剂量 还可能导致各种疾病，如癌症。虽然适当的转铁蛋白调节显然对正常 关于细胞功能，我们对转录因子如何实现对基因网络的如此精确的控制缺乏了解。 具体地说，控制转录因子如何与细胞中正确的一组靶点结合的原则仍然不清楚。 例如，转录因子通常不结合基因组内包含其目标序列基序的所有位点，并且 许多转铁蛋白结合位点不包含规范序列基序。此外，Tf以多个 细胞类型可以结合到每种细胞类型中的不同位置。这表明蜂窝环境起着至关重要的作用 在确定转铁蛋白的结合部位时。为了提高我们预测和模拟转铁蛋白结合和活性的能力，以及 因此，决定细胞命运，我建议剖析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结合方面是最重要的，但特定序列的存在 染色质修饰和协同结合伙伴对于促进转铁蛋白结合非常重要，尤其是在 更低的TF表达水平，即使是可以访问核小体封闭DNA的先驱因子也是如此。我要测试一下这个 通过三个具体目标提出假设。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 同一大细胞群体和单个细胞中的蛋白质水平和染色质可及性分布。在目标2中，我 将确定特定染色质修饰的存在如何影响TF在不同的 单元类型。在目标3中，我将直接将转录因子表达的可变性与其协同结合联系起来 具有不同染色质可及性的合作伙伴在高度异质性的共同 并确定这对细胞命运有何影响。这些研究将提供详细的 分析各种因素在调节转铁蛋白结合中的作用，使我们能够更准确地制定 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 特定的细胞状态，以及扰乱TF活性如何导致疾病状态。这些研究将会进行 在斯坦福大学遗传学系，我将在那里接触到最新的下一代测序 技术以及其他世界级的资源。通过实施这个项目，我将在毕业后取得更大成就 通过学习和开发尖端技术来研究基因调控潜在机制的培训 基因组技术来回答以前很难用现有方法解决的问题。

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.