Dissecting principles of transcription factor binding

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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.