My long-term research goal is to read the gene regulation instructions written in our genome sequence. I would like to be able to predict gene expression levels in a given cell type from genome sequence alone and understand how the gene regulation instructions are differentially interpreted in diverse cell types. The ability to decipher the relationship between genome sequence and gene expression would have a wide reaching impact on human health. For example, very precise control of gene expression could be achieved through engineered regulatory sequences and gene regulatory mutations could be identified and easily interpreted in disease and then potentially fixed. Before achieving this loft goal, insights from directed studies in important biomedical systems must be gained. To dissect cell type-specific differences in gene regulation and how genome sequence mediates these changes, the proposed research focuses on gene regulation in response to estrogens. Estrogen signaling provides a good model for studying gene expression responses that are specific to different cell types. Estrogens produce diverse effects in a number of tissues;however, the molecular basis underlying physiological outcomes remains poorly understood. In previous work, I identified two human cell lines that exhibit very dissimilar responses to estrogen treatment, including disparity in the genes whose expression is affected and the locations of estrogen receptor binding. Through the use of multiple functional genomics assays, I hope to shed light on the mechanisms and therefore DNA sequences that underlie these cell type- specific responses. I will approach cell type-specific estrogen signaling from two angles: 1) Identify factors that interact with estrogen receptor to drive estrogen signaling in a cell type-specific manner;2) Dissect DNA sequence constraints on cell type-specific estrogen receptor-bound enhancers. To study interacting factors I will create a list of candidate factors through analysis of DNase hypersensitivity and DNA sequence motifs. I will then test these candidates for co-occurrence with ChIP-seq and determine necessity and sufficiency by making knock out and stable over-expression cell lines. In order to dissect sequence constraints on cell type-specific estrogen responsive enhancers, I will first find active estrogen receptor bound enhancers and the genes they target using ChIA-PET, a method that analyses long-range chromatin interactions on a genome scale. I will then develop a technique to study the behavior of cell type-specific enhancer sequences in isolation that takes advantage of high-throughput sequencing. The combination of these approaches should give a robust picture of the events that dictate cell type-specificity and how genome sequence encodes cell type dependent gene regulation. The short term goals of this research proposal set me on a path towards achieving my long-term career goals of becoming a tenure-track faculty member studying gene regulation in relation to genome sequence. During the transitional award I will learn new functional genomics techniques and analysis methods, including DNase hypersensitivity mapping and ChIA-PET. I will also become an active member of the nuclear receptor community through the attendance of large conferences and individual meetings. In addition to the scientific training that I will receive during this award, I plan to learn the managerial and communication skills necessary to becoming a successful mentor. I will gain experience managing a technician, contributing to the mentorship of a graduate student and participating in collaborations with other labs. These skills, along with scientific training, should prepare me for the transition from postdoctoral fellow to independent investigator.
This study aims to understand why the presence of estrogen impacts distinct tissues in different ways. Estrogen mimics are used in the treatment of breast cancer and osteoporosis, often with serious side effects. Knowledge of the mechanisms that cause tissues to respond differently to estrogens will shed light on these side effects and provide new pathways and targets for developing more effective estrogen-based therapies.
|Gertz, Jason; Savic, Daniel; Varley, Katherine E et al. (2013) Distinct properties of cell-type-specific and shared transcription factor binding sites. Mol Cell 52:25-36|