Impact of enhancer sequence and interacting factors on estrogen receptor-mediated

7.项目总结/摘要 我的长期研究目标是阅读写在我们基因组序列中的基因调控指令。我 希望能够仅从基因组序列预测给定细胞类型中的基因表达水平， 了解基因调控指令如何在不同的细胞类型中得到不同的解释。的能力 破译基因组序列和基因表达之间的关系将具有广泛的意义。 对人类健康的影响。例如，可以通过以下方法实现对基因表达的非常精确的控制： 工程化的调控序列和基因调控突变可以被识别并容易地解释， 疾病，然后可能被修复。在实现这一崇高目标之前， 必须建立生物医学系统。 剖析细胞类型在基因调控方面的特异性差异以及基因组序列如何介导这些差异 变化，拟议的研究重点是对雌激素的基因调控。雌激素信号 为研究不同细胞类型的基因表达反应提供了良好的模型。 雌激素在许多组织中产生不同的作用;然而， 生理结果仍然知之甚少。在以前的工作中，我发现了两种人类细胞系， 对雌激素治疗表现出非常不同的反应，包括表达差异的基因， 受影响的雌激素受体和结合的位置。通过使用多功能基因组学 我希望能阐明这些细胞类型背后的机制和DNA序列， 具体的回答。 我将从两个角度探讨细胞类型特异性雌激素信号传导：1）识别与雌激素信号传导相互作用的因素。 雌激素受体以细胞类型特异性方式驱动雌激素信号传导; 2）解剖DNA序列 对细胞类型特异性雌激素受体结合增强子的限制。为了研究交互因素，我将创建一个 通过对DNA酶超敏性和DNA序列基序的分析，列出候选因素。然后我将测试 这些候选者与ChIP-seq共现，并通过敲除确定必要性和充分性。 稳定的过表达细胞系。为了剖析特定于细胞类型的序列约束， 雌激素反应增强子，我将首先找到活性雌激素受体结合增强子和基因， 使用ChIA-PET靶向，ChIA-PET是一种在基因组规模上分析长距离染色质相互作用的方法。我会 然后开发一种技术来研究细胞类型特异性增强子序列的行为， 高通量测序的优势。这些方法的结合应该会给出一个强有力的画面 决定细胞类型特异性的事件以及基因组序列如何编码细胞类型依赖性基因 调控 这个研究计划的短期目标使我走上了实现长期职业目标的道路 成为一名终身教职员工，研究基因组序列相关的基因调控。期间 在过渡期我将学习新的功能基因组学技术和分析方法，包括DNA酶 超敏反应绘图和ChIA-PET。我也将成为核受体的活跃成员 通过参加大型会议和个人会议，促进社区的发展。除了科学 在此期间，我将接受培训，我计划学习必要的管理和沟通技巧 成为一个成功的导师我将获得管理技术人员的经验， 并参与与其他实验室的合作。这些技能，沿着科学 培训，应该准备我从博士后研究员到独立调查员的过渡。

7. Project Summary/Abstract 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 lofty 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.