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

7.项目摘要/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 我希望能够仅从基因组序列预测给定细胞类型的基因表达水平 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。一种能力 破译基因组序列与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，对基因表达的非常精确的控制可以通过 设计的调控序列和基因调控突变可以在 疾病，然后有可能被修复。在实现这一崇高目标之前，从重要的定向研究中获得的见解 必须获得生物医学系统。 剖析基因调控中特定细胞类型的差异以及基因组序列如何调节这些差异 随着雌激素的变化，拟议的研究重点是基因调控对雌激素的反应。雌激素信号转导 为研究针对不同细胞类型的基因表达反应提供了一个很好的模型。 雌激素在许多组织中产生不同的影响；然而，潜在的分子基础 生理结果仍然鲜为人知。在之前的工作中，我鉴定了两个人类细胞系 对雌激素治疗表现出非常不同的反应，包括表达的基因的差异 影响和雌激素受体结合的位置。通过使用多功能基因组学 通过分析，我希望阐明这些细胞类型背后的机制，从而阐明DNA序列- 具体的回应。 我将从两个角度探讨细胞类型特定的雌激素信号：1)确定与 雌激素受体以细胞类型特异性的方式驱动雌激素信号；2)剖析DNA序列 细胞类型特异性雌激素受体结合增强子的限制。为了研究相互作用的因素，我将创建一个 通过对DNA酶敏感性和DNA序列基序的分析，列出了候选因素。然后我会测试一下 这些候选与CHIP-SEQ同现，并通过敲打来确定必要性和充分性 获得稳定过表达的细胞系。为了剖析特定于细胞类型的序列约束 雌激素反应增强子，我会首先找到活性的雌激素受体结合增强子和它们的基因 目标使用chia-PET，这是一种在基因组规模上分析远程染色质相互作用的方法。这就做 然后开发一种技术来研究细胞类型特定的增强子序列在分离中的行为 高通量测序的优势。这些方法的结合应该会给出一幅稳健的图景。 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基因 监管。 这项研究提案的短期目标使我走上了实现长期职业目标的道路 成为一名研究与基因组序列相关的基因调控的终身教职员工。在.期间 过渡奖I将学习新的功能基因组学技术和分析方法，包括DNA酶 超敏反应标测和CHIA-PET。我也将成为核受体的活跃成员 通过出席大型会议和个人会议，促进社区发展。除了科学上的 在这个奖项期间，我将接受培训，我计划学习必要的管理和沟通技能 成为一名成功的导师。我将获得管理技术人员的经验，为指导工作做出贡献 并参与了与其他实验室的合作。这些技能，以及科学 培训，应该为我从博士后研究员过渡到独立调查员做好准备。

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.