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

描述(申请人提供)：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说明。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，并了解基因调控指令在不同细胞类型中是如何差异解释的。破译基因组序列和基因表达之间关系的能力将对人类健康产生广泛而深远的影响。例如，通过工程调控序列可以实现对基因表达的非常精确的控制，基因调控突变可以被识别并在疾病中很容易解释，然后可能被修复。在实现这个LOFT目标之前，必须从重要的生物医学系统的定向研究中获得见解。为了剖析细胞类型特异性基因调控的差异以及基因组序列如何调节这些变化，拟议的研究重点放在雌激素反应的基因调控上。雌激素信号转导为研究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，生理结果背后的分子基础仍然知之甚少。在之前的工作中，我鉴定了两个对雌激素治疗表现出非常不同反应的人类细胞系，包括其表达受到影响的基因和雌激素受体结合位置的差异。通过使用多种功能基因组学分析，我希望阐明这些细胞类型特异性反应背后的机制，从而阐明DNA序列。我将从两个角度探讨细胞类型特定的雌激素信号：1)识别与雌激素受体相互作用的因素，以细胞类型特定的方式驱动雌激素信号；2)剖析细胞类型特定的雌激素受体结合增强子的DNA序列约束。为了研究相互作用因素，我将通过分析DNA酶超敏反应和DNA序列基序来创建候选因素列表。然后，我将测试这些候选基因与CHIP-SEQ的同现性，并通过建立敲除和稳定的过度表达细胞系来确定必要性和充分性。为了剖析细胞类型特定的雌激素反应增强子的序列约束，我将首先使用chia-PET找到活性的雌激素受体结合增强子及其靶向基因，chia-PET是一种在基因组规模上分析染色质远程相互作用的方法。然后，我将开发一种技术，利用高通量测序的优势，在分离中研究细胞类型特定增强子序列的行为。这些方法的结合应该给出一幅关于决定细胞类型特异性的事件以及基因组序列如何编码依赖于细胞类型的基因调控的强健图景。这项研究提案的短期目标让我走上了一条实现我长期职业目标的道路，那就是成为一名研究与基因组序列相关的基因调控的终身教员。在过渡期奖期间，我将学习新的功能基因组学技术和分析方法，包括DNA酶超敏图谱和CHIA-PET。我还将通过出席大型会议和个别会议，成为核受体社区的积极成员。除了在这个奖项期间我将接受科学培训外，我还计划学习成为一名成功的导师所必需的管理和沟通技能。我将获得管理技术人员的经验，为研究生的指导做出贡献，并参与与其他实验室的合作。这些技能，加上科学培训，应该会为我从 从博士后研究员到独立调查员。

DESCRIPTION (provided by applicant): 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.