Development of a nuclease-mediated technology to validate chromatin hubs

开发核酸酶介导的技术来验证染色质中心

• 批准号: 8308770
• 负责人: PEGGY J Farnham
• 金额: $ 27万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-24 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8308770
• 关键词: Address; B-Lymphocytes; Binding; Binding Sites; Biology; Cell Line; Cells; ChIP-seq; Chromatin; Complex; Data; Data Set; Development; Elements; Expressed Sequence Tags; Gene Expression Profile; Gene Targeting; Genes; Genome; Genome engineering; Genomics; Hela Cells; Histones; Human Genome; In Situ; Knowledge; Maps; Mediating; Methods; Nucleic Acid Regulatory Sequences; Pattern; Phase; Population; Promoter Regions; RNA; Regulation; Regulatory Element; Reporter; Research; Research Personnel; Side; Signal Transduction; Technology; Transcript; Transcription Coactivator; Validation; Zinc Fingers; base; design; genome-wide; insight; mutant; nuclease; technology development; transcription factor; Address; B-Lymphocytes; Binding; Binding Sites; Biology; Cell Line; Cells; ChIP-seq; Chromatin; Complex; Data; Data Set; Development; Elements; Expressed Sequence Tags; Gene Expression Profile; Gene Targeting; Genes; Genome; Genome engineering; Genomics; Hela Cells; Histones; Human Genome; In Situ; Knowledge; Maps; Mediating; Methods; Nucleic Acid Regulatory Sequences; Pattern; Phase; Population; Promoter Regions; RNA; Regulation; Regulatory Element; Reporter; Research; Research Personnel; Side; Signal Transduction; Technology; Transcript; Transcription Coactivator; Validation; Zinc Fingers; base; design; genome-wide; insight; mutant; nuclease; technology development; transcription factor

DESCRIPTION (provided by applicant): Although enormous progress has been made in mapping transcription factor binding sites throughout the genome and continuing to expand our knowledge of the global binding patterns of transcription factors is very important, simply collecting genome-wide datasets will not be sufficient to answer all crucial questions about genome biology. A number of methodological problems now need to be addressed. In particular, we must develop new methods for the functional analysis of complex regulatory elements. We have identified regions of the genome that are bound by a large number of transcription factors but are not likely to be promoter regions because they are located far from all known genes, predicted genes, expressed RNAs, and H3K4me3 signals; we have called these regions chromatin hubs. We propose to develop a method by which the hubs can be investigated in their normal genomic context. Most approaches designed to study the function of a complex regulatory element involve reporter analyses and cannot reveal effects on long-range regulation. In contrast, our technology will be based on pairs of zinc finger nucleases (ZFNs) and/or transcription activator-like effector nucleases (TALENs) that will allow precise deletion of critical regulatory regions of the human genome. In essence, we will develop an approach that will allow the study of a complex element by specifically deleting it from the human genome, thus enabling analysis of an entire set of closely packed transcription factor binding sites in ther natural genomic context. Our technology will not only be invaluable for the study of chromatin hubs (as described in our proposal) but will also be appropriate for an "in situ" characterization of the function of any complex regulatory region. Recent genomic research has focused on documenting expressed transcripts and identifying transcription factor binding sites. The next phase of genomics requires the development of technologies that will allow the investigator to understand how cis regulatory regions influence the transcriptome. Our studies will help to integrate different types of genomic data and provide new insights into genomic regulation.

描述（由申请人提供）：尽管在整个基因组的转录因子结合位点上取得了巨大进展，并继续扩展我们对转录因子的全球结合模式的了解非常重要，但仅仅收集全基因组范围的数据集就不足以回答所有有关基因组生物学的关键问题。现在需要解决许多方法论问题。特别是，我们必须开发新方法来进行复杂调节元素的功能分析。我们已经确定了受大量转录因子约束的基因组区域，但不太可能是启动子区域，因为它们远离所有已知基因，预测基因，表达的RNA和H3K4ME3信号。我们称这些区域为染色质集线器。我们建议开发一种可以在其正常基因组环境中研究轮毂的方法。旨在研究复杂调节元件功能的大多数方法都涉及记者分析，并且无法揭示对远程调节的影响。相比之下，我们的技术将基于锌指核酸酶（ZFN）和/或转录激活剂样效应子核酸核酸酶（TALES）的成对，这些核酸酶（TALENS）将允许精确缺失 人类基因组的关键调节区域。从本质上讲，我们将开发一种方法，该方法将通过从人类基因组中特异性删除复杂元件来研究复杂元素，从而在其他自然基因组环境下对整个紧密包装的转录因子结合位点进行分析。我们的技术不仅对于研究染色质中心（如我们的提案中所述）是无价的，而且还适用于对任何复杂调节区域功能的“原位”表征。最近的基因组研究重点是记录表达的转录本和识别转录因子结合位点。下一阶段的基因组学需要开发技术，这将使研究人员能够了解CIS调节区域如何影响转录组。我们的研究将有助于整合不同类型的基因组数据，并为基因组调节提供新的见解。