Development of a nuclease-mediated technology to validate chromatin hubs

描述(申请人提供)：尽管在绘制整个基因组中的转录因子结合位点方面已经取得了巨大的进展，继续扩大我们对转录因子的全球结合模式的知识是非常重要的，但仅仅收集全基因组的数据集并不足以回答关于基因组生物学的所有关键问题。现在需要解决一些方法问题。特别是，我们必须开发新的方法，对复杂的监管要素进行功能分析。我们已经确定了基因组中与大量转录因子结合但不太可能是启动子区域的区域，因为它们距离所有已知基因、预测基因、表达的RNA和H3K4me3信号都很远；我们称这些区域为染色质中心。我们建议开发一种方法，通过这种方法可以在正常的基因组环境中研究中枢。大多数旨在研究复杂监管要素功能的方法都涉及记者分析，无法揭示对长期监管的影响。相反，我们的技术将基于锌指核酸酶(ZFN)和/或转录激活物样效应核酸酶(TALEN)对，这将允许精确缺失 人类基因组的关键调控区域。本质上，我们将开发一种方法，通过从人类基因组中具体删除一个复杂的元素来研究它，从而能够在其他自然基因组环境中分析一整套紧密结合的转录因子结合位点。我们的技术不仅对染色质中心的研究(如我们的建议中所述)非常有价值，而且还适合于对任何复杂调控区域的功能进行“原位”表征。最近的基因组研究集中在记录表达的转录本和鉴定转录因子结合位点。基因组学的下一阶段需要开发技术，使研究人员能够了解顺式调控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，并为基因组调控提供新的见解。

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.