CAREER: Conservation of cohesin-containing cis regulatory modules in the human and mouse lineages

小鼠被用作一种灵活的遗传系统，用于探索疾病是如何发生的，包括在人类中。然而，对患有这种疾病的小鼠有效的治疗方法几乎不适用于其他患有这种疾病的动物。这显然是因为，即使基因途径在不同物种之间看起来相似，但它们的调控或网络连接是不同的。其中一些变化可能是由反转录转座子元件引起的，这些元件的存在已被证明与人类和小鼠细胞的粘附素相关调控模块的变化有关。用特殊试剂修改测序技术使我们能够开始绘制人类和小鼠的调节区；结果显示，调节位点的变化率很高，只有大约一半的位点显示了人和小鼠细胞系之间的保守序列，即使在发现保守的地方，预期的转录因子也不会在相似的条件下结合。更复杂的是，大多数基因网络由多个转录因子(TF)调控，这些转录因子必须以特定的顺序和组合结合；这种结合通常位于距离基因相当远的位置。这项研究旨在剖析的因素之一是，在遥远的位置将DNA环回，使其接近，并用一种调节蛋白将其锁定在适当的位置。了解这些调节元件TF组合是什么，导致变化的机制是什么，以及它们的调节作用如何被调节，将在疾病治疗等领域有重要的应用，包括更好的靶向治疗。这项研究需要强大的计算技能以及对细胞生物学状态和分析技术细节的了解；除了指导将在开展研究中发挥主导作用的研究生之外，高中生将被招募参加计算机化暑期新兵训练营，本科生将被招募到带薪暑期实习，沉浸在研究方法和问题中，重点是让更多的女性和服务不足的少数群体参与积极的研究体验。在这个项目中，PI将使用最近开发的方法来探索转录因子与CTCF和匹配的人和鼠细胞中的粘附素复合体的共同结合。这些位置代表了基因组中3D相互作用的锚定区域。初步工作表明，除了两个基因组中已经很高的顺式调节模块的周转之外，这些位点还有广泛的周转。这一发现与之前发表的工作形成了鲜明对比，该工作表明，在低分辨率下，这两个物种之间的物理3D结构具有高度的保守性。PI以前已经开发出计算方法，证明了转录因子的复杂共定位，并表明这些模式在小鼠中是保守的。这些模式被证明即使在潜在的序列分歧的情况下也保持着相似的调控特性。在这里，PI将从这两个物种的粘附素位置的保守性方面探索这些保守的模式。作为这项工作的延伸，该项目将重点研究逆转座子在两个谱系中粘连蛋白位点扩展中的作用。最后，该项目将整合来自CHIA-PET和HIC数据的3D结构信息，以确定粘附素位点的得失对特定环和拓扑域的保守的影响。这项工作是从一种考虑保守性的新方法延伸出来的，也就是考虑调控模式的保守性，而不仅仅是考虑序列背景。正因为如此，PI能够通过比较来研究整个老鼠和人类的基因组，而不是只研究按序列比较的一小部分基因组。因此，该项目将为这两个基因组的调控结构提供新的见解，并阐明通过循环和结构域结构进行基因调控的各个方面，这些方面以前是不可能考虑的。该项目的进展情况将在http://boylelab.org/.上公布。

The mouse has been used as a flexible genetic system for exploring how diseases occur, including in humans. However, the treatments that work for mice having a condition almost never work in other animals with that condition. This apparently occurs because, even when gene pathways seem similar across species, their regulation, or network wiring, is different. Some of these changes may be caused by retrotransposon elements, whose presence has been shown to be correlated with changes in the cohesin-associated regulatory modules of human and mouse cells. Modifying sequencing technologies with special reagents has allowed us to start mapping regulatory regions in both human and mouse; results show that there is a high rate of change at regulatory sites, with only about half of the sites showing conserved sequence between human and mouse cell lines and, even where conservation is found, the expected transcription factors don't bind under similar conditions. A further complication is that most gene networks are regulated by multiple transcription factors (TFs) that must bind in a particular order and combination; often this binding is at sites quite distant from the genes. Looping of the DNA at distant sites back around to bring it close, and 'locking' it in place with a regulatory protein, is one of the factors this research aims to dissect. Understanding what these regulatory element TF combinations are, what mechanisms lead to change, and how their regulatory actions can be modulated will have important applications in such areas as the treatment of disease, including better targeted therapies. This research requires strong computational skills as well as understanding the biological states of cells and the technical details of the assays; in addition to mentoring the graduate students who will take leading roles in carrying out the research, high school students will be recruited to participate in computational summer boot camps and undergraduates will be recruited to paid summer internships for immersion in the research methods and questions, with a strong emphasis on bringing more women and under-served minorities into active research experiences. In this project the PI will use recently developed methods to explore the co-binding of transcription factors with CTCF and the cohesin complex in matched human and mouse cells. These sites represent anchor regions for 3D interactions in the genome. Initial work suggests that there is extensive turnover of these sites beyond the already high turnover of cis-regulatory modules in the two genomes. This finding stands in sharp contrast to previously published work showing, at low resolution, there is a high level of conservation of the physical 3D structure between the two species. The PI has previously developed computational methods demonstrating a complex co-localization of transcription factors and shown that these patterns are conserved in mouse. These patterns were shown to maintain similar regulatory properties even with underlying sequence divergence. Here the PI will explore these conserved patterns in terms of conservation at cohesin sites in the two species. As an extension of this, the project will focus on the effect of retrotransposons in the expansion of cohesin sites in the two lineages. Finally, the project will integrate 3D structural information from ChIA-PET and HiC data to determine the effects of the gains and losses of cohesin sites on the conservation of specific loops and topological domains. This work extends from a new way of considering conservation, that of conservation of regulatory patterns instead of considering only sequence context. Because of this, the PI is able to study the entire mouse and human genomes in comparison instead of only the small fraction of the genomes comparable by sequence. As a result, the project will provide new insights into the regulatory structure of the two genomes and shed light on the aspects of gene regulatory control through looping and domain structure that would previously have been impossible to consider. Progress from this project will be available at http://boylelab.org/.