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

小鼠已被用作探索疾病如何发生的灵活遗传系统，包括在人类中。然而，对患有某种疾病的小鼠有效的治疗方法几乎从未对患有这种疾病的其他动物有效。这显然是因为，即使基因通路在物种之间看起来相似，它们的调控或网络布线也是不同的。这些变化中的一些可能是由反转录转座子元件引起的，其存在已被证明与人类和小鼠细胞的粘附素相关调节模块的变化相关。使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控区;结果显示，调控位点的变化率很高，只有大约一半的位点显示人类和小鼠细胞系之间的保守序列，即使发现保守性，预期的转录因子在相似条件下也不会结合。更复杂的是，大多数基因网络都受到多个转录因子（TF）的调控，这些转录因子必须以特定的顺序和组合结合;这种结合通常发生在距离基因相当远的位点。将DNA在远处的位点绕回来使其靠近，并用调节蛋白将其“锁定”在适当的位置，是这项研究旨在剖析的因素之一。了解这些调节元件TF组合是什么，什么机制导致变化，以及如何调节它们的调节作用将在疾病治疗等领域具有重要的应用，包括更好的靶向治疗。这项研究需要强大的计算能力，以及了解细胞的生物学状态和分析的技术细节;除了指导将在研究中发挥主导作用的研究生外，高中生将被招募参加计算夏季靴子营地，本科生将被招募参加带薪暑期实习，沉浸在研究方法和问题中，重点是让更多的妇女和服务不足的少数民族参与积极的研究经验。 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cohesin复合物在匹配的人类和小鼠细胞中的共结合。这些位点代表基因组中3D相互作用的锚区域。初步的工作表明，有广泛的营业额，这些网站超出了已经高营业额的顺式调控模块在两个基因组。这一发现与先前发表的工作形成鲜明对比，在低分辨率下，两个物种之间的物理3D结构具有高度的保守性。PI先前已经开发了计算方法，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。这些模式被证明保持类似的监管性质，即使潜在的序列分歧。在这里，PI将探讨这些保守的模式，在两个物种中的粘附素网站的保护。作为该项目的延伸，该项目将重点关注逆转录转座子在两个谱系中的粘附素位点扩展中的作用。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获得和损失对特定环和拓扑结构域的保护的影响。这项工作从一种新的方式来考虑保护，即保护的监管模式，而不是只考虑序列上下文。正因为如此，PI能够比较研究整个小鼠和人类基因组，而不是仅通过序列比较基因组的一小部分。因此，该项目将为两个基因组的调控结构提供新的见解，并通过以前无法考虑的循环和结构域结构阐明基因调控控制的各个方面。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/.