Comparative and functional analysis of conservation and rearrangement of topologically associating domains across mammals.

项目总结 染色体重排是种间和种内遗传变异的一大来源，是主要的 人类疾病的始作俑者。尽管重新排列断点的位置现在可以映射到高 要解决这些问题，解释这些事件的进化或临床影响仍然具有挑战性。取决于 在它们发生的地方，重排可以扰乱基因组功能隔间的组织，众所周知 作为拓扑关联结构域(TADS)。在TADS中，附近的基因座(即启动子和增强子)相互作用 与TADS外的基因座之间的相互作用受到TAD界限的限制。 TAD边界的破坏可导致异位基因调控、异常表型和遗传 精神错乱。因此，人们只能完全理解染色体重排的功能结果。 当在基因组拓扑学的背景下研究时。来阐明一些进化方面的影响 基因组重组，我们最近研究了长臂猿基因组，这经历了快速和最近 人类和其他类人猿的核型进化。在长臂猿基因组中，我们观察到TADS 保持遗传和表观遗传的完整(基因组假洗牌)，因为进化断点 几乎完全与TAD边界重叠。与人类和其他哺乳动物的比较表明， 这些TAD边界在进化上是保守的，表明TAD边界的建立早于 甚至可能促成了进化断裂的发生。受我们在以下方面的初步发现的启发 Gibbon，我们建议使用广泛的比较和功能方法来分析多个物种 Boreoeutheria树上自然高度重新排列的基因组，并描述了遗传背景， 表观遗传状态，以及它们TAD边界的进化保守。我们将确定错误的洗牌是否 是基因组进化的一种循环机制，我们将确定与 进化的脆弱性，因为这些区域和状态可能与人类疾病相关(目标1)。此外，我们 将决定跨不同分支的TAD边界的保护水平。总体而言， 这些注释将是帮助解释临床和/或进化相关的有价值的资源 重新安排。然后我们将使用进化激励的方法来删除高度保守的子集 并使用CRISPR/Cas9在细胞系和小鼠中评估分支特异性TAD边界 它们缺失对DNA相互作用、染色质状态和基因表达的影响(目标2)。最后，通过 分析亲缘关系密切的物种之间的差异基因表达和染色质构象 结构不同的基因组，我们将评估染色体重排可以改变的程度 短程和长程功能相互作用并有助于差异基因表达(目标3)。总体而言，这 研究将阐明与进化基因组重组相关的表观遗传学变化，并将有助于 阐明基因组重排导致病理的机制。

PROJECT SUMMARY Chromosomal rearrangements are a great source of inter- and intra-specific genetic variation and are major contributors to human disease. Although position of rearrangement breakpoints can now be mapped at high resolution, interpreting the evolutionary or clinical implications of these events remains challenging. Depending on where they occur, rearrangements can disrupt the organization of genomic functional compartments, known as topologically associating domains (TADs). Within TADs, nearby loci (i.e. promoters and enhancers) interact frequently with each other, while interactions with loci outside TADs are prevented by TAD boundaries. Disruption of TAD boundaries can result in ectopic genes regulation, aberrant phenotypes, and genetic disorders. The functional outcomes of chromosomal rearrangements, therefore, can only be fully understood when studied in the context of genome topology. To shed light on some of the evolutionary implications of genome reorganization, we recently studied the gibbon genome, which has experienced rapid and recent karyotype evolution with respect to human and the other apes. In the gibbon genome, we observed that TADs remained genetically and epigenetically intact (genomic false-shuffle), because evolutionary breakpoints overlapped almost exclusively with TAD boundaries. Comparison with human and other mammals shows that these TAD boundaries are evolutionary conserved, indicating that TAD boundary establishment predated, and may have even contributed to, occurrence of evolutionary breakage. Motivated by our preliminary findings in gibbon, we propose to use a broad comparative and functional approach to assay multiple species with naturally highly rearranged genomes across the Boreoeutheria tree, and characterize the genetic context, epigenetic state, and evolutionary conservation of their TAD boundaries. We will determine if the false shuffle is a recurring mechanism of genome evolution and we will identify chromatin states associated with evolutionary fragility, as these regions and states could be relevant to human disease (Aim 1). Additionally, we will determine the level of conservation for TAD boundaries across different clades. Overall, the combination of these annotations will be a valuable resource to aid the interpretation of clinically and/or evolutionarily relevant rearrangements. We will then use an evolutionary-motivated approach to delete a subset of highly conserved and clade-specific TAD boundaries using CRISPR/Cas9 in cell lines and mouse, to assess the functional consequences of their deletion on DNA interaction, chromatin state, and gene expression (Aim 2). Finally, by analyzing differential gene expression and chromatin conformation between closely related species with structurally different genomes, we will evaluate the extent to which chromosomal rearrangements can alter short- and long-range functional interaction and contribute to differential gene expression (Aim 3). Overall, this study will elucidate the epigenetics changes associated with evolutionary genome reorganization and will help elucidating mechanisms by which genome rearrangement can lead to pathology.