Determining the influence of 3D chromatin structure on human evolution

项目总结/摘要 三维基因组结构对人类特异性基因调控进化的重要性， 表型和物种形成仍然在很大程度上未知。在这里，我打算以一种高- 拓扑相关结构域（TADs）的影响，这是基因组的 自相互作用DNA的邻域和CCCTC结合因子（CTCF）结合基序， 有助于促进DNA循环，对细胞活力和进化。我假设有一个子集 在整个进化过程中，边界对于维持重要的基因调控至关重要 并且人类谱系中衍生的CTCF结合位点可能导致基因 表情变化。我将通过首先进行高通量的 在CRISPR删除屏幕中，我将删除一组超过300个进化上保守的基因， 在人单倍体细胞系中的人特异性DNA边界，以确定这些DNA中的任何一个是否 边界对于细胞活力是必不可少的（目标1）。其次，我将确定人类特异性CTCF 结合基序在人类中获得或丢失，或者通过比较 来自人类、黑猩猩和两种已灭绝的古人类的基因组序列：尼安德特人 还有丹尼索瓦人我将进一步询问这些新的CTCF网站的重要性的子集 通过CRISPR/Cas9编辑人类细胞来模仿灭绝基因组的基因组并使其表型 基因表达变化和染色质构象的变化。这样就有可能 确定这些变化是否导致基因调控和基因组循环的差异（目标2）。 结合起来，这个提议将在进一步理解3D染色质如何 结构可以影响表型，特别是因为它与人类进化有关。

PROJECT SUMMARY/ABSTRACT The importance of 3D genome structure on the evolution of human specific gene regulation, phenotypes, and speciation remains largely unknown. Here, I plan to characterize in a high- throughput manner the impact of topologically associated domains (TADs), which are genomic neighborhoods of self-interacting DNA, and CCCTC binding factor (CTCF) binding motifs, which help facilitate DNA looping, on cell viability and evolution. I hypothesize that a subset of TAD boundaries are critical for maintaining important gene regulation throughout evolution and that derived CTCF binding sites in the human lineage may have led to gene expression changes. I will interrogate this hypothesis by first performing a high-throughput CRISPR-deletion screen in which I will delete a set of over 300 evolutionarily conserved and human specific TAD boundaries in a human haploid cell line to determine if any of these boundaries are essential for cell viability (Aim 1). Second, I will identify human specific CTCF binding motifs that are gained or lost in humans or have orientation changes by comparing genome sequences from human, chimpanzee, and two extinct archaic hominids: Neanderthal and Denisovan. I will further interrogate the importance of a subset of these novel CTCF sites by CRISPR/Cas9 editing human cells to mimic that of an extinct genome and phenotype them for changes in gene expression changes and chromatin conformation. It will then be possible to determine if these changes caused differences in gene regulation and genome looping (Aim 2). Combined, this proposal will take a crucial step in further understanding how 3D chromatin structure can affect phenotypes especially as it relates to human evolution.