Determining the influence of 3D chromatin structure on human evolution

项目摘要/摘要 3D基因组结构在人类特定基因调控进化中的重要性， 表型和物种形成在很大程度上仍不清楚。在这里，我计划用一种高度的- 吞吐量方式是拓扑相关结构域(TADs)的影响，这些结构域是基因组 自我相互作用的DNA和CCCTC结合因子(CTCF)结合基序的邻域，这 有助于促进DNA循环，对细胞活力和进化产生影响。我假设TAD的一个子集 边界对于在整个进化过程中维持重要的基因调控至关重要 而人类谱系中衍生的CTCF结合位点可能导致了基因 表情发生了变化。我将通过首先执行高吞吐量来验证这一假设 CRISPR-删除屏幕，在其中我将删除一组300多个进化保守的和 人类单倍体细胞系中人类特异性TAD边界的研究 边界对细胞存活至关重要(目标1)。第二，我将确定人类特有的CTCF 通过比较人类获得或丢失的结合基序或具有取向变化的结合基序 来自人类、黑猩猩和两个已灭绝的古人类的基因组序列：尼安德特人 还有丹尼索万。我将进一步询问这些新的CTCF位点的子集的重要性 CRISPR/Cas9通过编辑人类细胞来模仿已灭绝的基因组并对其进行表型分析 用于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了基因调控和基因组循环的差异(目标2)。 总而言之，这项提议将在进一步了解3D染色质如何 结构可以影响表型，特别是当它与人类进化有关时。

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.