Friday, October 25, 2024
10/25/2024
PROJECT SUMMARY/ABSTRACT The lion’s share of phenotypic divergence between humans and our closest evolutionary relatives is thought to occur due to changes in noncoding regions of the genome, many of which are cis-regulatory elements such as enhancers and promoters. However, we currently have a very limited understanding of how lineage-specific noncoding changes have shaped human evolution. Here, I propose to study how variation in putative cis- regulatory elements drove the phenotypic divergence we observe between our own lineage (modern humans) and our closest relatives: Neanderthals and Denisovans (archaic humans). I hypothesize that certain lineage- specific variants in cis-regulatory regions confer differential gene regulatory activity between archaic and modern humans, thereby altering the expression of their targets and playing a profound role in the emergence of lineage-specific phenotypes. I will focus on noncoding variants that have reached fixation in one lineage but are absent from other human lineages, as these variants could underlie lineage-specific phenotypes and some of them may have been driven to fixation by positive selection. Using high-coverage archaic human genomes from three Neanderthals and one Denisovan, and existing catalogs of modern human genetic variation, I have identified 57,403 fixed single-nucleotide variants that are unique to archaic humans. Here, I propose to functionally characterize in an unbiased and high-throughput manner the cis-regulatory effect of each variant within four cell types that are relevant to the divergence between recent human groups. Specifically, I will perform massively parallel reporter assays (MPRAs) in neural progenitor cells, osteoblasts, skin fibroblasts, and adipocytes to quantitatively measure the differences in cis-regulatory activity conferred by each of these archaic-derived variants relative to their modern human counterpart. The results from these experiments, along with previous data from our lab measuring the cis-regulatory effect of modern human-derived variants, will enable me to use a powerful sign test to estimate lineage-specific selection acting on entire pathways observable phenotypes (Aim I). Finally, I will identify variants in the library of 57,403 that are associated with the regulation of early neurodevelopmental genes and insert them with CRISPR/Cas9 into induced pluripotent stem cells, which I will subsequently derive into cortical organoids. With single-cell RNA-sequencing and immunostaining, I will compare the organoids with edited archaic variants to unedited organoids to determine gene expression changes, variable cell migration, cell population composition, and broader morphological differences (Aim 2). Together, this work will provide the first catalog of the regulatory effects of variants that emerged and spread in archaic human evolution and will shed light on their role in shaping lineage-specific human phenotypes.
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