The regulatory impact of transposable elements (TEs) on differential gene expression in closely related species during cellular differentiation - Abstract Environmental factors contribute to the susceptibility of developing heart disease, but the same factors do not equally increase the disease risk for all people. Understanding how almost identical DNA between individuals provides so much variation in physiological and pathological presentation is essential to predicting a person’s risk of heart disease correctly. Differential gene regulation is a strong candidate for explaining these observed variations. Transposable Elements (TEs) comprise fifty percent of primate genomes but have long been considered “junk” DNA. However, recent studies have shown that TEs play a role in gene regulation in terminally differentiated cells between humans and chimpanzees, whose genomes share 98% similarity. Other studies have also demonstrated that differential silencing of TEs between humans and chimpanzees does not contribute to differential gene expression between the species' induced pluripotent stem cells (iPSCs). A knowledge gap exists regarding the developmental stage when TEs might begin to explain species-specific gene expression differences. I hypothesize that TEs have played a significant role in generating differences between primates through species-specific gene regulation throughout cellular differentiation. Aim 1 is to define the roles of TEs in species-specific gene regulation as iPSCs differentiate into cardiomyocytes. I will differentiate and collect human and chimpanzee iPSCs as they progress from pluripotency to mesoderm, cardiac mesoderm, and cardiomyocytes. Cellular phenotypes will be resolved through RNA sequencing, and differential gene expressions between species will be determined. To assess which regions of DNA have active (H3K27ac) and repressive (H3K9me3) chromatin modifications, Cleavage Under Targets and Tagmentation (CUT&Tag) will be performed in both species. I will overlap DNA regions with active and repressive histone modifications to the TE annotation file, RepeatMasker, to identify TEs in regulatory proximity to differentially expressed genes. Aim 2 is to discover the roles of TEs in differential gene expression between closely related species during the early embryological event of mesoderm specification. Human and chimpanzee iPSCs will be differentiated into the mesoderm, endoderm, and ectoderm lineages. The contribution of TEs to differences in early regulatory events between species will be assessed through the approaches discussed above, comparing mesoderm to the other germ layers to determine lineage-specific effects. Through the abovementioned project, I will gain skills in iPSC cell culture, differentiation techniques, and assays to assess epigenetic gene regulation. I will also develop expertise in utilizing computational approaches for processing RNA-sequencing data and developing pipelines to compare expression and gene regulatory features across species, necessitating the identification of orthologous regions and genes for direct comparison. These skills will prepare me for my long-term goal of becoming an independent genomics and cardiovascular research scientist.
