Characterizing Alkbh1 Function in Human Embryonic Stem Cells

Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics

First Advisor

Xiao, Andrew

Abstract

AbstractCharacterizing ALKBH1 function in Human Embryonic Stem Cells Joseph James Balowski 2025 Among the most remarkable biological processes is the ability of a single fertilized egg to give rise to the multitude of distinct cell types that make up a multicellular organism. Understanding how diverse cell types are created using a single shared genome is one of the core projects of science. This differential utilization of the genome is accomplished by epigenetics—the chemical modification of genomic DNA or the proteins associated with it—and by epitranscriptomics, involving modifications to RNA that affect its stability, translation, or function. These modifications are catalyzed by “writers” (enzymes that deposit the mark), recognized by “readers” (which detect and act upon the mark), and sometimes removed by “erasers” (enzymes that reverse the modifications). One such epigenetic factor is AlkB-homolog 1, or ALKBH1. ALKBH1 is primarily known as a demethylase of nucleic acids. In mouse embryonic stem cells, Alkbh1 perturbation has been shown to affect pluripotency and differentiation. In this dissertation, I characterize the transcriptomic and epigenomic effects of perturbing ALKBH1 in primed human pluripotent stem cells (hESCs). ALKBH1 knockout, knockdown and overexpression cell lines were created and assessed by RNA-sequencing, histone CUT&RUN, MBD-seq, and ChIP-DNA/RNA-seq. Aklbh1 perturbation in primed hESCs does not produce an overt phenotype, as cells grow normally, are morphologically typical, maintain pluripotency, and retain their ability to differentiate. A molecular phenotype can be observed by bulk RNA-seq, and while it is both mild and complex, emergent themes of mitochondrial perturbation and altered metabolism can be observed. ALKBH1 ChIP-RNA-seq shows enrichment for mitochondrial RNAs, especially tRNAs. It is hypothesized that ALKBH1 is changing the modification state of these RNAs, which may in turn be perturbing mitochondrial translation and be causative of the transcriptomic phenotype. Thus, while epigenomic activities of ALKBH1 have been reported in other systems and may be at play here as well, in primed human embryonic stem cells, current available evidence points to epitranscriptomic activities predominating. Further work is needed to support this model, and to determine the role of ALKBH1 in other aspects of stem cell biology and organismal development.

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