The impact of APOBEC3A-mediated mutagenesis on gene regulation in cancer

Washington University, Saint Louis MO

Investigators

Abstract

Project Summary/Abstract This research proposal is intended to facilitate training toward the applicant's long-term career goal of becoming an independent investigator in gene regulation in cancer. This research training plan will encompass predoctoral and postdoctoral training necessary to gain advanced technical skills in cancer biology research and to develop skills in scientific communication, grantsmanship, and mentorship. The research objective in the F99 phase is to determine the role of APOBEC3A-mediated mutagenesis in altering transcriptional regulation in cancer. APOBEC3A (A3A) is an endogenous mutagen that catalyzes the deamination of cytosine (C) to uracil (U) in single-stranded DNA and RNA. The genomic mutation patterns generated by A3A are the second most common mutation patterns in cancer, thus understanding the impact of A3A-mediated mutagenesis is essential. A3A expression leads to widespread changes to gene expression, however most differentially expressed genes do not contain A3A-induced mutations. This project will investigate the hypothesis that A3A activity alters pre- transcriptional regulation through DNA mutagenesis and post-transcriptional regulation through RNA editing. Preliminary data show that A3A activity alters chromatin accessibility in a subset of differentially expressed genes, yet these chromatin accessibility changes also lack A3A-induced mutations. DNA methylation is an important epigenetic mark that regulates gene expression in part by coordinating chromatin accessibility. Aim 1a will define the effects of A3A activity on genome-wide methylation patterns and determine if these changes are specific to A3A-induced damage or if other base-damaging agents elicit the same changes. Integrating these data with gene expression and chromatin accessibility data will establish how A3A-induced methylation changes alter chromatin function. To determine the mechanisms by which A3A generates changes to methylation, chromatin accessibility, and gene expression, we will use CRISPR-Cas9 base editing to target A3A activity to a specific locus and evaluate the effects in the presence and absence of functional DNA repair. Aim 1b will investigate how A3A induces changes to the transcriptome through RNA editing. Preliminary data show that hundreds of transcripts have altered stability during A3A expression. A3A RNA editing generates missense mutations in the transcripts of three RNA binding proteins (RBP) important for RNA stability. Aim 1b will determine the effects of these missense edits on RBP function to define how A3A-mediated RNA editing impacts stability across the transcriptome. The K00 research will continue investigation of transcriptional regulation in cancer by focusing on how three-dimensional organization is altered in cancer cells. These studies will integrate higher- order chromatin sequencing and microscopy with advanced bioinformatics. Together, the F99 and K00 training plans will support the applicant's development of advanced technical skills in mechanistic cancer research and the professional training required to become a leading investigator in transcriptional regulation in cancer.