Friendly fire: identifying mechanisms regulating expression of the genome-editing enzyme, APOBEC3A

Policelli, Paige (2025) Friendly fire: identifying mechanisms regulating expression of the genome-editing enzyme, APOBEC3A. University of Southampton, Doctoral Thesis, 222pp.

Record type: Thesis (Doctoral)

Abstract

Aberrant activation of the apolipoprotein-B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A) cytidine deaminase has been implicated as a major source of C>T and C>G mutations in cancer; in particular in carcinomas of the head and neck, bladder, lung, oesophagus, cervix and breast. Over the last decade, impressive efforts to characterise the mutagenic activity of APOBEC3A have emerged, although the current understanding of the mechanisms that regulate APOBEC3A expression are limited. In this thesis, the identification of mechanisms controlling APOBEC3A gene expression that may become dysregulated and trigger aberrant APOBEC3A expression in cancer, are investigated in keratinocytes from which squamous cell carcinomas arise and APOBEC-mediated mutagenesis is common.

To do this, two complementary approaches were performed: (1) prediction of transcription factors responsible for regulating APOBEC3A expression from single cell RNA sequencing data with validation in vitro using short-interfering RNAs. From this, Grainyhead-like transcription factor 3 (GHRL3) was revealed to have a role in regulating APOBEC3A expression within differentiating keratinocytes, additionally validated in two head and neck squamous cell cancer cell lines. Furthermore, GRHL3 was demonstrated to act downstream of interferon regulatory factor 6 (IRF6), a key mediator of keratinocyte differentiation downstream of NOTCH/Receptor-interacting serine/threonine protein kinase 4 (RIPK4). (2) The coupling of epigenomic and circularised chromosome conformation capture (4C) analyses to identify cis-acting regulatory elements that interact with the APOBEC3A genomic locus. Candidate silencers and enhancers were identified to occupy the same topologically associated domain (TAD) upon which APOBEC3A centrally resides. Furthermore, CTCF-orientation analysis and DNA-FISH revealed potential chromatin loops that may facilitate their colocalisations. These findings significantly enhance our understanding of APOBEC3A regulation and offer new clues as to how it may switch from anti-viral factor to promoter of oncogenesis.