Genome Engineering in the Study of APOBEC3 Gene Function

Abstract

The “apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like” (APOBEC) gene family defines a set of related Cytidine Deaminases. The best characterised of these are ones that function in the process of mRNA editing. Members of the family are also known to use DNA as their preferred substrate, acting in a consensus sequence, and mediating Cytidine deamination leading to a C to T transition. Recently, this process has been recognised as a major mutational mechanism in several cancer types, where it has been further suggested that members of the APOBEC3 family, and potentially APOBEC3H, that cause this. In order to understand the role of APOBEC3H further, we have used CRISPR-Cas9 gene editing to show that APOBEC3H is a p53 regulated target gene. This has involved making a p53 knockout version of the MCF7 breast cancer cell line, using CRISPR-Cas9 to delete part of the third coding exon of the gene. With this line, it has been possible to confirm that APOBEC3H expression is dependent on p53. In further work, CRISPR-Cas9 was used to make an APOBEC3H knockout in the HCT116 colon cancer line. Having established two independent APOBEC3H knockout lines in HCT116, RNAseq analysis was carried out to compare gene expression of mutant and wild-type cells in both the control and p53 activated setting. Analysis of the RNAseq data has shown that knockout of APOBE3H leads to an attenuated p53 response, suggesting the possibility that APOBEC 3H is co-regulator of p53 regulated gene expression. This is further indicated by the demonstration that over-expression of APOBEC3H in HCT116 wild-type cells enhances the expression of p53 target genes following p53 activation. Taken together, these data identify APOBEC3H as a p53 regulated gene, with a potential role in modulating the p53 response, and potentially identify a new mechanism for APOBEC3H action in cancer.