Award details

Investigating regulation and function of the cytosine deaminase APOBEC3A during cell cycle re-entry

Reference	BB/V010271/2
Principal Investigator / Supervisor	Dr Tim Fenton
Co-Investigators / Co-Supervisors	Professor Michelle West
Institution	University of Southampton
Department	Cancer Sciences
Funding type	Research
Value (£)	523,904
Status	Current
Type	Research Grant
Start date	03/01/2022
End date	30/06/2024
Duration	30 months

Abstract

The APOBEC (apolipoprotein-B mRNA editing catalytic polypeptide) enzymes are polynucleotide cytosine deaminases that perform important roles in metabolism, innate and adaptive immunity via their ability to edit RNA and single stranded DNA. This proposal focuses on APOBEC3A (A3A), an interferon-inducible gene with activities against both exogenous viruses and retroelements, which has recently been implicated as the major driver of the A3 mutational signature in cancer genomes; a profusion of C to T and C to G mutations at TpC sites. In this project we will build upon our novel findings that A3A expression and RNA editing activity are potently induced when keratinocytes are stimulated to re-enter the cell cycle and that A3A deletion causes a dramatic increase in keratinocyte proliferation, implicating it in cell cycle control. The robust induction of A3A by growth factor starvation/stimulation we have identified, coupled with the A3A knockout cells we have generated presents a unique opportunity to study endogenous A3A regulation and function. We will use CRISPR-Cas9 screening to identify regulators of A3A expression, circular chromatin conformation capture (4C) to study cis-acting regulators of A3A induction and pharmacological and genetic approaches to map the pathways involved. To dissect the role of A3A during cell cycle re-entry we will use global approaches (RNA-seq of total and polysome-associated RNAs) to gain a comprehensive picture of A3A RNA editing and its potential roles in regulating gene expression. This will be supported by functional experiments comparing WT and A3A KO keratinocytes, with findings validated in organotypic raft cultures to build a new understanding of A3A regulation and function in epithelia. This work will provide valuable insight into fundamental processes including the regulation of cell cycle and gene expression, with direct relevance to pathologies including viral infection, inflammation and cancer.

Summary

One way in which our bodies fight viral infections is to attack viral genomes (either DNA or RNA, depending on the virus) when viruses enter our cells and attempt to replicate. One protein involved in this immune response, APOBEC3A (A3A) modifies cytosine, one of the four building blocks of DNA and RNA, causing errors (mutations) and breaks in viral genes. A3A is not normally found at high levels in our cells except in certain specialised immune cells called macrophages. It is switched on in response to infection and plays an important role in protecting us from a range of viruses, however it is also activated in inflammatory conditions including eczema and psoriasis. Although A3A helps to defend us from viral infections, this protection comes at a cost, as we and others have shown that it can turn against our own genes, generating mutations that cause cancer. Numerous studies have confirmed that this process occurs in a large proportion of cancers, particularly those arising in the tissues (epithelia) that line the mouth and throat, lung, breast, bladder and cervix. Not only does A3A mutate our DNA during cancer development but it appears that A3A can continue to act in this rogue fashion while patients are receiving chemotherapy; driving drug-resistance and ultimately, treatment failure. This knowledge has stimulated initiatives in academia and industry to develop A3A inhibitors; drugs that could block this mutagenic activity in patients receiving chemotherapy, thereby preventing tumours from becoming resistant to the therapy. While this approach holds the potential to improve outcomes for millions of cancer patients, there is much we do not yet know about the way in which A3A is controlled and about the functions that it performs in the normal, healthy epithelial cells from which A3A-mutated cancers develop. Without this knowledge, we have little idea of what triggers rogue A3A activity, or what the side-effects of inhibiting A3A activity might be in patients. In this proposal, we set out a series of experiments to address these questions, based on three key findings that we have made from studying A3A in cultured human epithelial cells. 1) We have discovered that by mimicking a wound-healing response in epithelial cells, we can switch the A3A gene on to levels far higher than those previously seen in these cells and that remains at very high levels as these cells replicate their DNA, a time at which the DNA is potentially vulnerable to rogue A3A activity. 2) By deleting the A3A gene in epithelial cells, we have uncovered a previously unknown role for A3A in regulating the rate at which these cells divide, a role that is critical to understand if we are to anticipate the effects of targeting A3A for cancer therapy. 3) Recent studies have demonstrated that A3A can modify many cellular messenger RNAs, the intermediate transcripts that allow our genes to be translated into proteins but the significance of this activity remains unclear. We observe a very strong induction of this RNA-editing activity when we activate A3A in epithelial cells. Based on these novel observations, we will use our unique tools to identify in detail how the A3A gene is switched on in epithelial cells that have been stimulated to divide and will establish the role that A3A plays in regulating this process. We will conduct a comprehensive survey of A3A-mediated RNA editing events and will test our hypothesis that this activity allows A3A to change the rate at which key proteins are made. This project will result in a step-change in our knowledge of A3A regulation and function, its importance in normal epithelial biology and in pathologies ranging from inflammation to viral infections and cancer. This knowledge will be vital if we are to successfully harness A3A as a drug target. It will also address a fundamental question regarding the role of A3A-mediated RNA editing in controlling how our genes are expressed.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/V010271/1 Investigating regulation and function of the cytosine deaminase APOBEC3A during cell cycle re-entry

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