title: ZC3H14, a new player in the early response to DNA double strand breaks
Creators: name: Kieffer, Shaylee
Publisher: name: University of Galway
Year: 2024
Subjects: subject: ZC3H14
subject: DNA double strand breaks
subject: Biological and Chemical Sciences
subject: Biochemistry

description: The primary goal of all organisms is to pass down intact genetic information to the next generation, despite mutagenic threats from both endogenous and exogenous sources. To prevent accumulation of DNA damage, cells have developed complex mechanisms that detect and respond to different forms of lesions. These complex pathways are collectively called the DNA damage response (DDR). Impaired responses to DNA damage drive oncogenesis, neurodegeneration, and immunodeficiencies. Both DNA damaging agents and drugs which target specific repair proteins are a mainstay of modern current therapeutic approaches. Thus, to understand the aetiology of disease, the impact of current therapeutic interventions, and to develop further precision medicines, it is vital to understand the complex series of pathways involved in the DDR. One of the major protein kinases involved in signal transduction within the cells’ response to double strand breaks (DSBs) is Ataxia-telangiectasia mutated (ATM). Previous research from Prof. Noel Lowndes’ laboratory identified a zinc-finger protein, ZC3H14, as a DNA damage-dependent ATM-interacting partner. This is an unexpected interaction as ZC3H14’s canonical roles are in mRNA processing, including regulation of poly(A) tail length and mRNA nuclear export. Similarly to ATM and other DDR proteins, ZC3H14 has both neurological and links to cancer when it its function is impaired. This novel data implicates ZC3H14 in the DDR, as well as placing it in a disease-critical pathway. Unpublished data has placed ZC3H14 in the ‘Early’ response to DSBs, which occurs minutes after break formation. Cells lacking ZC3H14 showed striking repair defects: cells become sensitive to ionising radiation (IR); depletion of ZC3H14 results in a persistent increase in the damage markers γH2AX foci and ATM-pS1981; and knockdown of ZC3H14 ablated irradiation-induced foci (IRIF) of ‘Early’ DNA damage response proteins downstream of MDC1. In addition, ZC3H14 co-immunoprecipitated with both ATM and MDC1. Preliminary data suggests that ZC3H14 is more involved in NHEJ than HR, despite its placement in the response upstream of pathway choice. Here, we aim to deepen our understanding of the role of ZC3H14 in the DDR using bioinformatic, cellular and molecular geology, and biochemical methodology. We generated ZC3H14KO cells, allowing us to study the placement of ZC3H14 in the DDR pathway, and to confirm the defect of DDR protein recruitment via independent methods. These cells show an increase in endogenous damage and impaired foci formation. We designed GFP-ZC3H14 domain mutants that truncate the N-terminal PWI-like domain or the five tandem C-terminal zinc finger domains in order to explore the mechanisms of these domains on ZC3H14’s role in repair. We were able to determine that ZC3H14 is a damage-specific substrate of ATM, and that it localises in proximity to damage-activated ATM-pS1981 in vivo. Despite these interactions, we were unable to visualise ZC3H14 at damage foci, and hypothesise that due to ZC3H14’s heavy localisation to nuclear speckles, there may be only a subset of ZC3H14 at the speckle’s periphery that is phosphorylated and responsive to damage. As well, ZC3H14 has a large intrinsically disordered domain which could contribute to its localisation. Interestingly, we used the newly released AlphaFold3 server to predict that ZC3H14 could contain a disorder-toorder region which has an alpha-helixes rich folded structure in the presence of DNA and other DDR proteins, but not nuclear speckle proteins. All of this points to a damageactivated phosphorylated form of ZC3H14 which has some as-yet unsolved role at DSBs. This role is likely in regulating the recruitment of MDC1 or RNF8, but could also have to do with phase separation of IRIF condensates, or provide a link between mRNA processing and DNA repair. The project aims to characterize a novel role for a zinc-finger protein, ZC3H14, in the DNA damage response (DDR): its interactions with Ataxia-telangiectasia mutated (ATM), its effect on cell survival, its impact on formation of irradiation-induced foci (IRIF), its mechanism of action, and its signature in human diseases. ZC3H14 has not been previously implicated in DNA repair. Many proteins within this pathway can be targeted for synthetic lethality, to be taken advantage of both for molecular tools for further study, and for druggable targets with the advent of personalised medicines.