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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - PARPin (Regulation of (ADP-ribosyl)ation signalling in the DNA damage response: elucidating the function of a novel PARP1/ARTD1 interactor)

Teaser

Summary

The integrity of our DNA is constantly challenged by various types and sources of damaging agents that can cause genome instability, which is a common hallmark of many different cancer types and human pathologies. To protect DNA from damage, organisms have evolved a cellular defence mechanism termed the DNA damage response (DDR). The DDR includes a diverse set of molecular machines called proteins that act to sense DNA lesions and effectively repair the damage, restoring the health of the genetic code.

Defects in the DDR can cause, or contribute to, a range of pathologies, including cancer, ageing, immune deficiencies and neurological disorders. As such, deepening our understanding of the DDR is of fundamental importance. To improve our understanding of the DDR, we need to discover all the factors that make up this cellular defence mechanism. More importantly, we need to discover the function of each of these factors and how they interact or â€˜talkâ€™ with other repair factors to ensure genome stability. Expanding our understanding of the DDR will lead to better targeted therapies for cancer and other DDR-related pathologies.

In this project, the main objective was to characterise a novel factor involved in the DDR, to begin to understand how this factor regulates genome stability. Through this, our aim was to better understand the function of the clinically-relevant DNA repair enzyme PARP-1. In response to DNA damage, PARP-1 creates an SOS signal at DNA breaks called poly(ADP-ribosylation). In response to this SOS signal, repair proteins are mobilised to the DNA break to help with the process of repair. Therefore, PARP-1 plays a critical role in DNA repair by initiating the SOS signal. In other words, the research objective was to understand how PARP-1 is able to make the SOS signal in response to DNA breaks. Importantly, inhibiting this SOS signal with PARP inhibitors selectively kills certain cancer types, including breast and ovarian cancers, emphasising the clinical importance of the research objective.

Work performed

As mentioned above, PARP-1 is a DNA repair enzyme that modifies target proteins with a post-translational modification called poly(ADP-ribosylation) or PARylation. The main substrates of PARP-1 are histones and itself (called â€˜auto-modificationâ€™). However, it was unknown how PARP-1 achieved substrate specificity. We hypothesised that this might occur through an adaptor protein which would, in essence, tell PARP-1 what to modify. We therefore looked at the genomes of simpler organisms to assess whether there were hints of a co-evolved protein that might function together with PARP-1. We found the uncharacterised protein called C4orf27 as a good candidate in the human genome. We discovered that C4orf27 localises to DNA breaks in a manner dependent on the presence of PARP-1, but not on its enzymatic activity. Furthermore, the two proteins interact in cells, suggesting that C4orf27 might have an integral role in PARP-1 function. Importantly, in our efforts to understand the function of C4ofrf27, we could show that C4orf27 promotes PARP-1-dependent ADP-ribosylation of one of its key targets, the histone proteins, and prevents hyper auto-modification of PARP-1. Thus, we re-named C4orf27 as histone PARylation factor (HPF1). Our discovery showed that HPF1 is a first-in-class substrate specificity factor for PARP-1, or indeed any of the PARP family of proteins.

Another interesting discovery we made is that cancer cells with HPF1 removed are extremely sensitive to a drug that blocks PARP-1 enzymatic activity, called PARP inhibitor. PARP inhibitors are chemotherapeutic drugs currently in the clinic that are used to kill cancer cells with mutations in the BRCA1 and BRCA2 genes. However, cells deleted of HPF1 still have functional BRCA1 and BRCA2 proteins, which raises the exciting possibility that loss of HPF1 in BRCA1/2-mutated cancer cells may sensitise them further to PARP inhibitors. Further work is required to test this possibility.

Lastly, to begin to explore the physiological role of HPF1, we have now generated genetic models which will be the focus of future work in the host laboratory. Collectively, together with on-going structural and biochemical work and our published work, this will help determine whether HPF1 would be a good target for therapeutic strategies against cancer.

Final results

We have discovered a new gene product in the human genome that is involved in the DDR and genome stability. Importantly, we have discovered a fundamental and likely evolutionarily conserved mechanism of PARP-1 regulation. Our work has opened a new area of research within the chromatin biology, DNA repair and ADP-ribosylation fields. Moreover, given that PARP-1 has multiple functions beyond DNA repair, e.g. transcription and chromatin regulation, we fully expect that HPF1 will impact multiple important PARP-1-dependent biological pathways in cells. Indeed, soon after our first discovery, we could show further novel aspects of HPF1 function. We found that HPF1 switches the amino acid substrate preference of PARP-1, from aspartate/glutamate residues to serine residues. This activity is remarkable and also incredibly rare in biology, underlining the necessity to investigate this mechanism further in the future. Importantly, serine ADP-ribosylation has now been established as the major product formed by PARP-1 after DNA damage. Thus, from our initial discovery of HPF1, we have been able to establish the key components of the enzymatic cycle of serine ADP-ribosylation and its functional importance. The host laboratory is now primed to take advantage of these exciting discoveries in the future.

On a broader level, we are in the early stages of understanding the role of HPF1 as a therapeutic target in cancer therapies. Our finding that loss of HPF1 sensitises cancer cells to PARP inhibitor paves the way for future work to determine the relationship between HPF1 and cancers in which BRCA1 and BRCA2 genes are mutated. At present we do not know whether HPF1 would be amenable to small molecule drug targeting or whether its expression is differentially regulated in specific cancers. However, our initial findings provide an excellent platform from which to explore these further.