Defining the function of histone ADP-ribosylation in DNA repair and genome integrity

DNA is continually being exposed to a variety of agents that induce DNA damage resulting in tens-of-thousands of DNA lesions per cell every day. As such, an intricate set of pathways known as the DNA damage response (DDR) detect DNA damage when it occurs and activate mechanisms for its repair. These pathways are critical for our health and well-being and their dysfunction can lead to a variety of clinical symptoms including cancer, neurodegeneration, immune-deficiencies and premature ageing. Therefore, understanding how cells respond to and repair DNA damage will provide information about the underlying causes of these conditions and, importantly, how they can be treated. This strategy is exemplified by inhibition of ADP-ribosyltransferases (ARTs), a class of enzymes that detect DNA damage and attach ADP-ribose units onto proteins at damage sites to promote DNA repair. Inhibitors of these enzymes are currently being used successfully to treat ovarian cancer and have the potential to treat other pathologies associated with defects in the DDR. However, despite the importance of ART inhibitors in the clinic, our knowledge of how these enzymes regulate DNA repair is limited. Furthering this understanding will underpin refined strategies that exploit ART inhibitors to treat diseases associated with DDR dysfunction and provide a paradigm for how ARTs regulate other critical processes including cell growth and differentiation, gene expression and programmed cell death.

The proteins modified at DNA lesions by ARTs in response to DNA damage are particularly ill-defined and the basis of how this regulates the repair process is only poorly understood. This situation is epitomized by histones, the proteins that package DNA into the nucleus of the cell. These proteins are known targets for ARTs. However, the sites modified on histones in response to DNA damage and how this regulates DNA repair remains unknown. This lack of mechanistic insight is due, in part, to the absence of an appropriate experimental platform in which both ARTs and histone genes can be manipulated to directly test hypotheses of how modification of specific sites on histones by ARTs regulates DNA repair in a cellular context. We have established that these criteria are uniquely met in the model organism Dictyostelium, providing the opportunity to identify novel DNA repair factors and concepts in this system that will subsequently be applied to humans.

Our current work has developed an experimental pipeline in Dictyostelium to identify histone ADP-ribosylation sites in the cell and to genetically manipulate histone genes to block their modification. The aim of this research is to exploit this unique approach to test how these modifications regulate DNA repair. We will comprehensively catalogue the histones, and the amino acid residues in them, that are modified by ARTs in response to DNA damage. We will then exploit the genetic tractability of Dictyostelium to disrupt the specific histone ADP-ribosylation events identified to establish their importance in regulating DNA repair. This will provide a robust experimental platform to identify novel repair proteins that are recruited to ADP-ribosylated histones in Dictyostelium. Having identified these factors, we will subsequently characterize how the equivalent proteins regulate DNA repair in the humans. In addition to providing an increased understanding of how cells promote DNA repair to prevent mutagenesis, these studies will provide information to facilitate the design of specific therapeutic agents to target DNA repair pathways to treat a variety of diseases including cancer.

We address three outstanding questions regarding ADP-ribosylation of histones in regulation of double strand break (DSB) repair: a) Which histones are modified in response to DSBs in vivo and at what sites? b) What is the consequence of defective histone ADP-ribosylation on DSB repair? c) What factors interact with ADP-ribosylated histones and how do they regulate DNA repair in humans?

A major bottle-neck for the ADP-ribosylation field is the lack of an experimental model in which ADP-ribosylation sites can be mutated at endogenous histone loci to decipher the molecular basis of how these modifications regulate DNA repair. The ability to manipulate histone genes in Dictyostelium offers a unique platform to address these questions. Established mass spectrometry (MS) approaches will identify specific sites on Dictyostelium histones ADP-ribosylated in response to DSBs in vivo. Gene replacement technology will introduce mutations at these sites identified by MS into endogenous histone genes, to generate histone ADP-ribosylation defective strains. The ability of these strains to perform DSB repair will be assessed using standard assays including plasmid integration assays to determine NHEJ and HR efficiencies, sensitivity to DNA damage, enrichment of repair factors at damage sites. These strains offer a rigorously controlled platform to identify factors that interact with ADP-ribosylated histones, which can then be extended to humans. Immunoprecipitation of nucleosome complexes from wild-type and histone ADP-ribosylation defective strains will identify factors that specifically interact with ADP-ribosylated histones by comparative MS. The orthologues of novel proteins identified in Dictyostelium will be verified by enrichment at DSBs in human cells, disrupted by CRISPR/Cas9 genome editing, and the functional consequences on human DSB repair assessed using standard assays, including repair efficiencies, enrichment of repair factors at DSBs etc.

The immediate beneficiaries of this work will be the scientific community by providing reagents and methodologies of importance to the DNA repair, ADP-ribosyltransferase and Dictyostelium research communities. These experiments will consolidate Dictyostelium as a model to study DNA repair and this information will be exploited to increase our understanding of how ADP-ribosylation of histones regulates DNA repair in humans.

In the longer term, this work will provide information that impacts on human health and wellbeing. Given that DNA repair pathways are targeted in chemo and/or radiotherapy regimens inducing DNA double strand breaks, this work will provide conceptual advances that can be exploited to improve these treatments. NL has established links with industrial partners such as AstraZeneca to understand how targeting specific ART combinations can impact on DNA repair efficiency. These studies will identify and highlight novel therapeutic strategies to specifically inhibit double strand break repair pathways. This approach will be applicable to other forms of DNA damage, widening the range of therapeutic targets. It also has the potential to identify novel biomarkers for activation of specific DNA repair pathways in tumour cells. In the longer term this will contribute to the development of precision medicine for cancer with therapies tailored to the individual by identifying and targeting relevant repair pathways active within the tumour cells.

Specific beneficiaries include:

i) Academic Sector:
- This work will contribute to worldwide academic advancement by improving our understanding of DNA repair in addition to how ADP-ribosylation regulates fundamental biological processes.
-Innovative methodologies developed in Dictyostelium will be transferable to other organisms and improve technologies in Dictyostelium involving genetic manipulation (e.g. gene disruption and mutant generation)
-Understanding the molecular basis of DNA repair will impact on genome editing technologies that can be used either as a research tool, or in gene replacement therapy.
-This work will provide a training for the postdoc in a variety of scientific and transferable skills. It will also provide future research projects to train/teach undergraduate/postgraduate researchers.

ii) Commercial sector
- Providing commercial companies with information to develop therapies that specifically target malignant cells. Understanding DNA repair can be exploited in the clinic through a synthetic lethal strategy to kill cells with defects in a defined genetic background.
- Providing companies with the information required to define tumours responsive to PARP inhibition.
- Inappropriate DNA repair is a causative factor of age-related disease, offering the opportunity to exploit inhibitors of these pathways to ameliorate their symptoms.
- Providing information for companies wishing to screen for gene mutations in novel DNA repair genes that contribute towards malignancy, or resistance of tumours to clinical intervention.
- Establishing Dictyostelium as a model to study the impact of inhibition of histone modification will facilitate its use in screening for pharmaceuticals impacting on histone modifications.
iii) Public sector
- Influence of policy decisions regarding the effects of DNA damage during stem cell propagation and embryonic development.
- Contribution to the development of policies to protect the general public from the long term effects of DNA damage.

iv) Wider public
- Developing novel, targeted cancer therapies will impact on the lives of future cancer patients.
- The development of tailored information regarding life style, diet etc. to protect from the effects of DNA damage during ageing. This is particularly important in light of the increasing numbers of elderly people, allowing lifestyle choices at an early age to increase the quality of life in later years.