Defining a fundamental role for histone methylation in preventing DNA damage-induced replication catastrophe

Duplication of the cell's genome is essential for the continuation of life, and this must occur in an efficient and well-timed manner to ensure that each daughter cell gets the correct amount of genetic material. The presence of damage within the cell's DNA can slow or stall the DNA replication machinery, preventing the timely completion of genome duplication. This can result in either cell death, if the unrepaired damage is too great, or transfer of the damage into the daughter cells when the cell divides, which can cause mutations. The accumulation of DNA damage and mutations over time is a fundamental part of the process in which normal healthy cells undergo transformation into tumour cells. It is therefore clear that problems with repairing damage that occurs during DNA replication are a significant cause of human disease and cancer.
To prevent genetic damage from persisting, cells have evolved a highly complex network of cellular pathways that recognise and repair DNA damage. One of the most important DNA repair pathways that functions during DNA replication is the Fanconi Anaemia (FA) pathway. The FA pathway is named after the rare human disease, Fanconi Anaemia, which is associated with severe clinical symptoms such as growth retardation, limb malformation, brain abnormalities, progressive bone marrow failure leading to anaemia and an increased predisposition for developing cancer. To date, it has been found that mutations in any one of 16 different genes, all working within the same pathway, can give rise to FA.
The study of rare human syndromes, such as FA, has dramatically increased our knowledge about how DNA repair pathways protect the cell from genetic damage, and the consequences when this process fails. However, despite our progress, we still do not fully understand some fundamental aspects of how these pathways function or indeed whether additional factors exist that are yet to be discovered. Indeed, patients suffering from an FA-like disorder have been identified in whom the disease-causing mutation has yet to be discovered.
In this respect, we have identified a new protein (PFAP; Putative Fanconi Anaemia Protein) that functions to help repair DNA damage that arises during DNA replication. Interestingly, the defects caused by loss of PFAP are strikingly similar to those observed in cells from FA patients, suggesting that PFAP functions within the FA pathway, and may even be mutated in patients exhibiting FA-like symptoms. In addition, we have discovered that PFAP binds to an enzyme called HMT-1 (Histone Methyltransferase 1), whose primary role is to modify the structure of proteins surrounding DNA (called chromatin) through a process called methylation. Strikingly, loss of HMT-1 gives rise to the same problems as we observed in cells lacking PFAP, indicative of a biochemical link between 'chromatin methylation' and the FA repair pathway.
Whilst we know that chromatin methylation can alter the way cells respond to certain types of DNA damage, it is unknown how it affects the FA pathway. Therefore, this application aims to investigate this, using three different approaches. Firstly, we aim to establish where PFAP functions within the FA pathway and ascertain whether PFAP is mutated in cells derived from patients exhibiting an FA-like disorder. Secondly, we aim to understand how PFAP controls DNA replication in the presence of DNA damage. Lastly, we aim to determine if chromatin methylation carried out by PFAP/HMT-1 contributes to how replication-associated DNA damage is repaired.
The proposed research on this fundamental and highly conserved aspect of the cellular response to DNA damage has the potential to make a significant long-term impact on improving human health and wellbeing. In addition to furthering our understanding of how normal cells transform into tumour cells, it could also aid the diagnosis of FA-like disorders and/or facilitate the development of new chemotherapeutic drugs to treat cancer.

'Replication stress' is a term that encompasses any type of abnormality that obstructs replication, the common result of which is increased genome instability. In addition to the well-established link between genomic instability and tumour development, it is becoming increasingly evident that replication stress is also an important pathological process underlying the development of human disease. It is thought that failed replication caused by unrepaired DNA damage leads to increased cell death, particularly in highly proliferative tissues. This is exemplified by the human syndrome Fanconi Anaemia (FA), which is characterised by developmental defects, progressive bone marrow failure, tumour predisposition and a hypersensitivity to agents that induce replication stress e.g. Mitomycin C (MMC). The study of human disorders such as FA and the cellular pathways affected in these patients has significantly furthered our understanding of the molecular mechanisms that have evolved to protect against the deleterious effects of replication stress.
Here we have identified PFAP (Putative Fanconi Anaemia Protein), a member of a histone methyltransferase complex, as a novel suppressor of replication stress. Cells lacking PFAP exhibit a strikingly similar phenotype to that associated with defects in the FA DNA repair pathway, such as a hypersensitivity to MMC, damage-induced replication abnormalities and chromosomal breakage following exposure to MMC. Taken together, these data suggest that PFAP functions to coordinate the repair of replication-associated DNA damage in conjunction with the FA pathway. Our finding that PFAP depleted cells also have reduced levels of histone methylation provides the first evidence of a mechanistic link between epigenetic histone modification and the maintenance of genome stability.
Our proposal focuses on understanding how PFAP promotes the repair of damaged replication forks and whether this is mediated through histone methylation.

This work aims to improve our understanding of how cells respond to replication stress and how defects in this process give rise to genome instability and human disease. It is known that a number of inherited disorders exhibiting developmental abnormalities and cancer predisposition (e.g. Fanconi Anaemia (FA)) are associated with elevated levels of replication stress and chromosomal fragility. This research project focuses on investigating the role of BOD1L and Setd1A as novel regulators of the FA pathway.

Beyond academia and related research fields, our preliminary data detailed within, in combination with the results arising from the proposed work, have the potential to make a significant long-term impact on the health sector, and improve 'quality of life' and 'lifelong health and wellbeing'. These impacts will be achieved through close collaborations with clinicians and researchers expert in the fields of replication stress and genome instability.

Firstly, this research project focuses on investigating the role of BOD1L and Setd1A as novel regulators of the FA pathway. Based on our preliminary data, it is conceivable that inherited mutations in either of these genes could give rise to a human disorder that has a very similar clinical and cellular phenotype to that of FA. I have already initiated a collaboration with Prof. D. Schindler (University of Wuerzburg) to determine whether BOD1L and/or SETD1A are mutated in patients exhibiting symptoms consistent with Fanconi Anaemia. Therefore, this proposal may aid our ability to achieve a molecular diagnosis of FA and/or related disorders, as well as assisting in their clinical management, and thus impacting on human health.

Interestingly, BOD1L has been reported to be mutated in a proportion of cases of Myelodysplastic Syndrome (MDS), a human disease often associated with the onset of severe anaemia that can often progress to bone marrow failure and/or acute myeloid leukemia (symptoms that are very reminiscent of FA). Given that many of the genes mutated in FA are known tumour suppressor genes (e.g. BRCA2 (FANCD1), PALB2 (FANCN) and RAD51C (FANCO)), it is conceivable that both BOD1L and SETD1A are also involved in preventing tumourigenesis through their ability to promote genome stability. To gain more insight into this, I have initiated a collaboration with Dr. M. Raghavan (QE Hospital, Birmingham Haematology Dept) to sequence the BOD1L gene in a large cohort of MDS cases. As a consequence, the screening for BOD1L gene mutations in MDS may provide novel clinical approaches to determining both prognosis and responsiveness to therapy.

Finally, our proposed research investigating the potential role of the BOD1L-Setd1A complex in suppressing replication stress may form the basis for the development of enzymatic inhibitors of Setd1A to be used as a tool to induce catastrophic replication stress in tumour cells. Increased replication stress is a common underlying cause of genome instability associated with many different tumour types. This phenotype is now being exploited by academic and pharmaceutical laboratories, who are developing inhibitors that function to specifically increase the levels of replication stress/genome instability within tumour cells to such a point that they are unable to survive. Indeed, the successful use of PARP inhibitors to treat homologous recombination-defective tumours has paved the way for the development of other novel anti-cancer agents that work on the principle of synthetic lethality. The ongoing development of epigenetic inhibitors as novel anti-cancer agents targeting other methyltransferases supports a targeted future collaboration with pharmaceutical laboratories to develop Setd1A inhibitors, with the ultimate aim of developing novel therapeutic anti-cancer strategies.
Taken together, it is clear that the work proposed here could have a significant long term impact on human health and well-being through one or more different routes.