Primary Supervisor: Dr Martin Higgs, Institute of Cancer and Genomic Sciences
Secondary supervisor: Professor Tim Barrett, University of Birmingham
PhD project title: Studying how loss of lysine methylation affects DNA repair in neuronal cells
University of Registration: University of Birmingham
Background: Our DNA is under constant attack from DNA damaging agents, which pose a significant threat to organismal viability and development, cellular homeostasis, neurological function, and haematopoiesis. Given its importance, cells have evolved multiple DNA damage response (DDR) proteins to repair DNA damage. The activity of these factors is controlled by a complex network of post-translational modifications (PTMs).
My group is interested in one DDR-associated PTM, lysine methylation, and the enzymes that catalyse this. Our focus is to understand how these ‘lysine methyltransferase’ enzymes control the DDR and help maintain genome stability. Studies from my lab have demonstrated that one particular lysine methyltransferase is involved in repairing specific DNA lesions (1). We have also demonstrated that patients harbouring loss-of-function (LoF) mutations in this enzyme exhibit severe neurological defects, and cells from these patients exhibit defects in the DDR (2). We therefore predict that this enzyme plays a vital role in repairing DNA damage in neurons.
Aims and experimental approaches: The aim of this studentship is to characterise how loss of lysine methylation impacts repair of other DNA lesions, and investigate the consequences in neurons. The project has 3 main objectives:
1) Uncover how specific methyltransferases repair DNA lesions: We will first create cell lines lacking lysine methyltransferases using CRISPR-Cas9. Using these cells in combination with patient-derived LCLs bearing LoF mutations, the role of these enzymes in repairing DNA double strand breaks and oxidative lesions will be examined using a combination of cell survival assays, immunofluorescence, ELISA and comet assay.
2) Model the impact of methyltransferase mutations on enzyme function: Using established lysine methylation assays, the impact of various LoF mutations on the catalytic activity of these enzymes will be examined biochemically. In parallel, global levels of methylation will be assessed in patient LCL cell lines using ChIP and immunoblotting. Alongside this, we will investigate whether mutated methyltransferase variants interact with other co-factors by immunoprecipitation/pulldown.
3) Assess the importance of lysine methylation in repairing DNA damage in neurons: Our collaborators (Kleefstra laboratory) have already generated neurons encoding patient-associated mutations using CRISPR-Cas9. To investigate whether these mutations affect DNA repair in these cells, we will analyse levels of cell cycle re-entry, genome stability, DNA repair and apoptosis after inducing DSBs and/or oxidative stress. This will be achieved using flow cytometry, immunoblotting, immunofluorescence and cell survival assays.
This PhD project will offer extensive training opportunities in a variety of integrated techniques, generate novel data on how lysine methylation regulates the DNA damage response, and lead to a greater understanding of neuronal DNA repair.
- Higgs et al., 2018. Histone methylation by SETD1A protects nascent DNA through the nucleosome chaperone activity of FANCD2. Molecular Cell; 71(1): 25-41.
- Kummeling et al., 2020. Characterization of SETD1A haploinsufficiency in humans and Drosophila defines a novel neurodevelopmental syndrome. Molecular Psychiatry; https://doi.org/10.1038/s41380-020-0725-5.
BBSRC Strategic Research Priority: Understanding the Rules of Life: Neuroscience and behaviour
Techniques that will be undertaken during the project:
This PhD encompasses a wide variety of training opportunities in several techniques:
- co-immunoprecipitation and pulldowns
- in vitro activity and binding assays
- ELISA and comet assays
- fluorescent and visual microscopy
- immunoblotting and immunofluorescence
- histone extractions/ChIP
- molecular cloning and site-directed mutagenesis
- somatic and neuronal cell culture and differentiation
- flow cytometry.
Contact: Dr Martin Higgs, University of Birmingham