Studying the regulation of DNA repair in neuronal cells
Principal Supervisor: Dr Martin HiggsLink opens in a new window
Co-supervisor: Professor Tim Barrett
PhD project title: Studying the regulation of 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 development, neurological function, and organismal survival. To counteract this, cells have evolved complex DNA damage response (DDR) pathways to repair this damage. A critical part of DNA repair is the control of repair proteins by post-translational modifications.
My group is interested in one DDR-associated histone PTM, lysine methylation, and the enzymes that catalyse this. Our focus is to understand how these ‘lysine methyltransferase’ enzymes control DNA repair.
Studies from my lab have demonstrated that one particular lysine methyltransferase is required to repair several types of DNA lesions (1-2), including DNA double-strand breaks. We have also shown that this enzyme plays a direct role in methylating a vital DNA repair protein called MRE11, controlling its activity and repair functions. Interestingly, patients harbouring mutations in either this methyltransferase or MRE11 exhibit neurological and neurodevelopmental defects (3,4). This suggests that neuronal function relies heavily on these lysine methyltransferase enzymes, perhaps due to their roles in DNA repair.
Aims and experimental approaches: The aim of this studentship is to characterise how lysine methylation of MRE11 controls DNA repair in neurons.
The project has 3 main objectives:
1) Establish cellular model systems: First, we will delete MRE11 or the lysine methyltransferase from existing iPSC/hESC cells using CRISPR-Cas9. In parallel, we will clone existing wild-type, patient-derived or methyl-deficient MRE11 variants into viral vectors and use these to create complemented iPSCs. We will then verify the localisation and expression of these variants and derive these iPSC cells into neurons for following experiments. Alongside this, we will create immortalised neuronal SH-SY5Y lines lacking this lysine methyltransferase using CRISPR-Cas9 and/or RNAi, along with versions re-expressing the enzyme of interest.
2) Uncover how lysine methylation repairs double-strand breaks in neurons: Using these cellular systems, we will investigate the role of these enzymes in expression of neuronal cell markers, as well as neuronal growth, survival, and rates of apoptosis. We will next investigate how MRE11 methylation contributes to the repair of DNA double strand breaks (physiological and induced) in these cells using immunofluorescence for known DNA damage markers, as well as immunoblotting and comet assays.
3) Investigate the impact of loss of lysine methylation on transcription: Since MRE11 is implicated in maintaining transcription under various stresses (5), we will use RNA-seq to investigate whether loss of MRE11 methylation affects neuronal transcription. We will also investigate the impact of DNA damage on this process and investigate the roles of any common differentially expressed genes in the DDR.
This PhD project will offer extensive training opportunities in a variety of integrated genomics techniques, generate novel data on how lysine methylation regulates the DNA damage response in neurons, and lead to a greater understanding of the control of neuronal DNA repair.
1) Higgs et al., 2018. Histone methylation by SETD1A protects nascent DNA through the nucleosome chaperone activity of FANCD2. Molecular Cell; 71(1): 25-41.
2) Bayley et al. H3K4 methylation by SETD1A/BOD1L facilitates RIF1-dependent NHEJ. Mol Cell;
3) Kummeling et al., 2020. Characterization of SETD1A haploinsufficiency in humans and Drosophila defines a novel neurodevelopmental syndrome. Molecular Psychiatry; in press. doi: 10.1038/s41380-020-0725-5.
4) Taylor, Groom and Byrd, 2004. Ataxia-telangiectasia-like disorder (ATLD)-its clinical presentation and molecular basis. DNA Repair; 3(8-9):1219-25
5) Forey et al., 2021. A Role for the Mre11-Rad50-Xrs2 Complex in Gene Expression and Chromosome Organization. Mol Cell; 81(1):183-197.
BBSRC Strategic Research Priority: Understanding the rules of life – Neuroscience and Behaviour, and Integrated Understanding of Health - Ageing.
Techniques that will be undertaken during the project:
This PhD encompasses a wide variety of training opportunities in several techniques:
- CRISPR-Cas9 gene editing and RNAi
- somatic and neuronal cell culture and differentiation
- ELISA and comet assays
- fluorescent and visual microscopy
- immunoblotting and immunofluorescence
- histone extractions/ChIP
- molecular cloning and site-directed mutagenesis
- flow cytometry
- qPCR and chromatin immunoprecipitation