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Studying epigenetic regulation of DNA repair in neuronal cells

Primary Supervisor: Dr Martin Higgs, Institute of Cancer and Genomic Sciences

Secondary supervisors: Professor Tim Barrett

PhD project title: Studying epigenetic regulation of DNA repair in neuronal cells

University of Registration: University of Birmingham

Project outline:

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. Since DNA damage occurs on chromatin rather than naked DNA, post-translational modifications (PTMs) of histones play a vital role in controlling the DDR.

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 oxidative damage. We have also demonstrated that patients with loss-of-function (LoF) mutations in this enzyme have severe neurological defects, and blood cells from these patients exhibit defects in DNA repair (3). Because neurons are particularly susceptible to oxidative DNA damage, we therefore predict that this enzyme plays a vital role in repairing such damage in neurons.

Aims and experimental approaches: The aim of this studentship is to characterise how histone lysine methylation controls the repair of oxidative DNA damage in neurons.

The project has 3 main objectives:

1) Establish cellular model systems: Our collaborators (Schubert and Kleefstra laboratories; Holland) have already generated iPSC cells encoding LoF mutations in our enzyme of interest using CRISPR-Cas9. These cells will be cultured and derived into neurons in vitro. 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 oxidative damage in neurons: Using these cellular systems, we will investigate the precise role of these enzymes in repairing oxidative lesions by examining cell survival, cell cycle, genome stability, DNA repair and apoptosis after oxidative damage. This will be achieved using flow cytometry, immunoblotting, immunofluorescence, cell survival, ELISA and comet assays. Key findings will be verified using iPSC-derived neurons and patient-derived cells.

3) Investigate the impact of loss of lysine methylation in neurons: Using our cellular systems, the impact of loss of this epigenetic enzyme on neuronal transcription will be assessed using transcriptional profiling by RNAseq and RT-qPCR. Key targets will be validated in different cellular systems, and cross-referenced with targeted ChIP data.

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 how epigenetics controls neuronal DNA repair.

References:

  • Higgs et al., 2018. Histone methylation by SETD1A protects nascent DNA through the nucleosome chaperone activity of FANCD2. Molecular Cell; 71(1): 25-41.
  • Bayley et al., H3K4 methylation by SETD1A/BOD1L facilitates RIF1-dependent NHEJ. Molecular Cell (under review).
  • Kummeling et al., 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 & 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
  • RNA-seq
  • qPCR and chromatin immunoprecipitation

Contact: Dr Martin Higgs,University of Birmingham