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Investigating the role of protein lysine methylation in the DNA damage response

Principal Supervisor: Dr Martin R. Higgs, Institute of Cancer and Genomic Sciences

Co-supervisor: Dr. Marco Saponaro, Institute of Cancer and Genomic Sciences

PhD project title: Investigating the role of protein lysine methylation in the DNA damage response.

University of Registration: University of Birmingham

Project outline

Background: Our DNA is under constant attack from DNA damaging agents, which are a continuing threat to genomic integrity. To counteract this, multiple DNA damage response (DDR) proteins have evolved to detect DNA damage, activate cell cycle checkpoints, and promote the repair and resolution of DNA lesions. The activity of these factors is controlled by a complex network of post-translational modifications (PTMs).

My group is interested in one particular DDR-associated PTM, lysine methylation, and the enzymes that catalyse this. These ‘lysine methyltransferases’ play vital roles in normal cellular function and organismal development, during transcription, and in preventing cellular transformation. Our main focus is on how these enzymes control the DDR and help maintain genome stability.

Aims and methodological approaches: This PhD will specifically investigate how members of the KMT2 family of lysine methyltransferases function in the DDR. The project has three main objectives:

1) Uncover the role that specific methyltransferases play in the DDR: To achieve this objective, cellular sensitivity assays using DNA damage-inducing agents will be carried out in cells depleted of these enzymes by RNAi. In parallel, the levels of genome instability in these cells will be analysed by light microscopy, and the recruitment of known DDR factors to sites of DNA damage will be assessed by immunofluorescence.

2) Examine how lysine methyltransferases regulate known DDR factors through Lys methylation: To assess this, lysine methylated DDR proteins will be isolated using pulldown/immunoprecipitation assays, and the precise site(s) of methylation will be uncovered using mass spectrometry. In vitro activity assays will then be used to determine whether these proteins are directly methylated.

3) Investigate how Lys methylation affects the interplay between transcription stress and DNA damage. To uncover how these KMT2 methyltransferases also regulate transcription after DNA damage, we will perform genome-wide transcriptional profiling of cells lacking specific KMT2 proteins before and after DNA damage, in collaboration with Dr. Marco Saponaro.

Together, these integrated approaches will lead to a greater understanding of how lysine methylation helps to maintain the integrity of the genome.

References:

  • BOD1L is required to suppress deleterious resection of stressed replication forks. Higgs et al., 2015; Mol Cell: 59; 462-77.
  • Quantitative dissection and stoichiometry determination of the human SET1/MLL histone methyltransferase complexes. Van Nuland et al., 2013; Mol Cell Biol: 33; 2067-77.
  • Hijacked in cancer: the KMT2 (MLL) family of methyltransferases. Rao and Dou, 2015; Nature Reviews Cancer: 15; 334-346.
  • Mutation of cancer driver MLL2 results in transcription stress and genome instability. Kantidakis et al., 2016; Genes Dev: 15; 408-20.

BBSRC Strategic Research Priority: Molecules, Cells and Systems

Techniques that will be undertaken during the project:

The project will involve a wide variety of laboratory techniques, including:

  • Mass spectrometry
  • NGS sequencing
  • Co-immunoprecipitation and pulldowns
  • RNAi techniques
  • In vitro and in vivo activity and binding assays,
  • Fluorescent and visual microscopy
  • Immunoblotting and immunofluorescence
  • Molecular cloning
  • Cell culture
  • CRISPR-based mutagenesis.

Contact: Dr Martin R. Higgs, Institute of Cancer and Genomic Sciences