Principal Supervisor: Dr Theo KantidakisLink opens in a new window

Co-supervisor: Dr Boris Kysela

PhD project title: The histone methyltransferase MLL2 regulates ncRNA transcription by RNA polymerase III

University of Registration: University of Aston

Project outline:

One key characteristic of ageing and disease is the change in the gene expression of key biochemical pathways. We are interested in elucidating the consequences of mutations/depletion of key proteins that control specific pathways of gene expression, and understand how these contribute to transcriptional stress, DNA damage and cell viability.

Project

The histone 3 lysine 4 (H3K4) methyltransferase MLL2 emerged in the last 10 years as one of the most mutated genes in cancer genomes. We have previously reported the mechanism by which MLL2 mutation can contribute to disease by deregulating RNA polymerase II transcription and resulting in transcriptional stress, DNA damage and mutations (Kantidakis et al., 2016).

This project will investigate if MLL2 also binds Pol III and regulates its transcription. Pol III is responsible for producing several short, non-coding RNAs, including the tRNAs. We have recently shown how the individual tRNA expression is altered in different cancers (Sangha and Kantidakis, 2022). We now have preliminary data that the expression of tRNAs, and probably of other ncRNAs encoded by RNA polymerase III, is regulated by MLL2.

We will use a variety of standard and cutting edge molecular, cellular, biochemical and computational methods to investigate the role of MLL2 on Pol III transcription. We will take advantage of established human MLL2 knock-out (KO) or mouse conditional KO cell lines (Kantidakis et al., 2016) to explore: 1) The effect of MLL2 deletion on the expression of Pol III-transcribed genes, 2) The effect of MLL2 deletion on the epigenetic landscape of Pol III target genes, 3) The mechanism by which the above occur. By completing these objectives, we will acquire a better understanding of how this histone modifier, can control ncRNA expression through chromatin, and regulate multiple downstream processes, like the course and rate of protein translation and the production of functional proteins, that in turn affect ageing and disease.

References:

Kantidakis, T. et al., 2016. Mutation of cancer driver MLL2 results in transcription stress and genome instability. Genes Dev 30, 408–20.

Sangha, A.K. and Kantidakis, T., 2022. The Aminoacyl-tRNA Synthetase and tRNA Expression Levels Are Deregulated in Cancer and Correlate Independently with Patient Survival. Curr Issues Mol Biol 44, 3001–3017.

BBSRC Strategic Research Priority: Understanding the rules of life - Systems Biology and Integrated Understanding of Health - Ageing

Techniques that will be undertaken during the project:

Cell biology: cell culture, cell growth/viability and cell mobility/invasion assays, optical and fluorescence microscopy

Molecular biology: gDNA and RNA isolation, PCR, RT-qPCR, site-directed mutagenesis, molecular cloning

Biochemistry: protein isolation, Western blotting, co-immunoprecipitation (Co-IPs), chromatin immunoprecipitation (ChIP), DNA damage and apoptosis assays

Computational techniques: RNA-Seq and ChIP-Seq analysis, data analysis (DNA mutations and RNA expression), gene enrichment and pathway mapping

Contact: Dr Theo KantidakisLink opens in a new window