The role of histone methyltransferase MLL2 on tRNA expression

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 epigenetic proteins that control gene expression, and understand how these contribute to transcriptional stress, DNA damage and cell viability.

The histone 3 lysine 4 (H3K4) methyltransferase MLL2 emerged in the last 10 years as one of the most mutated genes in cancer genomes. It is responsible for the mono-, di- and tri-methylation of H3K4. 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).

RNA polymerase III (Pol III) is responsible for producing several short, non-coding RNAs, including the tRNAs. The expression of these ncRNAs is altered in ageing and disease, including cancer as we have recently shown (Sangha and Kantidakis, 2022). This project will build on our extensive experience on Pol III transcription regulation and chromatin epigenetics, to investigate if MLL2 can control gene transcription, and especially tRNA expression, by Pol III.

To achieve this, we will employ a variety of standard and cutting edge molecular, cellular, biochemical and computational methods. We will take advantage of established human MLL2 knock-out (KO) and 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 the above 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.

Techniques

• Cell biology: cell culture, optical and fluorescence microscopy, cell growth/viability and cell mobility/invasion assays, cell irradiation and DNA damage and apoptosis assays
• Molecular biology: gDNA and RNA isolation, PCR, RT-qPCR, site-directed mutagenesis, molecular cloning, CRISPR genome editing, RNA-Seq
• Biochemistry: fractionation and protein isolation, Western blotting, co-immunoprecipitation (Co-IPs), siRNA knock-downs, chromatin immunoprecipitation (ChIP) and Re-ChIP, Cut&Run assays
• Computational techniques: RNA-Seq and ChIP-Seq analysis, genomic data analysis (DNA mutations, DNA methylation, mRNA and miRNA expression), gene enrichment and pathway mapping