Novel functions of epigenetic modifiers KMT2C/KMT2D (MLL3/MLL4) on gene expression and genomic stability

Secondary Supervisor(s): Dr Boris Kysela

University of Registration: Aston University

BBSRC Research Themes:

• Understanding the Rules of Life (Systems Biology)
• Integrated Understanding of Health (Ageing)

Project Outline

One key characteristic of ageing and disease is the change in the gene expression and accumulation of somatic mutations. We are interested in elucidating the consequences of mutations in 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) methyltransferases KMT2D (also known as MLL2 or MLL4) and KMT2C (MLL3) emerged in the last 10 years as two of the most mutated genes in cancer genomes. Outside cancer, partial loss of a single allele of KMT2D or KMT2C result to Kabuki and Kleefstra syndromes respectively; both are rare genetic disorders marked by intellectual disability and frequently accompanied by a range of complex physical and clinical characteristics. Importantly, the mechanism(s) of how KMT2C and KMT2D contribute to these genetic disorders and disease is not clear.

The main function of KMT2C and KMT2D was thought to be the monomethylation of H3K4 at enhancer elements and therefore to impart a significant role on gene expression, especially during development. However, it was recently shown that actually KMT2D and KMT2C mediated monomethylation is not essential for enhancers and probably not their main role.

We have previously reported¹ that KMT2D functions at gene promoters and when mutated it deregulates RNA polymerase II transcription. As a result, the cells suffer from transcriptional stress, DNA damage and mutation.

We now want to expand our findings to KMT2C and examine the roles of both proteins. To this end, we have developed conditional knock-out (KO) mouse embryonic fibroblast (MEF) cell lines that can precisely excise the KMT2D or KMT2C gene, resulting in effective KO mouse cell lines². Moreover, we also have human HCT116 cells that lack KMT2D³ or only the methyltransferase domain of KMT2D⁴.

To achieve our goals, we will take advantage of the above cell lines and employ a variety of standard and cutting edge molecular, cellular, biochemical and computational methods to explore: 1) The effect of KMT2C or KMT2D KO on coding and non-coding RNA expression 2) The effect of KMT2C or KMT2D deletion on the epigenetic landscape of target genes 3) The effect of ionising radiation and genotoxic agents on cells lacking KMT2C or KMT2D 4) The mechanisms by which the above occur.

By completing the above objectives, we will acquire a better understanding of how these histone modifiers, can control gene expression and genome stability through chromatin modifications and regulate multiple downstream processes, that in turn affect ageing and disease.

References

1. Kantidakis, T. et al., 2016. Mutation of cancer driver MLL2 results in transcription stress and genome instability. Genes Dev 30 (4), 408–20.

2. Ashokkumar, D. et al., 2020. MLL4 is required after implantation, whereas MLL3 becomes essential during late gestation. Development, 147(12): dev186999.

3. Guo, C. et al., 2012. Global identification of MLL2-targeted loci reveals MLL2's role in diverse signaling pathways. Proc Natl Acad Sci USA, 109(43):17603-8.

4. Guo C, et al., 2013. KMT2D maintains neoplastic cell proliferation and global histone H3 lysine 4 monomethylation. Oncotarget, 4(11):2144-53.