Development and characterisation of a DNMT3A mutant cell line model of clonal haematopoiesis

Background DNMT3AR882H/C is a common mutation of the de novo DNA methyltransferase in clonal haematopoiesis of indeterminate potential (CHIP) and increases risk of progression to Acute Myeloid Leukaemia (AML).

This mutation increases the self-renewal capacity of haematopoietic stem and progenitor cells (HSPCs), conferring a survival advantage. The mechanism for this is incompletely understood. Mutated DNMT3A causes focal hypomethylation at specific genomic regions, particularly CpG shores and enhancers.

We hypothesise that the proportion of wild-type and mutant DNMT3A within its functional tetramer unit may result in variable genomic localisation, DNA-binding and methylation efficiency.

This focal differential methylation may alter expression of genes that support self-renewal and differentiation, possibly by modulating transcription factor binding at critical regulatory regions.

The Mansour lab developed a murine model of CHIP with a Dnmt3aR878C mutation (the murine equivalent of R882C) to study this further. Aims The first aim of this project is to characterise the 416B-Dnmt3aR878C cell line, molecularly and functionally.

Once validated, single-allele Dnmt3a epitope-tagged (wild-type and R878C) clones will be developed, to investigate DNA-binding and localisation of wild-type and mutant DNMT3A.

Methods This new DNMT3A CHIP model employs 416B cells, a Friend Murine Leukaemia Virus (FLV)-immortalized, diploid, murine haematopoietic progenitor cell line. Using Crispr/Cas9-mediated homologous recombination repair, Dnmt3aR878C was knocked-in to 416B cells. Isogenic 416B-Dnmt3aWT cells were cultured in tandem.

First, integration mapping will be used to determine sites of FLV-integration. Next generation RNA-sequencing will be used to determine the expression profile of 416B-Dnmt3aR878C vs 416B-Dnmt3aWT.

Differential methylation patterns between to isogenic lines will be determined using whole genome bisulfite sequencing (WGBS). Self-renewal and differentiation in vitro assays will determine functional capacity of these cells. Next, single-allele Dnmt3a epitope-tagged clones in 416B cells will be produced.

A plasmid containing 1Kb of Dnmt3aR878C or Dnmt3aWT with a 3xTy1-tag immediately prior to the stop codon will be cloned into a vector containing multiple selection markers and delivered into 416B cells. Two isogenic clones will be made: 416B-Dnmt3aR878C-3xTy1/WT; 416B-Dnmt3aR878C/WT-3xty1. ChIP-sequencing will be performed to determine variable DNA-binding and localisation of the DNMT3A isoforms.

The generation of Dnmt3a epitope-tagged clones will provide a powerful model from which to ask questions about DNMT3A CHIP.

In particular we will explore the mechanism by which variable differential methylation takes place across the genome and the impact that has on transcription factor binding, higher order chromatin and gene expression. This preliminary data will be used to develop a PhD fellowship proposal.