The role of DOT1L in MLL-AF4 leukaemia

• Main Author: Godfrey, Laura
• Other Authors: Roberts, Irene ; Milne, Tom
• Published: University of Oxford 2018
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.740984

DOT1L is a methyltransferase which has been shown to methylate H3K79 (Ng et al. 2002; Feng et al. 2002; Lacoste et al. 2002). DOT1L and H3K79me have been shown to be involved in active transcription and in particular has been shown to be important for MLL-AF4 leukaemia, where high levels of H3K79me are found at MLL-AF4 gene targets (Krivtsov et al. 2008; Bernt et al 2011). The exact mechanism of how DOT1L is recruited to MLL-AF4 gene targets leading to high levels of H3K79me is currently unknown, especially as it has been demonstrated that AF4 and DOT1L exist in mutually exclusive complexes (Leach et al. 2013; Yokoyama et al. 2004; Biswas et al. 2011). In addition to the recruitment mechanism of DOT1L, the function of H3K79me remains unclear. In this thesis, the recruitment mechanism of DOT1L at MLL-AF4 gene targets was investigated using the in vivo TetR-recruitment system (Blackledge et al. 2014). Using this, it was found that DOT1L complex members AF9, ENL and AF10 were sufficient for DOT1L recruitment, in addition to PAF1, a member of the PAF1 elongation complex. To investigate the function of H3K79me at MLL-AF4 gene targets, it was necessary to identify a set of gene targets which were dependent upon H3K79me for transcription. To do this, the DOT1L inhibitor, EPZ-5676, was employed to treat an MLL-AF4 leukaemia cell line (SEM) followed by Nascent RNA seq and ChIP rx seq. From this a set of hypersensitive MLL-AF4 gene targets were identified. Importantly, Capture-C, ATAC seq and ChIP seq revealed a subset of hypersensitive targets with a putative intragenic enhancer. Following EPZ-5676 treatment, Capture-C revealed a disruption in the interaction between the putative intragenic enhancer and promoter of some MLL-AF4 hypersensitive genes targets. This provides evidence for a novel, context dependent role of H3K79me which may be involved in enhancer-promoter interactions and function at a subset of hypersensitive MLL-AF4 gene targets.