Oral Presentation Australian Epigenetics Alliance Conference 2022

CRISPR-ChIP delineates a Menin-dependent oncogenic DOT1L complex in MLL leukaemia. (#9)

Omer Gilan 1 , Charles Bell 2 , Laure Talarmain 2 , Daniel Neville 1 , Daniel Ferguson 1 , Kathy Knezevic 2 , Marion Boudes 3 , Enid Lam 2 , Chen Davidovich 3 , Mark Dawson 2
  1. Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia
  2. Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
  3. Biomedicine discovery institute, Monash University, Clayton, VIC, Australia

The regulation of all chromatin-templated processes involves the selective recruitment of chromatin factors to facilitate DNA repair, replication, and transcription. Chromatin immunoprecipitation (ChIP) is a fundamental experimental method used to provide spatiotemporal evidence for the coordination of these chromatin-based events including the dynamic regulation of chromatin modifications at cis-regulatory elements. However, obtaining a global appreciation of all the factors that influence a specific chromatin event has remained challenging. Here, as a proof of concept we demonstrate the utility of coupling unbiased functional genomics with ChIP to identify the factors associated with active transcription. Specifically, we use CRISPR-ChIP to identify the major chromatin factors associated with recruitment of RNA Polymerase II (Pol II) and the catalysis of two evolutionarily conserved histone modifications: H3K4me3, present at the transcriptional start site and H3K79me2, present throughout the gene body of actively transcribed genes. With CRISPR-ChIP, we identify all the non-redundant COMPASS complex members required for H3K4me3 deposition, as well as the major components required for Pol II promoter recruitment and functional competency for productive transcription. Importantly, using CRISPR-ChIP in leukaemia cells driven by MLL translocations, we uncover a functional partitioning of H3K79 methylation into two distinct regulatory units: an oncogenic DOT1L complex, where the malignant driver directs the catalytic activity of DOT1L at MLL-fusion target genes and a separate endogenous DOT1L complex, where catalytic activity is directed by MLLT10 at actively expressed genes not controlled by the MLL-fusion protein. This functional demarcation has therapeutic implications and explains why Menin inhibition surprisingly controls methylation of H3K79 at a critical subset of genes that sustain MLL-fusion leukaemia. Overall, CRISPR-ChIP provides a powerful tool for the unbiased interrogation of the mechanisms underpinning chromatin regulation.