CRISPR-ChIP to identify mechanisms of chromatin regulation [ChIP-seq]

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 critical 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, getting 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 this method to identify the major chromatin factors associated with recruitment of RNA polymerase 2 and the catalysis of two evolutionarily conserved histone modifications; H3K4me3 present at the transcriptional start site and H3K79me2 present through the gene body of actively transcribed genes. With CRISPR-ChIP we identify all the non-redundant COMPASS complex members required for H3K4me3 and the major components of mediator and TFIID required for Pol II recruitment during the maintenance of gene expression. 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 which is separate from the 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 design: ChIP-seq for H3K4me3, H3K79me2, H3K27ac, Pol II and FLAG-MLLAF9