DOT1L provides transcriptional memory through PRC1.1 antagonism.

DOT1L and Menin are essential cofactors for the oncogenic activity of MLL fusion proteins (MLL-FPs) in leukaemia. However, the mechanisms underpinning the therapeutic effects of their inhibitors remain unclear. Here we identify a critical role for the non-canonical Polycomb repressive complex 1.1 (PRC1.1) in mediating the cellular responses to DOT1L and Menin inhibitors. Menin inhibition induces PRC1.1-dependent deposition of H2AK119ub to silence a subset of MLL-FP targets, whereas DOT1L inhibition results in a genome-wide increase in H2AK119ub. We show that enhanced PRC1.1 activity arises specifically from the progressive loss of DOT1L-mediated H3K79 methylation, independent of MLL-FP displacement or transcriptional repression. This regulatory crosstalk is conserved across cell types and is driven by direct biochemical antagonism between H3K79 methylation and PRC1 activity. Together, our findings establish DOT1L as a component of transcriptional memory co-opted in leukaemia and suggest it serves as the missing link balancing the opposing forces of the MLL-Polycomb axis.