MLL-AF4 binds directly to a BCL-2 specific enhancer and impacts H3K27 acetylation. Homo sapiens

Survival rates for children diagnosed with acute lymphoblastic leukemia (ALL) have drastically improved, but those carrying mutations in the Mixed Lineage Leukemia (MLL) gene continue to have a very poor prognosis. The most common MLL mutation in ALL is the t(4;11)(q21;q23) chromosome translocation that fuses MLL in frame with the AF4 gene producing MLL-AF4 and AF4-MLL fusion proteins. Previously, we showed that MLL-AF4 binds to the BCL-2 gene and directly activates it through DOT1L recruitment and increased H3K79me2/3 levels. We went on to show that MLL leukemias are particularly sensitive to treatment with the BCL-2 inhibitor venetoclax which synergizes with both standard chemotherapeutic agents as well as DOT1L inhibitors. Altered expression of other BCL-2 family members is a major cause of resistance to ventoclax, so here we perform a detalied analysis of MLL-AF4 regulation of the entire BCL-2 family. By measuring nascent RNA production, we find that of all the BCL-2 family genes, MLL-AF4 directly controls the transcription of both BCL-2 and MCL-1, but only BCL-2 shows a significant loss of steady state RNA levels. Interestingly however, both MCL-1 and BCL-XL protein levels are dependent on MLL-AF4 activity through an unknown and likely indirect mechanism. Finally, we analyze MLL-AF4 regulation of the BCL-2 gene in greater detail and using Capture C we identify a major BCL-2 specific enhancer consisting of two clusters of H3K27Ac. Loss of MLL-AF4 activity results in a reduction of H2K27Ac levels at the major BCL-2 enhancer, revealing a novel regulatory dependence on MLL-AF4 function at BCL-2. Overall design: Examination of MLL-AF4-mediated regulation at BCL2-family genes using nascent RNA-seq to observe gene expression changes following siRNA-mediated knockdown and treatment with the DOT1L inhibitor EPZ-5676. We also investigated changes in histone mark occupancy at the BCL2 promoter and enhancer following siRNA-mediated knockdown of MLL-AF4