Regulation Of Normal and Malignant Hoxa Gene Expression Through Higher H3K79 Methylated States

The clustered Hoxa genes Hoxa5-Hoxa10 are important for self-renewal and differentiation of hematopoietic stem and progenitor cells. These genes are often aberrant upregulated in human malignancies, one of the most striking examples of which are leukemias with rearrangements of the mixed lineage leukemia (MLL) gene. Since these key posterior Hoxa cluster genes are known to show graded down-regulation upon hematopoietic differentiation, we sought to assess the epigenetic changes associated with this tightly controlled, developmentally critical transcriptional program. We performed ChIP sequencing on lineage negative, Sca-1 + Kit + (LSK) cells where the posterior Hoxa5-10genes are highly expressed compared to granulocyte macrophage progenitors (GMPs) that show markedly lower expression of these transcripts. We observed a dramatic diminution in H3K79 di/tri methylation as cells differentiated from LSKs to GMPs. In contrast, we found minimal changes in H3K79 mono-methylation during the LSK-GMP transition suggesting that high expression of Hoxa genes is maintained through higher H3K79 methylated states in normal hematopoiesis. Strikingly, the removal of H3K79 me2/3 but not me1 was sufficient for repressive epigenetic mechanisms such as polycomb repressive complex 2 (PRC2)- mediated H3K27 tri-methylation to invade the Hoxa locus during the LSK to GMP differentiation. The decrease in H3K79 di/tri methylation upon hematopoietic differentiation may be the result of a graded decrease in expression of the DOT1L co-factor Af10 which we have recently found to regulate the transition from H3K79 mono to dimethylation.

Next we wanted to probe whether MLL-leukemias drive Hoxa gene expression through increased conversion of H3K79 methylation from the mono to the di/tri-methylated states. We performed ChIP sequencing for H3K79 me1/2/3 to identify the different states of H3K79 methylation across the epigenetic landscape of MLL-transformed cells. Strikingly we observed that in both murine and human leukemia, there was a dramatic hyper-conversion of H3K79 mono-methylation to di/trimethylation specifically at MLL-AF9 target genes. Af10 deletion in the MLL-AF9 leukemia significantly reduced H3K79me2/3 while retaining - and in some cases even increasing - H3K79 me1 at MLL-target genes. We then assessed chromatin accessibility at the Hoxa locus following nuclease digestion and observed that the diminution of H3K79me2/3 was enough to significantly reduce chromatin accessibility at the Hoxa locus despite the relative retention of H3K79me1. Similar to the LSK-GMP transition, reduction in H3K79 me2/3 but not H3K79me1 was accompanied by a dramatic increase in H3K27me3 in a very specific subset of genes including Hoxa5-10.

The genetic and epigenetic changes resulting from Af10 deletion significantly impaired MLL-leukemogenesis and sensitized leukemia cells to DOT1L inhibition. These results suggest that the expression of Hoxa genes in hematopoiesis is controlled through higher H3K79 methylated states, possibly aided by the DOT1L co-factor Af10. The MLL-AF9 fusion seems to hijack this mode of regulation through aberrant hyperconversion of H3K79 methylation to higher methylated states at critical oncogenic targets including the Hoxa genes. These data suggest that targeting the interaction between DOT1L and Af10 is a potential therapeutic strategy since it blunts the conversion of H3K79me1 to higher states of methylation by MLL-fusion proteins and thus significantly impairs MLL-mediated transformation.