Intrinsic Disorder, Epigenetics, and Leukemia - the MLL-AF9 SAGA

Abstract

The MLL gene is a target of chromosomal translocations found in human leukemia. One of the translocation partners is AF9. The portion of AF9 fused to MLL is the C-terminal ANC1 homology domain (AHD) of AF9. We have shown that this domain is intrinsically disordered. Upon interaction with its binding partners, it undergoes coupled binding and folding. Intriguingly, this domain has been shown to bind four different partners: AF4 (PolII phosphorylation, productive transcriptional elongation), DOT1L (catalyzes H3K79 methylation, gene activation), as well as BCoR and hPC3 (CBX8) which are transcriptional repressors. The binding of both activating and repressing partners suggests that this domain acts as a multi-functional switch. We have used NMR spectroscopy to solve structures of AF9 with all four binding partners, showing they all bind in a similar manner to AF9, leading to questions about how binding to specific partners is regulated and/or orchestrated.Interestingly, we found a linear repeat motif in DOT1L to which AF9 binds with high affinity. Such repeat motifs are seen frequently in intrinsically disordered proteins, but the functional consequences of binding to such elements has not been clear. We created point mutations in AF9 which disrupt binding to only one site or to both sites and incorporated these into MLL-AF9 for functional characterization. DOT1L is a non-processive enzyme, i.e. the local concentration of enzyme will define the level of H3K79 methylation, so we hypothesized that a mutation disrupting one binding site will impact H3K79me3 levels but not H3K79me2 levels whereas one affecting both sites will impact both. Indeed, this is what we observed in our ChIP-Seq analysis. This suggests, at least in some cases, these repeat motifs are a clever way to regulate the degree of enzymatic activity by increasing the local effective concentration.