Structure-Function Studies of the Recruitment of DOT1L, an H3K79 Histone Methyltransferase, to MLL-AF9 and its role in Leukemogenesis

Kuntimaddi, Aravinda, Biophysics - Graduate School of Arts and Sciences, University of Virginia
Bushweller, John, Department of Molecular Phys and Biological Physics, University of Virginia

The MLL gene is a common target of chromosomal translocations found in human leukemia. MLL-fusion leukemias consistently have poor outcomes and disrupt HOX gene regulation through the recruitment of transcriptional elongation factors. One of the most common translocation partners of MLL is AF9 (MLLT3). The C-terminal domain of AF9 has been shown to interact with multiple transcriptional regulators. This t(9;11) chromosomal translocation forms a chimeric MLL-AF9 protein containing the functionalities of both the N-terminal portion of MLL and the C-terminal part of AF9. The MLL-AF9 protein recruits DOT1L, a histone H3 lysine 79 (H3K79) methyltransferase which mono-, di- or tri methylates H3K79 (H3K79me1/me2/me3), leading to aberrant gene transcription. The role of the direct interaction between DOT1L and MLL-AF9 and its role in leukemogenesis was unclear.

We show that DOT1L has three AF9 binding sites with varying affinities, and that both partners are disordered and co-fold upon forming separate DOT1L-AF9 complexes. We present the NMR solution structure of the highest affinity DOT1L-AF9 complex. Based upon this structural knowledge, we generated structure-guided point mutations with graded effects on recruitment of DOT1L to MLL-AF9. ChIP-Seq analyses of H3K79me2 and H3K79me3 show that graded reduction of the DOT1L interaction with MLL-AF9 results in differential losses in H3K79me2 and me3 marks at MLL-AF9 target genes. Furthermore, the degree of MLL-AF9 hematopoietic transformation as seen in serial replating assays are dependent upon the level of DOT1L recruitment.

Additionally, the MLL-AF9 fusion protein recruits the CBX8 protein. CBX8 is a member of the PRC1 complex and reads the histone H3 lysine 27 tri-methylation mark (H3K27me3), which is characteristically involved in transcriptional silencing. Here, we present the NMR solution structure of CBX8 in complex with AF9 and we show that the CBX8-AF9 structure is nearly identical to that of the other AF9 complex structures. The novelty of this particular interaction is that it has extremely weak binding affinity and has increased backbone dynamics compared to that of the other AF9 complexes. It is still a conundrum as to why the MLL-AF9 protein recruits CBX8 and what role this interaction plays in leukemogeneis. We propose several structure-guided functional studies that will further shed light on the role of this direct interaction in MLL-AF9 leukemogenesis.

PHD (Doctor of Philosophy)
DOT1L, MLL-AF9, leukemia, structure biology, NMR, nuclear magnetic resonance, epigenetics, ChIP-Seq, intrinsically disordered proteins
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