Mechanistic Insights into Direct Interactions that Mediate the Activity of DOT1L Complex in MLL-Rearranged Leukemia

Author

Ali Cihan

Date of Award

2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Roeder Laboratory

Abstract

Acute leukemia can arise from the translocation-mediated fusion of the N terminus of the Mixed Lineage Leukemia (MLL) protein to various partner proteins. Although more than 60 different translocation-based fusion proteins have been identified, fusion partners such as AF9, AF10, and ENL constitute the majority of the MLL-rearranged leukemia cases; additionally, these fusion partners are also a part of the DOT1L (disruptor of telomeric silencing 1-like) complex. Aberrant histone H3 Lysine 79 (H3K79) methylation catalyzed by DOT1L was shown to be crucial for the maintenance of MLL-rearranged leukemia. DOT1L is the only known H3K79 methyltransferase, and further, a DOT1L inhibitor is currently being evaluated in clinical trials to treat Acute Myeloid Leukemia. Although cell-based studies have implicated the importance of interactions on the N terminus of the MLL fusion protein for leukemogenesis and disease maintenance, there have been very limited corresponding biochemical analyses demonstrating how these interactions of MLL regulate or contribute to abnormal levels of methylation of H3K79 by DOT1L. Additionally, both cell-based and in vitro studies suggested that H3K79 methylation is dependent upon histone H2B ubiquitylation (H2Bub). However, there is a lack of understanding of how these interactions stimulate MLL-fusion target genes. Besides its catalytic activity, DOT1L complex activity has been postulated to regulate transcriptional elongation on the grounds of its co-localization with Pol II, H3K79 methylation mark distribution on gene loci, and its association with subunits of SEC (Super Elongation Complex) such as AF4 (or AFF4), AF9 (or AF9-related ENL), and ELL1. However, any effect of DOT1L on other transcriptional steps is not known. Addressing these gaps, the present study employs a robust biochemical approach to underscore the importance of both MLL N terminus (MLLN) interactions and H2B ubiquitylation in the regulation of aberrant H3K79 methylation by MLL-fusion protein containing DOT1L complex in MLL-rearranged leukemia. Specifically, interactions between the PWWP (LEDGF) domain and histone H3 Lysine 36 tri-methylation; between the CXXC(MLLN) domain and unmethylated CpG of DNA; and between ubiquitylated H2B and DOT1L protein are found to stabilize the intrinsic nucleosome binding property of MLL. AF9, consequently augmenting the H3K79 methyltransferase activity of the MLL-fusion containing DOT1L complex. As a result, the leukemogenic MLL-fusion protein containing the DOT1L complex exhibits inherently higher methylation activity on nucleosome arrays compared to the natural DOT1L complex. Biochemically defined invitro assays helped to show the individual and synergistic effects of these interactions, offering valuable insights into the mechanisms underlying aberrant H3K79 methylation levels in MLL-fusion target loci. These interactions hold potential as therapeutic targets, paving the way for novel therapeutic strategies. Moreover, immobilized template assays reveal a direct interaction between TFIID and the DOT1L complex, with H2Bub and DOT1L protein enhancing the recruitment of TFIID to chromatin. These direct interactions implicate the DOT1L complex in transcriptional initiation. In summary, this study advances our understanding of the critical role played by the DOT1L complex in MLL-rearranged leukemia. By shedding light on the intricate biochemical mechanisms governing MLL fusion protein interactions and their impact on H3K79 methylation, the research opens new avenues for targeted interventions in the treatment of this disease.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Cihan, Ali, "Mechanistic Insights into Direct Interactions that Mediate the Activity of DOT1L Complex in MLL-Rearranged Leukemia" (2024). Student Theses and Dissertations. 756.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/756