Interrogating sequence-structure-function relationships in DNMT3A

Abstract

DNMT3A is a complex chromatin-modifying enzyme with a critical role in mammalian development and cancer biology. As one of two human de novo DNA methyltransferases, DNMT3A is responsible for establishing and maintaining proper gene expression profiles that allow the cell to differentiate properly during development. Dysregulation of this process in hematopoietic stem cells can lead to improper gene expression programs and diseases such as clonal hematopoiesis and Acute Myeloid Leukemia, as well as developmental disorders such as Tatton Brown Rahman syndrome. The role of DNMT3A in the genome has been extensively studied, and progress has been made towards understanding the role of DNMT3A at this genomic scale. However, at a molecular scale, many mysteries regarding the precise biochemical mechanisms by which DNMT3A performs its functions and is altered in disease remain. Specifically, the precise molecular mechanism by which the AML hotspot R882H mutation in DNMT3A causes its dominant negative effect remains unclear. In this dissertation, I will describe my work applying biochemical, genetic, and biophysical techniques to unravel the molecular basis of DNMT3A activity and its perturbation in disease. In chapter 1, I will begin by giving an overview of what is currently known about DNMT3A biology. Then, I will briefly review literature on the evolution and biochemistry of protein-protein interfaces, with a focus on homo-oligomers. This body of work provides important context for the oligomeric behavior of DNMT3A and particularly the R882H mutation. Then, I will provide an overview of deep mutational scanning, broadly defined. Deep mutational scanning is a powerful genetic technique that, driven by increases in the affordability of DNA sequencing and synthesis technology as well as implementation of CRISPR-based methods, has recently diversified in both questions addressed and methods. In chapter 2, I use base editor scanning to look for activating mutations to DNMT3A. In doing so, I uncover several activating mutations. These include a mutation at the ADD-MTase autoinhibitory interface, a cryptic splice site in the DNMT3A gene that causes a four amino acid deletion in the ADD-MTase linker, and a C-to-Y mutation in the ADD domain that based on existing structures is unlikely to perturb the known ADD-based autoinhibitory mechanism. In particular, this C-Y mutation may inform drug discovery efforts to design and understand small molecule DNMT3A activators. In chapter 3, I use deep mutational scanning, biochemistry, and Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) in collaboration with Shaunak Raval and Malvina Papanastasiou to uncover the molecular mechanism of the hotspot R882H mutation. First, we show that the R882H mutation causes the mutant protein to form aberrant oligomers, explaining its dominant negative effect. Then, we use a rescue mutant of DNMT3A R882H, L859F, to propose a model in which the R882H mutation pre-orders the residues that form the RD interface of DNMT3A into their binding-competent state, promoting oligomerization. L859F instead opposes this pre-ordering, rescuing the oligomeric state of the protein. This work both solidifies the oligomerization-promoting effect of the R882H mutation and provides evidence for its biophysical mechanism, which will be useful in drug discovery efforts to treat diseases associated with this mutation. Furthermore, this work describes the first report for this type of aberrant biochemical mechanism in cancer, providing a mechanistic framework that may be operative in other disease contexts as well. In chapter 4, I discuss unpublished work exploring the role of the N-terminal regulatory domains in DNMT3A oligomerization and activity. This preliminary data suggests that the PWWP domain may make contacts with the MTase domain that are important for enzyme function, and that these contacts may also be involved in the ability of DNMT3A to form the RD interface. I also show that these properties seem to be exclusive to the DNMT3A PWWP domain, through experiments subbing the DNMT3B PWWP domain for that of DNMT3A. Finally, in chapter 5 I discuss conclusions and future directions to build upon this dissertation.