Abstract

Hematopoietic stem cells (HSCs) maintain lifelong blood production by balancing self-renewal and multilineage differentiation. They are sustained by networks of transcription factors and epigenetic regulators that prime lineage-specific programs while simultaneously preventing activation of any one program up until the moment of lineage commitment. Despite extensive study, it remains unclear how these complex transcriptional programs are coordinated and how specific epigenetic regulators are deployed to reinforce specific cell fate decisions. Epigenetic regulators, MLL3 and MLL4, play a central role in pluripotent embryonic stem cell fate transitions, but their functions in multipotent somatic stem cells are less well characterized. MLL3 (also known as KMT2C) and MLL4 (also known as KMT2D) are histone methyltransferases that enucleate the Complex of Proteins Associated with Set1 (COMPASS) and monomethylate histone H3 lysine 4 residues (H3K4me1) at enhancers to promote transcription. While MLL3 and MLL4 share highly similar protein structures, characterized by clusters of N-terminal plant homeodomains (PHDs) and a conserved C-terminal SET methyltransferase domain, Mll3 and Mll4 loss-of-function mutations cause distinct HSC phenotypes. MLL4 inactivation impairs HSC self-renewal and enhances myeloid differentiation, while MLL3 inactivation enhances HSC self-renewal and impairs differentiation. Furthermore, MLL3 acts as a tumor suppressor in -7/del7(q) myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), whereas MLL4 is a tumor dependency in MLL-rearrangement AML. These data raise the questions of 1) whether MLL3 and MLL4 share redundant roles in HSCs, given their biochemical similarities, 2) how specific functional domains of MLL3 and MLL4 enable them to sustain hematopoiesis and suppress leukemogenesis, and 3) how MLL3 and MLL4 interface with other epigenetic regulators to coordinate HSC fate decisions. Through the work presented in this thesis, I have made several important discoveries that resolve mechanisms by which MLL3 and MLL4 coordinate HSC fate decisions in vivo. First, we discovered the redundant function of MLL3 and MLL4 in multilineage HSC differentiation by opposing a B-lymphoid-like default state. This establishes MLL3 and MLL4 as critical linchpins of HSC multipotency. Second, we showed that MLL3 restricts HSC mobilization and granulopoiesis in response to granulocyte-colony stimulating factor (G-CSF). This finding has key clinical implications as it suggests that G-CSF use may amplify the risk of therapy-related MDS/AML by disproportionately stimulating Mll3-mutant HSCs. Third, we characterized the key role of MLL3 PHD domains, yet not the SET methyltransferase domain, in restricting HSC self-renewal and myeloid transformation. This provides a conceptual framework to understand the MLL3 structure-function relationship in HSCs. Fourth, we uncovered distinct genetic interactions between MLL3 and MLL4 with other critical epigenetic regulators in MDS, such as TET2. This further contextualizes MLL3 and MLL4 into the complex epigenetic regulatory circuitry and serves to inspire additional studies on elucidating the epigenetic mechanisms in hematopoiesis and leukemogenesis. Together, these efforts paint a more nuanced picture of how MLL3 and MLL4 are deployed in HSCs to balance self-renewal and multipotency.

Committee Chair

Jeffrey Magee

Committee Members

Laura Schuettpelz, Grant Challen; Jeffrey Bednarski; Michael Meers

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

7-23-2026

Language

English (en)

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