Date of Award

2026

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell Biology, Neurobiology and Anatomy

First Advisor

Nancy Zeleznik-Le

Abstract

MLLT1 (also named ENL) is an epigenetic reader protein that binds to crotonylated or acetylated lysine residues of histone 3 through its N-terminal YEATS domain. Positive and negative regulatory complexes compete for binding to C-terminal MLLT1, and promote transcription elongation (SEC, Super elongation complex; DOT1L) or transcriptional repression (PRC1, Polycomb repressive complex 1). MLLT1 was initially identified as a chromosomal translocation partner to MLL(KMT2A) in a subset of MLL-rearranged leukemias. MLL-ENL fusion protein causes both lymphoid (predominantly B-ALL) and mixed lineage leukemias. MLLT1 has been implicated in leukemic stem cell survival in MLL-rearranged cell lines but its role in normal hematopoiesis has not been investigated. Mllt1 is expressed in multiple bone marrow cell subsets, with high expression levels in B-lineage progenitors, including Pro-B and Pre-B stages of B-cell development. We generated conditional Mllt1 knockout Mllt1fl/fl; Rosa26CreERT2/+ mice (Mllt1del) where Mllt1 gene is deleted upon tamoxifen treatment. We used this model to determine the function of Mllt1 in hematopoiesis. We observed a B-lineage defect in bone marrow of Mllt1del mice compared to control Mllt1fl/fl mice. We found a significant decrease in bone marrow pro-B, pre-B, immature B and transitional B cell populations in Mllt1del mice post Mllt1 deletion compared to control Mllt1fl/fl mice by multicolor immunophenotyping. Further, transitional B cells of spleen and peripheral blood B cells were reduced in Mllt1del mice compared to controls. Mllt1 deletion in in vitro cultured B-enriched progenitor cells from Mllt1fl/fl; Rosa26CreERT2/+ mice under lymphoid-primed condition resulted in significantly reduced numbers of B cells when compared to control cells treated with vehicle. In addition, expression of critical B-lineage transcription factors and genes including Il7r, Ebf1, Pax5, Blk, Sox4 which are also direct target genes of MLLT1, were decreased in the Mllt1del condition compared to vehicle. Gene set enrichment analysis (GSEA) of in vitro cultured cells from Mllt1del condition showed negative enrichment for gene sets related to B cell development when compared to control. Further, gene ontology analysis highlighted the genes involved in the primary signaling pathways of B-cell development such as JAK-STAT and PI3K-Akt signaling to be downregulated in the Mllt1-deficient in vitro cells. In vitro complementation of MLLT1 rescued the number of B cells in endogenous Mllt1 deleted condition compared to non-complemented Mllt1 deleted condition and specific YEATS domain mutants lacking reader and RNA-binding functions. Together, our in vivo and in vitro data demonstrate that Mllt1 is critical for maintenance of B-lymphopoiesis.

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