Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cell Biology
First Advisor
Krause, Diane
Abstract
Megakaryocytic-erythroid progenitors (MEP) are a population of bipotent hematopoietic progenitors that give rise to the megakaryocytic (Mk) or erythroid (E) lineages. The Mk lineage produces platelets, and the E lineage produces RBCs, that govern coagulation and oxygen transport, respectively. Both are critical components of the blood system, and maintaining an adequate and balanced pool of platelets and red blood cells is essential for life. As MEP stand as a final branchpoint before committing to Mk or E lineages, understanding the fate specification mechanism of MEP may provide insight into treating conditions that involve platelets and/or red blood cells.This thesis focuses on elucidating the role of selected transcription factors in the MEP fate specification process. Chapter 3 focuses on RUNX1 and shows that RUNX1 promotes Mk fate in MEP. This function is enhanced by serine/threonine phosphorylation of RUNX1. Phosphoserine-RUNX1 levels are highest in the Mk lineage compared to MEP or the E lineage while neither RUNX1 mRNA nor total protein levels differ between Mk and E committed progenitors. RUNX1 mutants that are non-phosphorylatable (RUNX1-4A) or phosphomimetic (RUNX1-4D) were tested for their effects on MEP fate specification and in the HEL cell line, a leukemic cell line that models MEP. It was found that the phosphomimetic mutant form of RUNX1 was more potent in inducing Mk fate in MEP and mature Mk signatures in HEL cell line than RUNX1-WT or the non-phosphorylatable mutant. Differential gene expression patterns induced by WT and mutant forms of RUNX1 were also explored in HEL cells, and the changes in gene expression were consistent with the observed phenotypes. The mechanism underlying RUNX1 phosphorylation in MEP was also explored, and CDK9 was discovered to be at least one of the kinases that phosphorylates RUNX1 in MEP. CDK9 inhibition affected MEP fate by promoting E fate specification. Chapters 4 and 5 are focused on the potential roles of NR4A1, MYB, and MYBL2 in MEP fate specification. While an effect of NR4A1 on MEP fate specification was not evident, NR4A1 potentially showed an as yet unexplored role in promoting Mk maturation. NR4A1 KD in CD34+ HSPC resulted in a decrease of polyploidization of maturing megakaryocytes. MYB KD promotes Mk fate, and MYBL2 KD inhibits colony formation. Neither MYB nor MYBL2 OE had reproducible effects on MEP fate specification. However, comparison of the binding sites of MYB and MYBL2 in K562 cells (n=1) revealed that MYB may bind to the promoter regions of some genes that are essential for erythropoiesis, which potentially explains KD of MYB promoting Mk fate. By enrichment analysis, genomic targets that were associated with MYB but not with MYBL2 were enriched for RNA splicing machinery genes, and genomic targets that were associated with MYBL2 but not with MYB were enriched for cell cycle machinery genes. Binding sites shared by MYB and MYBL2 also revealed some interesting target genes, including transcription factors and epigenetic regulators. Transcription factors work collectively to direct the fate specification of MEP. The work presented in thesis strongly suggests that RUNX1 is part of this network. While other investigated transcription factors (NR4A1, MYB, MYBL2) showed less of an effect on MEP fate specification, they play essential roles in the hematopoietic system and the data presented in this thesis will provide a basis for future studies.
Recommended Citation
Kwon, Nayoung, "Transcriptional Regulation of Fate Specification in Human Megakaryocytic-Erythroid Progenitors" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1505.
https://elischolar.library.yale.edu/gsas_dissertations/1505
