Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Nancy A. Speck

Abstract

Hematopoietic stem and progenitor cells (HSPCs) are generated de novo in the embryo from a subset of endothelial cells (ECs) known as hemogenic endothelial cells (HECs). HECs undergo an endothelial-to-hematopoietic transition (EHT) to form HSPCs which requires the transcription factor (TF) RUNX1. RUNX1 is widely regarded as the master regulator of EHT, but RUNX1 alone is insufficient to reprogram adult ECs to HSPCs. Understanding what makes an EC permissive to be specified as hemogenic by RUNX1, as well as the additional TFs that regulate the complex gene regulatory networks controlling HSPC ontogeny is required for efficient and robust generation of HSPCs ex vivo for therapeutic purposes. To uncover novel regulators of HSPC formation, we defined the transcriptomes and enhancer epigenomes of cells representing key developmental stages of HSPC ontogeny. We then constructed developmental-stage-specific transcriptional regulatory networks by linking enhancers to their target promoters and then predicting the bound TFs controlling the enhancer-promoter pair. We predicted known transcriptional regulators of EHT such as RUNX1, validating our overall approach, and identified putative novel TFs, including the ubiquitous TFs SP3 and MAZ. Deletion of either SP3 or MAZ resulted in a reduction in HSPC formation, confirming the role of these TFs in HSPC formation. Cooperation of SP3 and MAZ with RUNX1 was not investigated, but SP3 can directly interact with RUNX1. It is not known what makes an EC competent to be specified as hemogenic by RUNX1. We show that ectopic expression of RUNX1 alone can efficiently promote EHT and HSPC formation from embryonic ECs, but less efficiently from fetal or adult ECs. Efficiency correlated with baseline accessibility of TGFβ-related genes associated with endothelial-to-mesenchymal transition (EndoMT) and participation of AP-1 and SMAD2/3 to initiate further chromatin remodeling along with RUNX1 at these sites. Activation of TGFβ signaling improved the efficiency at which RUNX1 specified fetal ECs as HECs. Thus, the ability of RUNX1 to promote EHT depends on its ability to recruit the TGFβ signaling effectors AP-1 and SMAD2/3, which in turn is determined by the changing chromatin landscape in embryonic versus fetal ECs. This work provides insight into regulation of EndoMT and EHT that will guide reprogramming efforts for clinical applications. The emergence of HSPCs is controlled by the interplay of a multitude of factors including RUNX1, AP-1, SMAD2/3, SP3, and MAZ, all shown here to be critical regulators of HEC specification. This work lays the foundation for understanding the complex transcriptional regulatory network controlling HEC specification and HSPC emergence. Understanding the interplay of these factors, as well as other known regulators of HSPC emergence, will be imperative for generating HSPCs ex vivo for therapeutic purposes.

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Additional Files

Table_A1.xlsx (617 kB)
Table_A4.xlsx (128 kB)
Table_A5.xlsx (12270 kB)
Table_A6.xlsx (769 kB)
Table_A7.xlsx (331 kB)
Table_A8.xlsx (71090 kB)
Table_A9.xlsx (256 kB)
Table_B2.xlsx (72 kB)

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