CHARACTERIZING PLAG1 IN HUMAN HEMATOPOIETIC STEM CELLS

Hematopoietic stem cell (HSC) dormancy is understood as supportive of HSC function and their long-term integrity. While regulation of stress responses incurred as a result of HSC activation is recognized as important in maintaining stem cell function, little is understood of the preventative machinery present in human HSCs that may serve to resist their activation and promote HSC self-renewal. We demonstrate that the transcription factor PLAG1 is essential for long-term HSC function and when overexpressed endows a 15.6-fold enhancement in the frequency of functional HSCs in stimulatory conditions. Genome-wide measures of chromatin occupancy and PLAG1-directed gene expression changes combined with functional measures reveal that PLAG1 dampens protein synthesis, restrains cell growth and division, and enhances survival, with the primitive cell advantages it imparts being attenuated by addition of the potent translation activator, c-MYC. We find PLAG1 capitalizes on multiple regulatory factors to ensure protective diminished protein synthesis including 4EBP1 and translation-targeting miR-127, and does so independently of stress response signaling. Overall, our study identifies PLAG1 as an enforcer of human HSC dormancy and self-renewal through its highly context-specific regulation of protein biosynthesis, and classifies PLAG1 among a rare set of bona fide regulators of mRNA translation in these cells. Our findings showcase the importance of regulated translation control underlying human HSC physiology, its dysregulation under activating demands, and the potential if its targeting for therapeutic benefit. Thesis Doctor of Science (PhD) Blood production (hematopoiesis) is a stem cell-driven regenerative system that can repair damaged blood systems and therefore offer lifesaving treatments for devastating malignancies and immune disorders. Realizing the full potential of hematopoietic stem cells (HSCs) is encumbered by our inability to maintain HSCs with long-term functionality in clinical settings. Enhanced fundamental insights into the properties that define human HSC identity and fates can thus inform the rational design and advancement of HSC-based therapies. We discovered a regulator, PLAG1, that is essential for the long-term blood production function of human HSCs. When elevated PLAG1 amplified the absolute number of human HSCs 15-fold and improved their maintenance and function in clinically-relevant culture and transplantation settings. Using genome-wide technologies paired with functional assays we elucidated that PLAG1 employs a multi-pronged strategy to rewire protein production rates as a means to enhance HSC preservation and function. Our findings highlight regulation of protein production as an untapped strategy that could be incorporated into clinical settings to improve patient outcomes.