UC San Diego

Hematopoietic stem cells (HSCs) provide all the major blood cell lineages over the lifetime of an organism. These cells must tightly regulate the processes of quiescence, self-renewal, and differentiation to ensure proper hematopoietic homeostasis. Failure to do so leads to hematopoietic disorders such as leukemia and aplastic anemia. Patients afflicted with these diseases presently rely on donor-derived bone marrow transplants, which are difficult to obtain due to the low availability of immunocompatible donors. The discovery of induced pluripotent stem cells (iPSCs) provides hope for generating patient-specific HSCs. However, despite our growing knowledge of HSC biology, efforts to produce bona fide HSCs from iPSCs have resulted in improper hematopoiesis. To generate HSCs in vitro, we must comprehend how HSCs are being specified in vivo, a process that remains poorly understood. Deciphering the signaling mechanisms that control HSC specification will increase our chances of producing bona fide HSCs ex vivo that can be used to treat patients in a precise and reliable manner.

In order to identify novel genes and transcription factors involved in HSC specification, we performed a forward genetic screen to find zebrafish mutants defective in HSC formation. Adult males were subjected to N-ethyl-N-nitrosourea (ENU) mutagenesis to generate point mutations throughout the spermatogonia. Large-scale whole mount in situ hybridization (WISH) based screens were carried out on F3 generations to test the ability of the embryos to generate HSCs. Upon identifying mutant lines, we implemented next generation sequencing (NGS), specifically RNA-sequencing-based linkage mapping of single nucleotide polymorphism (SNP) haplotype blocks to determine the causal mutation. We identified a mutation in the gene, supt16h, a histone chaperone known to reorganize chromatin and assist in RNA elongation. To elucidate mechanistic action of supt16h on HSC specification, we employed biochemical, molecular, and NSG techniques. Our results demonstrated an unidentified relationship between supt16h and p53 during transcription to specify HSCs via Notch signaling modulation. This body of work has characterized an unsuspected role of Supt16h during HSC specification and provides a model that can be used to generate deeper insight into histone remodeling, P53 transcriptional regulation, and potentially contribute to advancing future iPSC-based transplantation therapies.