It is of critical importance that the rates of hematopoietic stem cell (HSC) differentiation and self- renewal are carefully regulated and kept in balance, because severe disease states arise when this balance is disrupted. Unfortunately, the mechanisms that maintain this balance are poorly understood, and this lack of understanding represents a major impediment to research progress, while also severely restricting the clinical potential of HSC-based therapeutic interventions. The goal of the research proposed here is to elucidate mechanisms that regulate HSC quiescence, self-renewal and differentiation, with a focus on the critical HSC regulatory factor, Yin Yang 1 (YY1). YY1 is a ubiquitous zinc finger transcription factor that is essential for HSC development in mice. Our recent publication showed that YY1-deficient HSCs fail to self-renew and fail to maintain a quiescent state. Furthermore, Stem Cell Factor (SCF)/c-Kit signaling, a critical regulatory pathway in HSC development, is significantly downregulated in YY1-deficient HSCs. YY1 occupies the distal enhancer and promoter sequences at the Kit locus and promotes Kit gene expression in HSCs. Thus, our compelling data implicates the SCF/c-Kit pathway as a critical downstream mediator for YY1 in regulating HSC self- renewal and quiescence. Our preliminary data also provide strong evidence that YY1-dependent repression of Structural Maintenance of Chromosomes (SMC) 1 and 3, core components of the cohesin complex, are critical to its ability to establish quiescence in HSCs. Importantly, defective HSC quiescence in Yy1 null mice is completely rescued by heterozygosity at Smc3. Collectively, our results support the hypothesis that YY1 regulates HSC self-renewal and quiescence by mechanisms that include activation of SCF/c-Kit signaling by controlling chromosome structural change at the Kit locus, and repression of cohesin. To investigate the hypothesis, an N-terminally truncated YY1 mutant, which lacks the transcriptional activation and co-activator recruitment functions of YY1, but which retains transcriptional repression and DNA looping functions of YY1, will be expressed in the bone marrow of Yy1-/- conditional knockout mice, which allows the YY1 mechanism of action to be evaluated with respect to SCF/c-Kit signaling, cohesin expression, HSC self-renewal and proliferation. In addition, a unique Yy1-/- SMC3+/- conditional knockout mouse strain will be used to investigate the mechanism(s) by which YY1-regulates cohesin and its importance in determining HSC self-renewal and differentiation. The proposed studies will elucidate as yet poorly characterized mechanisms and pathways in which YY1 participates and how its effects on HSC cell fate are mediated. These mechanisms are likely to include YY1-dependent effects on chromatin accessibility, higher-order chromatin/chromosome structure and/or activation/repression of target genes including Kit and Smc3.
The proposed research is critically relevant to public health because the results elucidate new, more efficient methods for culturing and manipulating hematopoietic stem cells (HSCs) in vitro, including methods for propagating and expanding HSCs populations. These methods will be invaluable for hematopoietic stem cell transplant and may lead to development of successful therapeutic applications for treating hematopoietic cancers and autoimmune diseases.