Biology and Biomedical Sciences: Molecular Genetics and Genomics
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
HSPA9 was previously identified as a candidate gene in a commonly deleted region (CDR) associated with myelodysplastic syndrome (MDS), a clonal hematopoietic stem cell disorder. Cytogenetic abnormalities occur in ~50% of MDS patients and an interstitial deletion or loss of chromosome 5 containing HSPA9 is the most common, occurring in up to 25% of patients. In order to understand the role of HSPA9 in hematopoiesis and disease development, we created an Hspa9 knockout mouse model. We characterized hematopoiesis of heterozygous mice (Hspa9+/-), which have a 50% reduction in Hspa9 expression, modeling the heterozygous loss of HSPA9 and 50% reduction in mRNA observed in MDS patients. Homozygous knockout of Hspa9 is embryonic lethal prior to fetal liver hematopoiesis, preventing further evaluation of hematopoiesis in Hspa9-/- mice.
Analysis of stem, progenitor and mature stages of hematopoiesis up to 18 months of age identified no significant differences in Hspa9+/- mice compared to Hspa9+/+ littermates in vivo. However, as early as 2 months of age, Hspa9+/- mice have a significant reduction in CFU-PreB colony formation in vitro, indicating a B-cell progenitor defect. This reduction in colony formation is hematopoietic-cell intrinsic and likely due to a functional B-cell progenitor defect, since B-cell progenitor frequencies in Hspa9+/- mice are normal. Gene expression array analysis revealed a reduction in gene expression pathways associated with proliferation and activation of B-lymphocytes. Gene expression analysis of hematopoietic progenitor cells from MDS patients also identified B-cell signaling pathways as the most down-regulated pathways. IL-7 added exogenously to CFU-PreB cultures was able to partially rescue the reduction in Hspa9+/- CFU-PreB colony formation, further indicating dysfunctional IL-7 signaling in Hspa9+/- B-cells.
To explore the contribution of Hspa9 to altered IL-7R signaling, we interrogated an IL-7 dependent cell line treated with an Hspa9 or non-targeting control siRNA. Knockdown of Hspa9 resulted in a significant growth defect in these cells and reduced Stat5 phosphorylation following IL-7 stimulation of cytokine-starved cells. Collectively, these data implicate Hspa9 in IL-7R signaling in B-cells. Further work will determine whether HSPA9 loss contributes to the reduction in B-cell progenitors and increased B-cell apoptosis observed in patients with MDS.
Krysiak, Kilannin, "The Role of Hspa9 in Mouse Hematopoiesis and IL-7 Receptor Signaling" (2014). All Theses and Dissertations (ETDs). 1314.