A Role for RUNX3 in Balanced Erythroid-Myeloid Differentiation
Balogh, Peter, Experimental Pathology - School of Medicine, University of Virginia
Goldfarb, Adam, Clinical Pathology, University of Virginia
Hematopoiesis is the life-long process of blood cell production that undergoes dynamic changes with ontogeny. In the embryo, primitive endothelial cells in the yolk sac undergo a cell-fate alteration to produce progenitor blood islands that generate primitive erythroid cells to aid in delivering oxygen to the growing organism. Blood island progenitors gradually gain lineage potency to produce other cells of the hematopoietic system. Multipotent progenitors start to arise from specialized zones in the circulatory system that allows them to travel to the fetal liver where the bulk of blood cell production occurs for the remainder of embryogenesis. Fetal liver progenitors develop the capability to generate all cells of the hematopoietic system, at which point they are designated bona fide hematopoietic stem cells. After birth, hematopoietic stem cells travel once again to seed the bone marrow, where they will reside for the duration of the life-span.
As hematopoietic stem cells age, they experience a gradual decline in function characterized by imbalanced lineage output, increased self-renewal, poor engraftment, and increased cytokine responsiveness. These changes manifest from cell-intrinsic shifts in cell cycle, gene expression, metabolism, as well as changes in the bone marrow microenvironment. Functional decline of aged hematopoietic stem cells leads to an increased risk of developing hematological malignancies. This connection is evident due to the fact that many molecular components that are perturbed in normal aging are key drivers of bone marrow disorders. Current approaches to understand the mechanisms of aging rely on global metrics of mutational burden, epigenetic drift, gene expression perturbations, and metabolic shifts. As such, there is a lack of clearly defined molecular mechanisms by which hematopoietic stem cell aging occurs.
In this study, we identified the transcription factor RUNX3 to be down-regulated in aged hematopoietic stem and progenitor cells, and describe a novel role for this factor in regulating myeloid lineage balance. Human and mouse RUNX3 transcript levels were diminished in aged hematopoietic stem and progenitor cells, and were correlated with diminished protein levels in aged human bone marrow specimens. The deregulation of RUNX3 appeared to be related to epigenetic alterations at the RUNX3 locus characterized in human cells by loss of activating histone acetylation marks at the enhancer and promoter, and in mouse cells by hypermethylation of the promoter. A functional defect in myeloid differentiation was demonstrated in vitro using colony formation assays and suspension culture, wherein erythroid and megakaryocyte differentiation was blocked, but granulocyte differentiation was spared. Committed erythroid progenitors also displayed defective maturation as evidenced by surface antigen expression and failure to express globin genes. RUNX3-deficient stem and progenitor cells grown in erythroid cell culture conditions experienced a significantly myeloid-skewed distribution of progenitor subtypes at the expense of erythroid and megakaryocyte progenitors. Changes in gene expression in RUNX3-deficient progenitors indicated a regulatory role for RUNX3 upstream of key erythroid factors GATA1 and KLF1. Due to the highly robust erythroid phenotype, individuals with unexplained anemia of the elderly were interrogated for RUNX3 expression and functional defects. RUNX3 transcripts were found to be significantly lower than healthy age-matched controls and erythroid colony formation was impaired at the bi-potent erythroid-megakaryocyte progenitor stage, suggesting that RUNX3 deficiency may contribute to bone marrow pathogenesis.
PHD (Doctor of Philosophy)
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