A network of transcription factors and associated complexes regulate the process of hematopoiesis and are required for maintenance and development of the hematopoietic program. Ing4, a tumor suppressor protein, was identified in a screen for epigenetic regulators of hematopoiesis in zebrafish as required for specification of hematopoietic stem and progenitor cells (HSPCs). Recent work has shown that Ing4 is inactivated in various cancer cells. This inactivation promotes stem cell-like qualities in malignant cells. Ing4 plays an inhibitory role in the NF-κB pathway, conferring, in part, Ing4's tumor-suppressor capability. Loss of Ing4 is correlated to diminished hematopoietic stem cell (HSC) specification in zebrafish and increased NF-κB target gene expression. NF-κB knockdown assays in zebrafish embryos suggest inhibition of NF-κB remediates loss of Ing4 expression, with HSC rescue efficacy varying directly with concentration of inhibitor.
Similarly, the necessity of Ing4 in murine hematopoiesis has been observed. Here, Ing4 deficiency impairs HSC function, while simultaneously enhancing the regenerative capacity of multipotent progenitor cells (MPPs). Characterization of bone marrow from Ing4-deficient mice shows abnormal hematopoiesis, with a striking decrease in MPPs as compared to wildtype mice (47.9% vs 19.3%).
Hematopoiesis under stress conditions is also altered in Ing4-deficient mice, as observed following competitive HSC transplantation. In a surprising finding, MPPs from Ing4-deficient mice showed a dramatic increase in peripheral blood multilineage chimerism compared to wildtype mice up to 9 months post-transplantation in a competitive transplant assay (19% vs. 59%). This supports the hypothesis that MPPs from Ing4-deficient mice have enhanced self-renewal capacity. Additionally, we have observed a subpopulation of Ing4-deficient MPPs that express lower levels of CD34, CD34+/mid. This population of CD34+/mid cells was also shown to express CD229 (85% positive), while very few WT MPPs express both CD34+/mid and CD229 (5.0%). Reduced levels of CD34 expression combined with CD299 are known to be markers of HSCs, and so we hypothesize that a subset of CD34+/midCD229+ MPPs in Ing4-deficient mice retain their self-renewal capacity. Taken together, our data suggest Ing4 typically functions as a suppressor of genes necessary for self-renewal and developmental potency of MPPs.
Additionally, cell cycle analysis combined with Ki-67 expression showed Ing4-deficient MPPs have enhanced ability to maintain quiescence, with 15.2% of cells found to be in G0 phase as compared to 6.5% of wildtype MPPs in G0. Finally, after 5-FU treatment, levels of MPPs in WT mice were similar pre- and post-treatment. Future experiments will seek to elucidate this observation in consideration of the pro-inflammatory environment.
These findings suggest Ing4 is a critical regulator of hematopoiesis, and these data provide important clues for further characterization of the pathways and functions of Ing4. Our data show that Ing4 deficiency promotes stem cell-like properties in MPPs, suggesting it has crucial regulatory functions in both stem cell self-renewal and maintenance.
Disclosures
No relevant conflicts of interest to declare.
Enrichment of Human Hematopoietic Stem/Progenitor Cells Facilitates Transduction for Stem Cell Gene Therapy