Date of Award

Summer 2020

Document Type

Open Access Dissertation

Department

Biological Sciences

First Advisor

Katie Kathrein

Abstract

The hematopoietic compartment is tasked with the establishment and maintenance of the entire blood program. Key to this process are hematopoietic stem cells (HSCs), which possess the unique ability to self-renew and differentiate to replenish blood cells throughout an organism’s lifetime. In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified chromatin remodeling protein and transcription repressor, Ing4, as a requirement for hematopoietic stem and progenitor cell (HSPC) specification. Ing4, a member of the inhibitor of growth (ING) family, is a subunit of the HB01-JADE-hEAF6 histone acetyltransferase complex responsible for most nucleosomal histone H4 acetylation in eukaryotes. Ing4 is known to form an inhibitory complex with RelA, the large subunit of the NF-kB protein complex, limiting expression of NF-kB target genes. NF-kB is a dimeric complex of Rel proteins that can activate, and sometimes repress transcription of cytokine genes. Thus, loss of Ing4 in mice causes hyper inflammatory responses because there is no inhibition of NF-kB. Ing4 has also been suggested to regulate a wide variety of cellular processes, including DNA repair, apoptosis, cell-cycle regulation, metastasis, angiogenesis, and tumor suppression. The role of Ing4 in hematopoiesis has not been established.

Here, we present data that shows Ing4 is critical for HSC gene regulation to direct both stem cell maintenance and differentiation in mice. Utilizing competitive transplantation, flow cytometry analysis of Ing4-deficient mouse bone marrow and peripheral blood, as well as colony-forming unit (CFU) assays, we have revealed that loss of Ing4 in mice resulted in a shift in the endogenous HSC sub-populations towards short term-HSCs (ST-HSCs)/long term-HSCs (LT-HSCs) and skewed HSC differentiation towards granulocyte-macrophage progenitors (GMPs). Furthermore, when sorted LT-HSC, ST-HSC, and MPP cells were transplanted into irradiated mice, Ing4-/- LT HSCs failed to reconstitute recipient BM, while Ing4-/- MPPs were greater contributors to multilineage engraftment compared to WT MPPs. Interestingly, MPPs are a population that have lost the self-renewal capacity of a stem cell, but our data suggests that under stress hematopoiesis, the loss of Ing4 likely results in reprogramming of MPPs to gain stem-like features. Further characterization of this HSC sub-population is underway. Moreover, the inability of Ing4-/- LT HSCs to engraft suggests an evolutionary requirement for Ing4 in normal hematopoiesis and gives us insight into how Ing4 modulates inflammatory pathways to regulate HSC function.

Through a number of in situ hybridization studies, we have previously shown that Ing4-deficient zebrafish embryos have decreased expression of HSC specific genes, runx1 and c-myb, in the aorta—gonad—mesonephros (AGM) region, while mRNA based overexpression of human ING4 results in increased expression of these same genes, suggesting that normal expression levels of Ing4 are required for HSC specification. As in mice, Ing4 deficiency in zebrafish results in increased expression of NF-kB target genes. Interestingly, simultaneous inhibition of NF-kB and Ing4 restores HSC formation in embryonic zebrafish. Based on these observed effects and the literature published on our mouse model, we conducted a mini screen of NF-kB inhibitors with known modes of action, to identify those that could compensate for the consequences of Ing4 loss of function. These experiments revealed that NF-kB inhibitors, MRT673007, Senexin, and Parthenolide significantly reduced cytokine expression in Ing4-deficient zebrafish and as expected, rescued HSC specification in this system.

Our findings support a novel role for Ing4 in blood development and highlight a potential molecular mechanism by which Ing4 regulates hematopoiesis through the NF-κB pathway. This provides further insight into the characterization of factors and pathways involved in hematopoiesis and the specific genetic defects in these regulators that can be targeted for therapeutic treatment of blood disorders.

Rights

© 2020, Melanie Rodriguez

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