Date of Award

10-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Shannon L. McKinney-Freeman, PhD

Committee

Suzanne J. Baker, PhD John V. Cox, PhD Joseph T. Opferman, PhD Erin Schuetz, PhD Tiffany N. Seagroves, PhD

Keywords

chromatin immunoprecipitation; co-regulation; PU.1; transcription factor

Abstract

We report that ectopic expression of Nfix in primary mouse HSPC extended their ex vivo culture from 20 to 40 days. HSPC overexpressing Nfix displayed hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation compared to controls. Ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPC as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL signaling. Blocking c-MPL signaling by removal of its ligand, thrombopoietin (TPO), or addition of a c-MPL neutralizing antibody negated the anti-apoptotic effect of Nfix overexpression on cultured HSPC. Furthermore, NFIX-FLAG was capable of binding to and transcriptionally activating a proximal c-Mpl promoter fragment. In sum, these data suggest that NFIX-mediated up-regulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo. Understanding the direct transcriptional targets or co-binding partners of NFIX would provide further insight into the mechanisms HSPC employ to maintain steady-state hematopoiesis or overcome stress hematopoiesis.

To this end, we combined global transcriptional profiling and genome-wide binding to identify direct transcriptional targets of NFIX in Nfix+/+ and Nfix-/- HPC5 cells, a primitive multi-potent hematopoietic cell line. We find that NFIX preferentially binds enhancer and promoter genomic regions. Integrative analysis revealed >500 differentially expressed genes of which 58% were direct NFIX targets. Many of these genes were downregulated in the absence of NFIX, indicating that NFIX functions primarily as a transcriptional activator in this context. PANTHER pathway analysis implicated NFIX in the regulation of apoptosis, myeloid cell differentiation and cell-cell adhesion. Using archived ChIP-seq data, we revealed significant co-localization of NFIX with other well-known hematopoietic transcription factors, including pSTAT1, RUNX1, RAD21, STAT3, ETO2, FLI1, GATA2, LYL1, LDB1 and PU.1. We showed NFIX and PU.1 together target genes regulating hematopoietic cell adhesion, cell death and differentiation in hematopoietic cells. Our data support a model in which NFIX collaborates with PU.1 to regulate differentiation and apoptosis in hematopoietic cells. In summary, we identified direct transcription targets and putative co-regulatory partners of NFIX.

In sum, the work here further characterizes the complex role of the NFI family member, NFIX. We show minor perturbations in PB lineages when transplanting NfixΔ/Δ HSPC. During secondary transplants, NfixΔ/Δ donor chimerism was similar to controls. Also, during steady-state hematopoiesis, we did not observe any overt phenotypes in the NfixΔ/Δ mice. We discovered that overexpressing Nfix ex vivo imparts cells with hypersensitivity to cytokines and resistance to apoptosis. These characteristics were due to an increase in c-MPL signaling and could be abolished if this signaling was blocked. We identified c-Mpl as the first transcriptional target of NFIX in a hematopoietic context. Finally, we have rigorously characterized the genome-wide binding of NFIX in a hematopoietic cell line as well as identified 291 putative transcriptional targets. This work provides more data towards illuminating the role of an NFI family member during hematopoiesis.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0002-5527-0783

DOI

10.21007/etd.cghs.2020.0513

Additional Files
2020-026-Walker-DOA.pdf (400 kB)
Declaration of Authorship
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