# Defining Nfix's role as a novel key regulator of hematopoietic stem cells

> **NIH NIH R01** · ST. JUDE CHILDREN'S RESEARCH HOSPITAL · 2020 · $403,875

## Abstract

Project Summary
Hematopoietic stem cell (HSC) transplantation is currently used >16,000 times/year in the United States to
treat hematologic disease, leukemia, and to restore hematopoiesis in cancer patients. HSC are thus one of the
most clinically exploited stem cell populations. Multiple recent studies demonstrate that the hematopoietic
stem and progenitor cells (HSPC) that restore hematopoiesis following bone marrow transplant (BMT) are
distinct from those that sustain native hematopoiesis. These findings place an imperative on better
understanding the selective regulation of HSPC repopulation. Thus, our laboratory seeks to dissect the cellular
and molecular mechanisms that regulate HSC repopulation. In a functional screen for genes whose depletion
perturbs HSC in vivo repopulation, we identified the Nuclear Factor I (NFI) gene family member, Nfix, as a
novel regulator of HSPC function post-BMT. Loss of Nfix severely curtailed the ability of HSPC to reconstitute
ablated mice. Further, Nfix-deficient HSPC display increased apoptosis post-BMT, lack CFU potential, and are
reduced in number in recipient bone marrow. As the expression of multiple apoptotic regulators is perturbed
in the presence and absence of Nfix in HSPC, we propose that Nfix is a key intrinsic regulator of HSPC survival
post-BMT. Here, we will test this hypothesis and also assess a role for Nfix in native hematopoiesis, according
to the following specific aims: 1) to test the hypothesis that Nfix regulates HSPC survival post-BMT, 2) to
identify the molecular targets of Nfix in HSPC, and 3) to test if Nfix is required during native hematopoiesis.
For Aim 1, we will employ mice deficient in key cell death pathway regulator genes to test if apoptosis is critical
to the loss of Nfix-deficient HSPC post-BMT. As Bcl-xL is downregulated in Nfix-deficient HSPC, we will also
test if restored Bcl-xL expression rescues the repopulating defect of Nfix-deficient HSPC. Finally, Nfix
overexpression greatly prolongs the ex vivo culture of primary hematopoietic cells. Thus, we will also test if
Nfix overexpression protects HSPC from apoptosis ex vivo. For Aim 2, we will leverage the transcriptomes of
HSC lacking Nfix, HSC overexpressing Nfix, and NFIX ChIP-seq binding patterns in primitive hematopoietic
cell lines to identify candidate downstream targets of Nfix. These candidates will be functionally validated via
shRNA-mediated gene knockdown in HSPC overexpressing Nfix for an obligate role in promoting the Nfix-
dependent survival of these cultures. We predict that genes necessary here will also play a key role down-
stream of Nfix in HSPC post-BMT. Finally, in Aim 3, we will analyze Nfix+/+, Nfixfl/+, and Nfixfl/flROSA26Cre-ERT2
bone marrow chimeras for perturbations in blood lineages, HSPC bone marrow compartments, and HSC
quiescence following Nfix deletion. Treatment with tamoxifen efficiently deletes Nfix from the hematopoietic
compartment in these mice. By defining Nfix's role as an HSC r...

## Key facts

- **NIH application ID:** 9849277
- **Project number:** 5R01DK104028-05
- **Recipient organization:** ST. JUDE CHILDREN'S RESEARCH HOSPITAL
- **Principal Investigator:** SHANNON L MCKINNEY-FREEMAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $403,875
- **Award type:** 5
- **Project period:** 2016-01-01 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9849277

## Citation

> US National Institutes of Health, RePORTER application 9849277, Defining Nfix's role as a novel key regulator of hematopoietic stem cells (5R01DK104028-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9849277. Licensed CC0.

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