Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Mar 8;6(5):1464-1473.
doi: 10.1182/bloodadvances.2021006182.

GATA2 deficiency elevates interferon regulatory factor-8 to subvert a progenitor cell differentiation program

Kirby D Johnson, Alexandra A Soukup  1 , Emery H Bresnick  1
Affiliations

GATA2 deficiency elevates interferon regulatory factor-8 to subvert a progenitor cell differentiation program

Kirby D Johnson et al. Blood Adv. .

Abstract

Cell type-specific transcription factors control stem and progenitor cell transitions by establishing networks containing hundreds of genes and proteins. Network complexity renders it challenging to discover essential versus modulatory or redundant components. This scenario is exemplified by GATA2 regulation of hematopoiesis during embryogenesis. Loss of a far upstream Gata2 enhancer (-77) disrupts the GATA2-dependent transcriptome governing hematopoietic progenitor cell differentiation. The aberrant transcriptome includes the transcription factor interferon regulatory factor 8 (IRF8) and a host of innate immune regulators. Mutant progenitors lose the capacity to balance production of diverse hematopoietic progeny. To elucidate mechanisms, we asked if IRF8 is essential, contributory, or not required. Reducing Irf8, in the context of the -77 mutant allele, reversed granulocytic deficiencies and the excessive accumulation of dendritic cell committed progenitors. Despite many dysregulated components that control vital transcriptional, signaling, and immune processes, the aberrant elevation of a single transcription factor deconstructed the differentiation program.

PubMed Disclaimer

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Elevated IRF8 causes aberrant myeloid differentiation potential of GATA2-deficient progenitor cells. (A) Experimental strategy for double mutant in vivo rescue system. Mice heterozygous for −77 enhancer deletion or Irf8tm1.2Hm were bred to generate E14.5 embryos. Fetal liver progenitor populations with select genotypes were analyzed by flow cytometry. (B) Representative flow cytometry analysis of progenitor populations from GMPs (Lin−Sca-1−cKit+CD34+CD16/32high). GPs and MPs are distinguished from multipotential GMPs by expression of Ly6C and from each other by M-CSFR (CD115). (C) Quantitation of the frequency of MPs and GPs within the Ly6C+ GMP populations from 7 litters (−77+/+Irf8+/+, n = 7; −77−/−Irf8+/+, n = 5; −77+/+Irf8−/−, n = 3: −77−/−Irf8+/−, n = 8; −77−/−Irf8−/−, n = 7). Relevant statistical comparisons for panel C are described in "Results." (D) Quantitation of colonies from sorted Ly6C− GMPs plated in M3434 methylcellulose media (STEMCELL Technologies) at 1200 cells/plate. Colonies were scored as mixed-lineage CFU-GM, or single-lineage granulocyte CFU-G, or CFU-M colonies. Ly6C− GMPs were obtained from 4 litters (−77+/+Irf8+/+, n = 6; −77−/−Irf8+/+, n = 8; −77+/+Irf8−/−, n = 6; −77−/−Irf8+/−, n = 5; −77−/−Irf8−/−, n = 6). Error bars for all plots represent mean ± standard error of the mean (SEM). *P < .05, **P < .01, ***P < .001, ****P < .0001; Welch’s unequal variance t tests.
Figure 2.
Figure 2.
GATA2-IRF8 axis differentially regulates fetal liver progenitor populations. (A) Quantitation of progenitor populations in E14.5 fetal livers obtained from 7 litters (−77+/+Irf8+/+, n = 7; −77−/−Irf8+/+, n = 5; −77+/+Irf8−/−, n = 3; −77−/−Irf8+/−, n = 8; −77−/−Irf8−/−, n = 7). (B) mRNA quantitation from sorted Ly6C− GMPs from 4 to 9 embryos obtained from 10 litters. Real-time PCR primers for quantitation of Irf8 transcripts selectively amplified the wild-type, but not mutant, allele. ND, not detected. Error bars for all plots represent mean ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001; Welch’s unequal variance t tests.
Figure 3.
Figure 3.
GATA2 suppression of IRF8 levels restricts common dendritic cell progenitor production. (A) Volcano plot of differentially expressed genes mined from transcriptome analysis of lineage-depleted wild-type and −77−/− fetal livers cultured for 3 days as previously described. GEO accession: GSE133606. DC genes included factors involved in DC differentiation and/or differentially expressed in DC progenitor populations. Gata2 downregulation, resulting from the −77 enhancer deletion, is also depicted. (B) Representative flow cytometry analysis of monocyte-dendritic cell progenitor (MDP) and common dendritic cell progenitor (CDP) populations. Quantitation of the frequency of CDPs and MDPs within the Flt3+M-CSFR+Ly6C− CMP pools (C) and of total cells per liver (D) from 7 litters (−77+/+Irf8+/+, n = 7; −77−/−Irf8+/+, n = 5; −77−/−Irf8+/−, n = 8; −77−/−Irf8−/−, n = 7; −77+/+Irf8−/−, n = 3). Error bars represent mean ± SEM. *P < .05, **P < .01, ***P < .001; Welch’s unequal variance t tests. Relevant statistical comparisons for panel C are described in "Results."
Figure 4.
Figure 4.
Enhanced dendritic cell differentiation of −77−/− CMPs requires IRF8. (A) Representative flow plots of dendritic cells differentiated from flow-sorted CMPs cultured with GM-CSF, IL-4, and TNFα. (B) Quantitation of dendritic cell populations using early (CD11c) and late (CD86) DC maturation markers. Data were obtained from 3 to 8 embryos (7 litters). The populations were pregated to exclude cell doublets and dead (4′,6-diamidino-2-phenylindole+) cells. Error bars represent mean ± SEM. *P < .05, **P < .01, ****P < .0001; Welch’s unequal variance t tests.
Figure 5.
Figure 5.
Model illustrating IRF8 function as an essential mediator within GATA2 genetic networks. Physiological levels of GATA2 establish a transcriptome (genes, black circles) with low Irf8 expression, which maintains a balance in myeloid and dendritic cell progenitors. Loss of the −77 enhancer and consequent reduction in GATA2 disrupts the network (gray circles), upregulating Irf8 and increasing proportions of MP, MDP, and CDP populations. Reducing Irf8, in the context of the −77 mutant allele, reversed granulocytic deficiencies and the excessive accumulation of dendritic cell progenitors.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Katsumura KR, Bresnick EH, GATA Factor Mechanisms Group. The GATA factor revolution in hematology. Blood. 2017;129(15):2092-2102. - PMC - PubMed
    1. Tsai SF, Martin DI, Zon LI, D’Andrea AD, Wong GG, Orkin SH. Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Nature. 1989;339(6224):446-451. - PubMed
    1. Tsai FY, Keller G, Kuo FC, et al. . An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature. 1994;371(6494):221-226. - PubMed
    1. Evans T, Felsenfeld G. The erythroid-specific transcription factor Eryf1: a new finger protein. Cell. 1989;58(5):877-885. - PubMed
    1. Tsai F-Y, Orkin SH. Transcription factor GATA-2 is required for proliferation/survival of early hematopoietic cells and mast cell formation, but not for erythroid and myeloid terminal differentiation. Blood. 1997;89(10):3636-3643. - PubMed

Publication types

MeSH terms

Substances