Patients with severe congenital neutropenia (SCN) display mutations in ELA2
, and HAX1
(Devriendt et al., 2001
; Klein et al., 2007
; Person et al., 2003
), but mutations in ELA2
predominate (Horwitz et al., 2007
). Modeling mutant Ela2 in primary murine cells has failed to mimic SCN (Grenda et al., 2002
), and Ela2−/−
mice have mature neutrophils (Belaaouaj et al., 1998
). In contrast, our data clearly show that expression of murine Gfi1N382S in otherwise normal murine Lin−
stem and progenitor cells blocks granulopoiesis in a manner strikingly similar to Gfi1N382S patient samples processed in a comparable manner. Although Gfi1−/−
mice and Gfi1N382S patients have a similar block to granulopoiesis (Hock et al., 2003
; Karsunky et al., 2002b
; Person et al., 2003
), our data reveal selective deregulation of putative Gfi1 targets by Gfi1N382S. Thus, deletion of murine Gfi1 engenders severe transcriptional changes that are apparently not relevant to Gfi1N382S–mediated human disease. Similarly, expression of Gfi1N382S in the 32D cell line results in profound G-CSF-induced cell death instead of differentiation (Zhuang et al., 2006
), which did not occur in primary Lin−
cells. Neither the 32D cell line nor Gfi1−/−
mice represent a self-sufficient model of Gfi1 mutant SCN.
Of the putative Gfi1 target genes we examined, only Csf1 and Csf1r were deregulated in Gfi1−/− bone-marrow cells, repressed by Gfi1, and derepressed by Gfi1N382S. It is currently unclear why Gfi1N382S regulates Csf1 differently than Cebpe, Egr1, Egr2, and Nab2. However, we have shown that Gfi1 binds to the Csf1 promoter in living U937 cells. In SCN patients and murine Lin− cells, Gfi1N382S may require an intact SNAG repressor domain to compete for corepressors utilized by Csf1 -promoter-bound wild-type Gfi1. Regulation of Csf1 and its receptor are obviously critical for Gfi1N382S action because antibody absorption of Csf1 in culture or genetic ablation of Csf1 released the Gfi1N382S block to granulopoiesis. Although inactivation of Csf1 did not perfectly restore myelopoiesis, the granulocytes formed from Gfi1N382S–expressing cells without Csf1 support the hypothesized dominant-negative activity of Gfi1N382S. Clearly, Gfi1 regulates multiple targets and the expression of Gfi1N382S may be used to selectively identify target genes that are critical to granulopoiesis and to the pathogenesis of SCN.
Previously, mutations found in humans with SCN, introduced into the corresponding murine cDNA, and expressed in primary murine cells failed to block the production of mature murine granulocytes. Given our results, it is perhaps not surprising that Gfi1N382S, which engenders the most severe mutant phenotype we observed, was identified in the patient population. Other zinc-finger amino acid substitutions, although not as potent as Gfi1N382S, are clearly capable of interfering with granulopoiesis. Although neutrophil elastase mutants do not successfully model SCN in mice, and thus highlight differences in myelopoiesis between rodents and humans, expression of GFI1N382S (and other Gfi1 mutants) in primary Lin− bone-marrow cells illustrates a central transcriptional program that controls neutrophil development and that is relevant to the pathogenesis of SCN.
Although Gfi1 may have an array of transcriptional targets in different cell types, the subset of these genes relevant to Gfi1N382S–induced SCN may be restricted to monopoietic genes in progenitors. That Gfi1 influences granulopoiesis by restricting the expression of monopoietic genes in neutrophils is currently a matter of debate. Several studies (Dahl et al., 2007
; Hock et al., 2003
; Laslo et al., 2006
) have suggested that monocytic genes are repressed by Gfi1, but recent work on Gfi1-protein degradation suggested that Gfi1 only accumulates in monocytic cells. Gfi1 may thus function to repress granulocytic genes in monocytes (Marteijn et al., 2006
). Although this possibility has not been excluded, neutrophils from Gfi1+/−
animals have deregulated transcription of secondary granule-protein-encoding genes (Khanna-Gupta et al., 2007
), suggesting that there is sufficient Gfi1 protein in neutrophils to regulate granulocytic genes. Our own data show that Gfi1 instructs granulopoiesis and that Gfi1N382S critically deregulates the expression of Csf1 to induce monopoiesis.
Gfi1 is rate limiting for granulopoiesis. Our data demonstrate that Lin− bone-marrow cells from Gfi1+/− mice generated substantially fewer granulocytic colonies than Gfi1+/+ littermates. Thus, physiological manipulation of Gfi1 dosage affects lineage fate. In addition, retroviral-vector-mediated expression of Gfi1 rescued a normal ratio of granulopoietic and monopoietic colonies from Gfi1−/− Lin− bone-marrow cells, whereas it ablated monocytic-colony formation in Gfi1+/+ Lin− bone-marrow cells. The amount of Gfi1 achieved in our experiments (and not deregulated expression) appears to influence lineage choice.
Both DNA-binding and transcription-repression functions appear critical for Gfi1 instruction of granulopoiesis. Gfi1 can apparently function as a molecular sink for the Pias3 SUMO E3 ubiquitin ligase and the U2AF26 splicing factor (Heyd et al., 2006
; Rodel et al., 2000
). Notably, these functions do not require DNA binding or an intact SNAG domain. If the biologically available amount of Pias3 or U2AF26 were the critical regulator of lineage fate, then either Gfi1N382S or Gfi1P2A should have bound these proteins and rescued Gfi1−/−
granulopoiesis, but they did not. In agreement with these data, Fiolka recently showed that mice with homozygous Gfi1P2A knockin alleles have Gfi1−/−
phenotypes (Fiolka et al., 2006
). On the other hand, we note that although granulopoiesis was restored in Gfi1P2A+N382S–expressing cells, the number of granulocytic colonies is not completely normal. Because neither Gfi1P2A nor Gfi1N382S mutations would be expected to disrupt Pias3 or U2AF26 interactions, the residual activity of the Gfi1P2A+N382S mutant might be explained by Gfi1 binding to these proteins. In contrast, Gfi1 antagonism of Pu.1 required the SNAG domain, but not DNA binding (Dahl et al., 2007
). We observed a lack of granulopoiesis in both Gfi1N382S- and Gfi1P2A–expressing Gfi1−/−
bone-marrow cells. If the major defect in Gfi1−/−
cells were overactive Pu.1, then the Gfi1N382S but not the Gfi1P2A mutant should have been capable of some rescue, but neither mutant rescued Gfi1−/−
granulopoiesis. These data emphasize the requirement for both Gfi1 DNA binding and transcription-repression functions to instruct granulopoiesis.
Our data support the hypothesis that a greater amount of Gfi1 resolves the fate of mixed-lineage cells (Laslo et al., 2006
). It is alternatively possible that Gfi1 functions through cell-cycle or cell-death pathways to limit monocytic-colony formation. However, cell-cycle analysis of Gfi1- or Gfi1N382S–expressing cytokine-stimulated Lin−
bone-marrow cells did not reveal substantial differences (data not shown). Moreover, Gfi1 was antiapoptotic in cytokine-stimulated bone-marrow progenitors; as already established in T cells (Grimes et al., 1996b
; Karsunky et al., 2002a
). Although Gfi1 was recently shown to block Pu.1-induced macrophage development in a cell line (Dahl et al., 2007
), our work demonstrates that forced expression of Gfi1 instructs granulopoiesis in a DNA-binding and SNAG-domain-dependent manner not explained by Pu.1 interactions. Instead, primary wild-type Lin−
cells yielded almost exclusively granulocytic colonies with dramatic diminution of monocytic colonies. When the same cells were stimulated with G-CSF in liquid culture, Gfi1-expressing cells had a uniform neutrophilic surface phenotype (7/4+
) and were morphologically more mature than vector-transduced cells. In addition, a striking reduction of not only monocytic (7/4−
) but also mixed lineage (7/4+
) phenotype cells occurred. In sum, Gfi1 is a rate-limiting granulocytic switch that represses critical monopoietic target genes (such as Csf1
) to resolve cell-fate decisions in progenitor cells.