Growth factor independence-1 (Gfi-1) plays a role in mediating specific granule deficiency (SGD) in a patient lacking a gene-inactivating mutation in the C/EBPϵ gene

Abstract

Neutrophil-specific granule deficiency (SGD) is a rare congenital disorder marked by recurrent bacterial infections. Neutrophils from SGD patients lack secondary and tertiary granules and their content proteins and lack normal neutrophil functions. Gene-inactivating mutations in the C/EBPepsilon gene have been identified in 2 SGD patients. Our studies on a third SGD patient revealed a heterozygous mutation in the C/EBPepsilon gene. However, we demonstrate elevated levels of C/EBPepsilon and PU.1 proteins in the patient's peripheral blood neutrophils. The expression of the transcription factor growth factor independence-1 (Gfi-1), however, was found to be markedly reduced in our SGD patient despite the absence of an obvious mutation in this gene. This may explain the elevated levels of both C/EBPepsilon and PU.1, which are targets of Gfi-1 transcriptional repression. We have generated a growth factor-dependent EML cell line from the bone marrow of Gfi-1(+/-) and Gfi-1(+/+) mice as a model for Gfi-1-deficient SGD, and demonstrate that lower levels of Gfi-1 expression in the Gfi-1(+/-) EML cells is associated with reduced levels of secondary granule protein (SGP) gene expression. Furthermore, we demonstrate a positive role for Gfi-1 in SGP expression, in that Gfi-1 binds to and up-regulates the promoter of neutrophil collagenase (an SGP gene), in cooperation with wild-type but not with mutant C/EBPepsilon. We hypothesize that decreased Gfi-1 levels in our SGD patient, together with the mutant C/EBPepsilon, block SGP expression, thereby contributing to the underlying etiology of the disease in our patient.

Gfi1 and Gfi1b: key regulators of hematopoiesis

“…45,46 In addition, diminished Gfi1 expression may contribute to the development of neutrophil-specific granule deficiency by causing reduced levels of secondary granule protein expression. 32 A condition similar to human SCN is encountered in mice that lack Gfi1 or that express the mutated form of Gfi1 found in a SCN patient. [47][48][49] This strongly suggests that the mutated Gfi1 form that causes SCN, functions in a dominant-negative manner over wild-type Gfi1.…”

“…5,15,26,27,[29][30][31] However, it should be noted that examples have been reported in which Gfi1 and Gfi1b can activate gene expression. 15,18,29,32 Gfi1 and Gfi1b also interact physically with other transcription factors through their zinc fingers to co-regulate gene expression (Figures 1 and 2). 17,29,[33][34][35][36] For example, Gfi1 interacts directly with ETS1 to repress the Bax gene through adjacent DNA binding sites.…”

“…23 It has been shown that in these cells Gfi1 cooperates with C/EBPe to control expression of genes that are implicated in terminal differentiation. 32 Gfi1 is not required for the differentiation of myeloid progenitors toward monocytes. However, in CD14 þ monocytes, the Gfi1 protein is expressed and binds to genes required for granulocytic differentiation and function (Table 1).…”

Granulocyte colony-stimulating factor-induced upregulation of Jak3 transcription during granulocytic differentiation is mediated by the cooperative action of Sp1 and Stat3

“…It has been shown previously that there is no change in the ability of Sp1 to bind DNA during the course of G-CSF induced differentiation of 32Dcl3 cells (Nuchprayoon et al, 1999), so Sp1 may depend on posttranslational modification or binding to inducible partners to preferentially activate transcription of target genes in response to a stimulus such as G-CSF. The promoters of CD11b (Chen et al, 1993), CD11c (Noti et al, 1996), CD14 (Zhang et al, 1994), CD18 (Rosmarin et al, 1998), as well as c-fes (Heydemann et al, 1996), myeloperoxidase (Piedrafita et al, 1996), lactoferrin (Khanna-Gupta et al, 2000), neutrophil elastase (Nuchprayoon et al, 1999), myeloid cell nuclear differentiation antigen (Kao et al, 1997), and human hematopoietic cell kinase (Hauses et al, 1998) all are preferentially activated in myeloid cells through mechanisms involving Sp1. If there is a requirement of an inducible partner for Sp1 to direct myeloid-specific transcription, then Stat3 may play that role.…”

“…Transient transfection of 32Dcl3 cells was achieved by electroporation, as described previously (Khanna-Gupta et al, 2000). Following electroporation, cells were left unstimulated or stimulated with 100 ng/ml of recombinant murine G-CSF (Stem Cell Technologies, Vancouver, BC, Canada).…”