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. 2007 May 15;109(10):4181-90.
doi: 10.1182/blood-2005-05-022004. Epub 2007 Jan 23.

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/EBPepsilon gene

Arati Khanna-Gupta  1 , Hong Sun, Theresa Zibello, Han Myung Lee, Richard Dahl, Laurence A Boxer, Nancy Berliner
Affiliations

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/EBPepsilon gene

Arati Khanna-Gupta et al. Blood. .

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.

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Figures

Figure 1
Figure 1
DNA sequence analysis of the C/EBPϵ gene in the SGD patient. (A) Genomic DNA isolated from a healthy individual (top panel, control) and from the SGD patient (bottom panel) was PCR amplified and sequenced using oligomers designed to capture the C/EBPϵ exons and intron-exon boundaries. The red arrow in the bottom panel indicates the heterozygous mutation in the SGD patient. (B) The genomic organization of the C/EBPϵ locus is indicated showing 3 exons (Ex 1, 2, and 3), 2 promoters (Pα and Pβ), 3 alternate translational start sites (ATG), and the highly conserved basic leucine zipper region (B-zip). (C) Amino acid sequence of the C/EBPϵ B-zip region. The heterozygous valine to alanine substitution in the SGD patient is indicated at position 218 in the basic region, which is underlined. The relevant leucine moieties are also underlined.
Figure 2
Figure 2
Transient cotransfection analysis of SGP promoter plasmids (HNC and LF) with wild-type and mutant C/EBPϵ expression plasmids in 32Dwt18 cells. 32Dwt18 cells were transiently cotransfected with HNC193 and LF477 (SGP) reporter plasmids (see “Patients, materials, and methods”) with 10 μg expression plasmids for wt C/EBPϵ (e) and mutant C/EBPϵ (V218A) either separately or together. Normalized luciferase values have been represented as a ratio of enzyme activity of SGP promoter plasmids plus C/EBPϵ expression plasmids to that of the SGP promoter plasmid without expression plasmids. The figure represents normalized mean ± SE obtained from 3 independent experiments, each performed in duplicate. Statistical analysis performed by Student t test revealed no statistical significance in the transactivation ability of wt and mutant plasmids. *P = .15; †P = .22.
Figure 3
Figure 3
Expression of C/EBPϵ in the SGD patient. (A) Real-time PCR analysis of C/EBPϵ expression in a normal sample and in the SGD patient was carried out in triplicate. Transcript levels were normalized to that of β-actin and expressed as a ratio of the signal observed in the normal sample (1). Error bars indicate standard error of the mean (SEM). (B) Western blot analysis of normal and SGD PMNs. NB4 and NB4 cells treated with ATRA serve as an additional control for C/EBPϵ expression.
Figure 4
Figure 4
Expression of PU.1 and Gfi-1 in the SGD patient. Real-time PCR analysis of (A) PU.1 and (B) Gfi-1 expression in normal versus SGD PMNs. Transcript levels of each mRNA were assessed in triplicate and normalized to that of β-actin and expressed as a percentage of the signal observed in the normal sample (100%). Due to the paucity of RNA, this experiment was performed only once in triplicate. Error bars indicate SEM. (C) Western blot analysis of PMNs from SGD and normal samples. Nuclear extracts prepared from peripheral blood neutrophils (PMNs) of 2 healthy volunteers and our SGD patient were subjected to Western blot analysis. Equal concentrations of protein were loaded in each lane. The blot was sequentially probed with antibodies for Gfi-1, PU.1, and β-actin.
Figure 5
Figure 5
A flow cytometric analysis of Gfi-1+/− and Gfi-1+/+ EML cells. Gfi-1+/− and Gfi-1+/+ EML cells were stained and analyzed using a FACSvantage flow cytometer and CellQuest software. (A) Cells (1.5 × 105) were then labeled with conjugated antibodies for 20 minutes at 4°C. All antibodies (B220, Sca-1, CD34, c-kit, Mac-1, Gr-1, and Ter119) used for flow cytometry including matched isotype controls were conjugated to phycoerythrin (PE) and purchased from eBiosciences. Error bars indicate SEM. (B) Wright-Giemsa staining of EML+/− and EML+/+ cells following terminal neutrophil maturation. Myeloid differentiation of EML cells was conducted in IMDM medium containing 20% horse serum, IL-3, SCF, and 10 μM ATRA for 3 days. The cells were then transferred to IMDM medium containing 20% horse serum and GM-CSF. Within a week, GM-CSF–dependent EPRO cells emerged. These cells were terminally differentiated by the addition of 10 μM ATRA for 3 days. Cells were cytospun on the days indicated and subjected to Wright-Giemsa staining.
Figure 6
Figure 6
Expression pattern of Gfi-1, PU.1, C/EBPϵ, and their downstream targets in ATRA-induced Gfi-1+/+ and Gfi-1+/– EPRO cells. (A) Real-time PCR analysis of ATRA-induced Gfi-1+/+ and Gfi-1+/− EPRO cells. EPRO cells were induced to terminally differentiate in the presence of ATRA. RNA samples were collected at the times indicated and subjected to real-time PCR analysis. Transcript levels of each mRNA were normalized to that of 18S rRNA and expressed as a percentage of the signal observed in the uninduced EPRO+/+ cells. This experiment was performed 3 times in triplicate. (B) Western blot analysis of ATRA-induced Gfi-1+/+ and Gfi-1+/− EPRO cells. Whole cell extracts prepared from ATRA-induced EPRO cells at the time indicated were subjected to Western blot analysis. Equal concentrations of protein were loaded in each lane. The blots were sequentially probed with antibodies for LF and β-actin, and NC and β-actin.
Figure 7
Figure 7
Transient transfection analysis of an SGP promoter (NC) harboring a Gfi-1 and a C/EBP site. (A) DNA sequence of the promoter of an SGP gene, neutrophil collagenase (NC). The first 193 bp of the human (HNC193) neutrophil collagenase promoter sequences representing the minimal promoter is illustrated. The 3 C/EBP sites (P, I, and D) are underlined in black and the conserved Gfi-1 sequence, in gray. The transcription start site is marked by a +1 sign and the translation start site ATG is indicated. (B) 32Dwt18 cells were transiently cotransfected with wild-type HNC193 (wt) or Gfi-1 mutant HNC193 (“Patients, materials, and methods”) and 10 μg expression plasmids for Gfi-1 and wt C/EBPϵ (e) separately or together. HNC193 (wt) was also cotransfected with the Gfi-1 expression plasmid and wt or V218A C/EBPϵ plasmids. Normalized luciferase values have been represented as a ratio of enzyme activity of HNC193 promoter plasmid plus C/EBP and/or Gfi-1 expression plasmids to that of HNC193 promoter plasmid without expression plasmids. The figure represents normalized mean ± SE obtained from 3 independent experiments, each performed in duplicate. Statistical significance was determined using Student t test, and the P values have been indicated.
Figure 8
Figure 8
Oligonucleotide pull-down assay using the Gfi-1/C/EBP probe in the HNC promoter. Nuclear extracts prepared from uninduced and ATRA-induced MPRO cells (a murine myeloid cell line for neutrophil maturation) were incubated with the biotinylated Gfi-1/C/EBP probe from the HNC promoter (A). The DNA-protein complexes were recovered using streptavidin-agarose beads and the bound proteins resolved by SDS-PAGE and Western blot analysis. The blot was probed sequentially with Gfi-1, C/EBPα, and C/EBPϵ antibodies (B). Oligonucleotide pull-down assays using the same probe and 293 extracts overexpressing Gfi-1, C/EBPα, and C/EBPϵ served as a positive control (C). (− indicates untransfected 293 cells; +, transfected 293 cells.)
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