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. 2003 Jul;34(3):308-12.
doi: 10.1038/ng1170.

Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2

Richard E Person  1 , Feng-Qian Li, Zhijun Duan, Kathleen F Benson, Jeremy Wechsler, Helen A Papadaki, George Eliopoulos, Christina Kaufman, Salvatore J Bertolone, Betty Nakamoto, Thalia Papayannopoulou, H Leighton Grimes, Marshall Horwitz
Affiliations

Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2

Richard E Person et al. Nat Genet. 2003 Jul.

Abstract

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic. We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.

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Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1
GFI1 mutations and hematopoietic phenotype. (a) Pedigrees of families with mutations in GFI1 and electropherograms of genomic DNA. Arrows indicate probands. (b) Mutations (pink) relative to zinc fingers, coordinating C (yellow) and H (blue), with BLAST alignment. (c) Peripheral blood film showing replacement of mature neutrophils with indistinct myeloid cells (Wright–Giemsa stain). (d) Cytospin of pooled GM, M colonies from peripheral blood cultures showing differentiation to macrophages (MΦ), absence of mature neutrophils and immature granular myeloid cells (arrows; Wright–Giemsa stain). (e) Flow cytometric analysis of peripheral blood from a normal adult (left) and the father in the pedigree with the N382S substitution (right) shows markedly fewer neutrophils (top; 56.7% to 10.5%), more CD33+ monocytic-like cells (top; 5.3% to 37.7%) and fewer naive CD4 T lymphocytes (middle; 32.5% to 17.4%) and B lymphocytes (bottom; 6.2% to 3.5%).
Figure 2
Figure 2
Transcriptional and DNA-binding consequences of mutations in GFI1. (a) Transient transfection assays in NIH3T3 cells testing repression by the indicated GFI1 cDNA on a TK–CAT reporter fused to dimerized Gfi1 consensus binding site. Data shown are the mean ± s.e. of three trials. Numbers indicate vector quantity (ng). Immunoblot of transfected cell extracts detected with antibodies to Gfi1 (α-Gfi1) and GAPDH (α-GAPDH) shows consistent protein expression (inset). WT, wild-type. (b) EMSA using in vitro synthesized (TnT system) proteins with 5′-32P-labeled double-stranded oligonucleotide containing Gfi1 consensus binding site supershifted with antibody to Gfi1 (α-Gfi1). Immunoblot of TnT-synthesized proteins shows consistent protein expression (inset).
Figure 3
Figure 3
ChIP assay showing Gfi1 targeting of ELA2. (a) PCR of ELA2 promoter in ChIP assay on indicated cell lines showing chromatin immunoprecipitation with two different specific or control antibodies. (b) EMSA with TnT-synthesized Gfi1 of oligonucleotide containing Gfi1 binding site identified in ELA2 at −2,714 to −2,740 (NE+), consensus Gfi1 binding site as a positive control and inactive A-T rich element in ELA2 at −300 to −326 (NE–) as a negative control. (c) Transient transfection assays in NIH3T3 cells testing repression of the indicated binding site (from b) fused to a TK–CAT reporter, in the presence or absence of expression of Gfi1. Data shown are the mean ± s.e. of three trials. Subscripts indicate the number of times the binding site is multimerized in the reporter construct. Each construct has a different basal level of expression, so activity in the presence of Gfi1 (red) is normalized to activity in its absence (blue). (d) Neutrophil elastase (NE) activity in peripheral blood from the father of the pedigree with the N382S substitution (pink) compared with that of two normal adult controls (green) superimposed on a standard curve using indicated quantity of purified enzyme (blue). Data shown are the mean ± s.e. of two measurements from 105 lysed mononuclear cells. (e) Quantitative RT–PCR of ELA2 expression, compared to GAPD control, in non-erythroid colonies formed from the peripheral blood of the three-year-old son in the pedigree with the N382S substitution versus his mother. Representative results with oligo(dT)-primed RNA are shown.
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