Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo

Abstract

Transcription factor GATA-1 reprograms immature myeloid cells to three different hematopoietic lineages-erythroid cells, megakaryocytes, and eosinophils. GATA-1 is essential for maturation of erythroid and megakaryocytic precursors, as revealed by gene targeting in mice. Here we demonstrate that deletion of a high-affinity GATA-binding site in the GATA-1 promoter, an element presumed to mediate positive autoregulation of GATA-1 expression, leads to selective loss of the eosinophil lineage. These findings suggest that GATA-1 is required for specification of this lineage during hematopoietic development. Mice lacking the ability to produce eosinophils should prove useful in ascertaining the role of eosinophils in a variety of inflammatory or allergic disorders.

Figure 1.

Targeting of the GATA-1 locus. (A, top) The murine GATA-1 locus contains a double GATA-site upstream of the hematopoietic exon IE. Other exons are designated with Roman numerals, including testis exon IT. The double-stranded sequence at the asterisked region is given below the schematic, with nucleotides to be deleted marked in boldface type. (Middle) After targeting, the double GATA-site is deleted, replaced by a floxed PGK-neo cassette. (Bottom) After cre recombinase-mediated excision, the “excised locus” has a minimal sequence consisting primarily of a single loxP site surrounded by two Not I sites. N, Not I site. The double-stranded sequence at the asterisked region is given below the schematic. (B) PCR genotyping of male mice carrying the wt or ΔdblGATA mutant (hem) allele. PCR products before (–) and after (N) digestion with Not I.

Figure 1.

Targeting of the GATA-1 locus. (A, top) The murine GATA-1 locus contains a double GATA-site upstream of the hematopoietic exon IE. Other exons are designated with Roman numerals, including testis exon IT. The double-stranded sequence at the asterisked region is given below the schematic, with nucleotides to be deleted marked in boldface type. (Middle) After targeting, the double GATA-site is deleted, replaced by a floxed PGK-neo cassette. (Bottom) After cre recombinase-mediated excision, the “excised locus” has a minimal sequence consisting primarily of a single loxP site surrounded by two Not I sites. N, Not I site. The double-stranded sequence at the asterisked region is given below the schematic. (B) PCR genotyping of male mice carrying the wt or ΔdblGATA mutant (hem) allele. PCR products before (–) and after (N) digestion with Not I.

Figure 2.

Peripheral blood from a wt male and a ΔdblGATA mutant male mouse. Wright-Giemsa staining of blood smears from a wild-type male (A) and hemizygous ΔdblGATA mutant (B) male. Red cells and platelets appear normal in the ΔdblGATA mutant male. Original magnification: 1,000×.

Figure 3.

Eosinophilia stimulated by an IL-5 transgene is not observed in ΔdblGATA mutant mice. (A) Schematic of cell populations in automated differential analysis of mouse blood. A heterozygous ΔdblGATA mutant female displays a normal-appearing differential plot (B). The IL-5 transgene imparts an increase in the number of eosinophils and large unclassified cells (C) in a male with a wt GATA-1 locus. This increase is not observed in a hemizygous ΔdblGATA mutant male harboring the same transgene (D). (E and F) Wright-Giemsa staining of bone marrow from mice harboring the IL-5 transgene. Numerous eosinophils with bright red granules are evident in a mouse with a wt GATA-1 locus (E) but not in a hemizygous ΔdblGATA mutant male (F). Original magnification: 600×.

Figure 3.

Eosinophilia stimulated by an IL-5 transgene is not observed in ΔdblGATA mutant mice. (A) Schematic of cell populations in automated differential analysis of mouse blood. A heterozygous ΔdblGATA mutant female displays a normal-appearing differential plot (B). The IL-5 transgene imparts an increase in the number of eosinophils and large unclassified cells (C) in a male with a wt GATA-1 locus. This increase is not observed in a hemizygous ΔdblGATA mutant male harboring the same transgene (D). (E and F) Wright-Giemsa staining of bone marrow from mice harboring the IL-5 transgene. Numerous eosinophils with bright red granules are evident in a mouse with a wt GATA-1 locus (E) but not in a hemizygous ΔdblGATA mutant male (F). Original magnification: 600×.

Figure 4.

RT-PCR analysis of gene expression in wt and ΔdblGATA mutant male mice. RT-PCR analysis was used to examine gene expression in the bone marrows of hemizygous ΔdblGATA mutant (n = 3) and wt (n = 3) male mice. Control reactions were performed with both wt and mutant samples with all primer pair combinations, either without RT or without cDNA template; in all these cases, no PCR product was generated (data not shown). EPOR, erythropoietin receptor; EPX, eosinophil peroxidase; GCSRFR, granulocyte colony stimulating factor receptor; GPIb, glycoprotein Ib; MBP, major basic protein; MPO, myeloperoxidase; vWf, von Willebrand factor.

Figure 5.

Production of mast cells from wt and ΔdblGATA mutant male mice. May-Grünwald-Giemsa (A and B) and toluidine blue stains (C and D) of cytospin preparations of bone marrow–derived mast cells, from wt (A and C) or ΔdblGATA mutant (B and D) male mice (original magnification: 1,000×). Toluidine blue-positive cells contain dark blue cytoplasmic granules. (E and F) Two-color FACS^® analysis of c-kit (x-axis) and high-affinity IgE receptor (y-axis) expression of bone marrow–derived mast cells from wt (E) or ΔdblGATA mutant (F) male mice. Mast cells are observed in both wt and mutant mice.