A "knockdown" mutation created by cis-element gene targeting reveals the dependence of erythroid cell maturation on the level of transcription factor GATA-1

Abstract

The hematopoietic-restricted transcription factor GATA-1 is required for both mammalian erythroid cell and megakaryocyte differentiation. To define the mechanisms governing its transcriptional regulation, we replaced upstream sequences including a DNase I hypersensitive (HS) region with a neomycin-resistance cassette by homologous recombination in mouse embryonic stem cells and generated mice either harboring this mutation (neoDeltaHS) or lacking the selection cassette (DeltaneoDeltaHS). Studies of the consequences of these targeted mutations provide novel insights into GATA-1 function in erythroid cells. First, the neoDeltaHS mutation leads to a marked impairment in the rate or efficiency of erythroid cell maturation due to a modest (4- to 5-fold) decrease in GATA-1 expression. Hence, erythroid differentiation is dose-dependent with respect to GATA-1. Second, since expression of GATA-1 from the DeltaneoDeltaHS allele in erythroid cells is largely restored, transcription interference imposed by the introduced cassette must account for the "knockdown" effect of the mutation. Finally, despite the potency of the upstream sequences in conferring high-level, developmentally appropriate expression of transgenes in mice, other cis-regulatory elements within the GATA-1 compensate for its absence in erythroid cells. Our work illustrates the usefulness of targeted mutations to create knockdown mutations that may uncover important quantitative contributions of gene function not revealed by conventional knockouts.

Figure 1

Structures of wild-type, neoΔHS, and ΔneoΔHS GATA-1 alleles. The wild-type locus contains alternative first exons (IT and IE) and a DNase I HS region (10). Exons are indicated by solid boxes. In the neoΔHS locus a loxP-flanked neomycin-resistance cassette replaces ≈8 kb lying between a BamHI site (B) and an artificial XhoI site (〈Xh〉) present in a λ phage genomic clone used in the construction. Cre-mediated excision of the neomycin-resistance cassette leaves a single loxP site (indicated by the solid triangle) in the ΔneoΔHS locus.

Figure 2

Anemia and abnormal erythropoiesis in neoΔHS embryos. (A) Embryos at E13.5. (B) Peripheral blood smears at yolk sac (E11.5) and fetal liver (E13.5) stages. Note binucleate primitive erythrocytes, indicated by the arrows on the left and arrows with P on the right. Note the presence of immature definitive erythroid cells (arrows with D) at E13.5.

Figure 3

Impaired erythroid colony formation of neoΔHS fetal liver cells. CFU-e and BFU-e colonies were cultured as described. Note the small size and pallor of mutant CFU-e colonies (A and B) and the increased nuclear/cytoplasmic ratio of stained erythroid cells (C and D). neoΔHS BFU-e are larger in size and less red in appearance (E and F). BFU-e contain less mature and dysplastic erythroid precursors and abundant megakaryocytes (G and H).

Figure 4

Decreased GATA-1 RNA and protein levels in neoΔHS erythroid cells. (A and B) Appearance of representative definitive erythroid colonies and cells obtained by in vitro differentiation of ES cells. Colonies were harvested at day 5 after disaggregation of embryoid bodies. Note the pallor of mutant colonies and the predominance of immature erythroid cells. (C) Semiquantitative RT-PCR analysis of GATA-1 RNA transcripts. Assays were performed as described (9) with 28, 30, and 32 cycles of PCR (shown by triangles) and primer pairs specific for GATA-1 and hypoxanthine phosphoribosyltransferase (HPRT) (as an internal control). Relative band intensities were quantitated by PhosphorImager analysis. (D). Western blot analysis of fetal liver erythroid cell nuclear proteins. Wild-type female (lane 1), heterozygous neoΔHS female (lanes 2 and 3), and hemizygous neoΔHS males (lanes 4 and 5) are indicated.