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The transcription factor GATA-1 is expressed in a subset of hemopoietic cells, where it mediates the cell-type specific expression of several genes. We have cloned the mouse and human GATA-1 genes. A region upstream to the first exon, and highly conserved between mouse and man, acts as an erythroid specific enhancer in transient assays, if linked to the GATA-1 or to the SV40 promoter. The activity of the enhancer is almost completely dependent on the integrity of a dimeric GATA-1 binding site.

The transcriptional binding protein NFE-1 (also called GF-1 and Ery-f1) is thought to play a necessary, but not sufficient, role in the regulation of differentiation-related gene expression in a subset of hematopoietic lineages (erythroid, megakaryocytic, and basophil-mast cell). In order to clarify the mechanism which underlies the lineage-specificity of the NFE-1 expression, as well as the relationship between the expression of this factor and growth factor responsiveness, we have evaluated the capacity of erythropoietin (Epo)-, granulomonocytic (GM)-colony stimulating factor (CSF)-, and granulocyte (G)-CSF-dependent subclones derived from the interleukin 3 (IL-3)-dependent cell line 32D, to express 1) NFE-1 mRNA, 2) NFE-1-related nuclear proteins, and 3) chloramphenicol acetyl transferase (CAT) activity when transfected with a CAT gene under the control of NFE-1 cognate sequences. NFE-1 mRNA was found to be expressed not only in cells with mast cell (IL-3-dependent 32D) and erythroid (Epo-dependent 32D Epo1) phenotypes, but also in cells with predominantly granulocyte/macrophage properties, such as the GM-CSF- (early myelomonocytic) and G-CSF- (myelocytic) dependent subclones of 32D. However, a gradient of expression, correlating with the lineage, the stage of differentiation, and the growth factor responsiveness of the cell lines, was found among the different subclones: Epo greater than or equal to IL-3 greater than GM-CSF greater than G-CSF. Binding experiments demonstrated NFE-1 activity in all cell lines except the G-CSF-dependent line. Function of the NFE-1 protein was assessed by the expression of the CAT gene linked to the SV40 promoter and a mutant (-175 T----C) HPFH gamma-globin promoter. High level CAT expression was seen only in the Epo1 cells although low level expression was also seen in the parent 32D. These results demonstrate that the specificity of the expression of NFE-1 for the erythroid--megakaryocytic--mast cell lineages is obtained by progressive inactivation of its expression in alternative lineages.

The -175 T greater than C mutation in the promoter of the A gamma- or G gamma-globin gene causes a 50-100 fold increase of the expression of the respective gene in adult erythroid cells (Hereditary Persistence of Fetal Hemoglobin). We show here that this mutation increases 3-9 fold the expression of a gamma-CAT reporter plasmid transfected into the erythroid cells K562, but not that of the same plasmid in non erythroid cells. The overexpression of the mutant is abolished by the mutation of the binding site for the erythroid specific factor NFE1; inactivation of the adjacent binding site for the ubiquitous factor OTF1 does not cause overexpression of the normal gamma-globin promoter. Previous results demonstrated that the -175 mutation slightly increases the in vitro binding of NFE1 and almost abolishes that of OTF1; the present functional data indicate that altered binding of NFE1, but not of OTF1, is responsible for the observed overexpression of the mutated promoter.

Using the electrophoretic mobility shift assay and the footprinting technique, we studied the binding of nuclear proteins from erythroid and non erythroid human cells to the promoter region of the human gamma-globin gene. Two regions (A and B) of the promoter are bound by proteins present in uninduced K562 cells, but not in induced K562 cells nor in fetal liver erythroblasts; a protein binding to region A is also present in a variety of lymphoid and myeloid cells. Region B is centered on an octamer sequence identical to that present in immunoglobulin promoter and enhancers and other eukaryotic promoters; a B region binding protein common to K562 and other cells efficiently binds the octamer containing region of the histone H2B gene, while different B region proteins are more specific for uninduced K562 cells and the gamma-globin octamer containing fragment. The possible role of these nuclear proteins in gamma-globin gene regulation and/or cell differentiation is discussed.

We have used the gel retardation and DNAase I assays to investigate the binding of nuclear proteins to the human beta-globin promoter. Upon incubation with beta-globin promoter fragments containing the duplicated CACCC boxes, nuclear proteins from human erythroid cells generate complexes yielding four retarded bands in acrylamide gels; the three slowest bands are common to both erythroid and non erythroid cells. The fast band is present only in K562 erythroleukemic cells induced to differentiation and hemoglobin accumulation and in fetal and adult erythroblasts, but absent in uninduced K562 cells. Binding occurs on a short DNA region including the proximal CACCC box, and is not significantly competed by excess gamma-globin fragments containing the CACCC box; the CACCC box appears to be essential for this binding, as shown by the failure of a fragment containing a natural beta-thalassemic mutation (-87, C----G) to bind significantly to nuclear factors. These data suggest that the erythroid specific CACCC binding factor might play a role in the developmental activation of beta-globin transcription.

The -198 T----C mutation in the promoter of the A gamma-globin gene increases 20-30 fold the expression of this gene in adult erythroid cells of patients (Hereditary Persistence of Fetal Hemoglobin, HPFH). We show here that this mutation creates a strong binding site, resembling a CACCC box, for two ubiquitous nuclear proteins, one of which is Sp1. The mutated promoter is four to five-fold more efficient than a normal gamma-globin promoter in driving expression of a CAT reporter plasmid when transfected into erythroid cells. The overexpression of the mutant is abolished by the introduction of an additional mutation disrupting the new binding site. No overexpression of the mutant is observed in non-erythroid cells, indicating that the ubiquitous factors bound on the mutated sequence must cooperate with erythroid specific factors.