ABSTRACT
GATA transcription factors are required for the differentiation of diverse cell types in several species. Recent evidence suggests that their biologic activities may be modulated through interaction with multitype zinc finger proteins, such as Friend of GATA-1 (FOG) and U-shaped (Ush). In cell culture, FOG cooperates with the hematopoietic transcription factor GATA-1 to promote erythroid and megakaryocytic differentiation. We show here that mice lacking FOG die during mid-embryonic development with severe anemia. FOG-/- erythroid cells display a marked, but partial, blockage of maturation, reminiscent of GATA-1- erythroid precursors. In contrast to GATA-1 deficiency, however, megakaryocytes fail to develop in the absence of FOG. Although the FOG-/- erythroid phenotype supports the proposed role of FOG as a GATA-1 cofactor in vivo, the latter finding points to a pivotal, GATA-1-independent requirement for FOG in megakaryocyte development from the bipotential erythroid/megakaryocytic progenitor. We speculate that FOG and other FOG-like proteins serve as complex cofactors that act through both GATA-dependent and GATA-independent mechanisms.
Subject(s)
Carrier Proteins/physiology , Erythropoiesis/physiology , Hematopoiesis/physiology , Megakaryocytes/physiology , Nuclear Proteins/physiology , Animals , Carrier Proteins/genetics , Cell Differentiation , Embryonic and Fetal Development , Erythroid Precursor Cells , Gene Deletion , Gene Targeting , Mice , Nuclear Proteins/genetics , Time Factors , Transcription FactorsABSTRACT
The hematopoietic transcription factor GATA-1 is essential for development of the erythroid and megakaryocytic lineages. Using the conserved zinc finger DNA-binding domain of GATA-1 in the yeast two-hybrid system, we have identified a novel, multitype zinc finger protein, Friend of GATA-1 (FOG), which binds GATA-1 but not a functionally inactive mutant lacking the amino (N) finger. FOG is coexpressed with GATA-1 during embryonic development and in erythroid and megakaryocytic cells. Furthermore, FOG and GATA-1 synergistically activate transcription from a hematopoietic-specific regulatory region and cooperate during both erythroid and megakaryocytic cell differentiation. These findings indicate that FOG acts as a cofactor for GATA-1 and provide a paradigm for the regulation of cell type-specific gene expression by GATA transcription factors.
Subject(s)
Carrier Proteins/biosynthesis , Cell Differentiation/physiology , DNA-Binding Proteins/biosynthesis , Erythroid Precursor Cells/cytology , Erythroid Precursor Cells/physiology , Gene Expression Regulation, Developmental , Megakaryocytes/cytology , Megakaryocytes/physiology , Nuclear Proteins/biosynthesis , Transcription Factors/biosynthesis , Zinc Fingers , 3T3 Cells , Amino Acid Sequence , Animals , Binding Sites , Carrier Proteins/chemistry , Cloning, Molecular , Conserved Sequence , DNA-Binding Proteins/chemistry , Embryo, Mammalian , Embryonic and Fetal Development , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Hematopoiesis , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Proteins/chemistry , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Saccharomyces cerevisiae , Transcription Factors/chemistry , Transcriptional Activation , TransfectionABSTRACT
Erythroid Kruppel-like factor (EKLF) is an erythroid-specific transcription factor that binds a CACCC motif found in the human beta-globin gene promoter. We have studied the promoter of the EKLF gene and identified binding sites for the transcription factors GATA-1 and CCAAT-binding Protein 1 (CP1). We show that both types of binding sites are required for full activity, and that the GATA motif at -60 is essential. The EKLF promoter can be directly activated in nonerythroid cells in cotransfection experiments by forced expression of GATA-1. These results suggest that EKLF is dependent on GATA-1 for its expression and lies downstream of, or coincident with, GATA-1 in a regulatory hierarchy in erythroid development.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Globins/genetics , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , 3T3 Cells , Animals , Base Sequence , Binding Sites , Cell Line , Cloning, Molecular , DNA Primers , DNA-Binding Proteins/biosynthesis , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Growth Hormone/genetics , Humans , Kruppel-Like Transcription Factors , Mice , Molecular Sequence Data , Restriction Mapping , Transcription Factors/biosynthesis , Transcription, Genetic , Transfection , Zinc Fingers/geneticsABSTRACT
Pretreatment of mammalian cell with DNA-damaging agents, such as UV light or mitomycin C, but not the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), results in the enhanced repair of subsequently transfected UV-damaged expression vectors. To determine the cellular factors that are responsible for this enhancement, we have used a modified gel retardation assay to detect the proteins that interact with damaged DNA. We have identified a constitutive DNA binding protein in extracts from primate cells that has a high affinity for UV-irradiated double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin, but not TPA or serum starvation, have higher levels of this damage-specific DNA binding (DDB) protein. These results suggest that the signal for induction of DDB protein can either be damage to the DNA or interference with cellular DNA replication. The induction of DDB protein varies among primate cells with different phenotypes: (1) virus-transformed repair-proficient cells have partially or fully lost the ability to induce DDB protein above constitutive levels; (2) primary cells from repair-deficient xeroderma pigmentosum (XP) group C, and transformed XP groups A and D, show constitutive DDB protein, but do not show induced levels of this protein 48 h after UV; and (3) primary and transformed repair-deficient cells from one XP E patient are lacking both the constitutive and the induced DDB activity. The correlation between the induction of the DDB protein and the enhanced repair of UV-damaged expression vectors implies the involvement of the DDB protein in this inducible cellular response.
