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GATA-1 regulation of the globin locus

To test whether human GATA-1 (hGATA-1) is involved in the transcriptional control of globin gene switching, transgenic mice were produced overexpressing hGATA-1. These were crossed with mice carrying a human beta-globin locus yeast artificial chromosome (beta YAC), and globin gene expression was analyzed in their progeny. Mice carrying both the hGATA-1 and the beta YAC transgenes have normal levels of gamma- and beta-globin mRNA, with no distortion in the rate or in the timing of gamma-to-beta switch, indicating that hGATA-1 is not involved in the developmental control of gamma- and beta-globin genes. In contrast, mice carrying the hGATA-1 and the beta YAC transgenes have 5- to 6-fold lower expression of the human epsilon globin gene compared with beta YAC mice lacking the hGATA-1 transgene. These results provide direct in vivo evidence that hGATA-1 is a specific repressor of human epsilon gene expression. These findings also suggest that binary transgenic mouse systems based on overexpression of transcriptional factors can be used to investigate the trans control of human globin gene switching. Systems such as the one described here should be useful in the study of any developmentally controlled human gene for which transgenic mice are available (Li, 1997).

Erythroid Kruppel-like factor (EKLF) is a zinc finger transcription factor required for beta-globin gene expression and is implicated as one of the key factors necessary for the fetal to adult switch in globin gene expression. In an effort to identify factors involved in the expression of this important erythroid-specific regulatory protein, the mouse EKLF gene has been isolated and the promoter region has been systematically analyzed. Initially, a reporter construct with 1150 base pairs of the EKLF 5'-region was introduced into transgenic mice and shown to direct erythroid-specific expression. The expression studies in erythroid cells were continued and a sequence element was identified consisting of two GATA sites flanking an E box motif. The three sites act in concert to elevate the transcriptional activity of the EKLF promoter. Each site is essential for EKLF expression indicating that the three binding sites do not work additively, but rather function as a unit. GATA-1 binds to the two GATA sites and evidence suggests the binding of another factor from erythroid cell nuclear extracts to the E box motif. These results are consistent with the formation of a quaternary complex composed of an E box dimer and two GATA-1 proteins binding at a combined GATA-E box-GATA activator element in the distal EKLF promoter (Anderson, 1998).

The tissue-specific transcription factor GATA-1 is a key regulator of red blood cell differentiation. One seemingly contradictory aspect of GATA-1 function is that while it is abundant in erythroid progenitor cells prior to the onset of overt differentiation, it does not significantly activate known GATA-1 target genes in those cells. In primary progenitor cells, GATA-1 protein is predominantly located in the cytoplasm, while induction of differentiation causes its rapid relocalization to the nucleus, suggesting that nuclear translocation constitutes an important regulatory step in GATA-1 activation. An ectopically expressed GATA-1/estrogen receptor fusion protein (GATA-1/ER) in red blood cell progenitors accelerates red blood cell differentiation, and concomitantly suppresses cell proliferation. Estrogen conditionally controls the nuclear translocation of GATA-1/ER, as well as its transcriptional activation. These phenotypic effects are accompanied by a simultaneous suppression of c-myb and GATA-2 transcription, two genes thought to be involved in the proliferative capacity of hematopoietic progenitor cells. Thus, GATA-1 appears to promote differentiation in committed erythroid progenitor cells both by inducing differentiation-specific genes and by simultaneously suppressing genes involved in cell proliferation (Briegel, 1996).

Erythrocyte development depends on the expression of the lineage-restricted trans-acting factor GATA-1. Despite predicted roles for this factor during early development, GATA-1-deficient cells in chimeric mice and embryonic stem cell cultures mature to a late proerythroblast stage and express at least certain genes that normally are thought to be regulated by GATA-1 (including erythroid Kruppel-like factor [EKLF] and the erythropoietin [Epo] receptor). Opportunities to test roles for GATA-1 in erythroid gene activation in these systems therefore are limited. In the present study, in an alternate approach to test the function of GATA-1, GATA-1 has been expressed together with the Epo receptor in myeloid FDCW2 cells and the resulting effects on cytokine-dependent proliferation and erythroid gene expression have been assessed. GATA-1 expression at low levels delays FDCW2ER cell cycle progression at the G1 phase specifically during Epo-induced mitogenesis. Upon expression of GATA-1 at increased levels, proliferation in response to Epo, interleukin-3 (IL-3), and stem cell factor is attenuated and endogenous GATA-1, EKLF and betamaj-globin gene expression is activated. Friend of GATA-1 (FOG) transcript levels are also enhanced, and ets-1 and c-mpl (but not Epo receptor) gene expression is induced. Finally, in FDCW2 cells expressing increased levels of GATA-1 and a carboxyl-terminally truncated Epo receptor, Epo (with respect to IL-3 as a control) was shown to markedly promote globin transcript expression. Thus, novel evidence for select hierarchical roles for GATA-1 and Epo in erythroid lineage specification is provided (Seshasayee, 1998).

An unresolved question relating to the current understanding of erythroid cell-specific gene expression asks how a limited number of transcriptional factors cooperate to direct high-level expression, mediated by cis-regulatory elements, yet separated over large distances within globin loci. GATA-1, the major erythroid transcription factor, activates transcription in a synergistic fashion with two Kruppel family factors: the ubiquitous protein Sp1 (Drosophila homolog: Buttonhead) and the erythroid-restricted factor EKLF (erythroid Kruppel-like factor), both of which recognize GC and/or GT/CACC motifs. Binding sites for both GATA-1 and these Kruppel proteins (especially Sp1) are found in close association in the promoters and enhancers of numerous erythroid cell-expressed genes and appear to cooperate in directing their expression. GATA-1 interacts physically with Sp1 and EKLF; interactions are mediated through their respective DNA-binding domains. GATA-1 and Sp1 synergize from a distance in constructs designed to mimic the architecture of globin locus control regions and downstream globin promoters. The formation of GATA-1-SP1 complexes has been demonstrated in vivo by the ability of Sp1 to recruit GATA-1 to a promoter in the absence of GATA-binding sites. These experiments provide the first evidence for functionally important protein-protein interactions involved in erythroid cell-specific expression and suggest a mechanism by which DNA loops might be formed or stabilized between locus control regions and globin promoters and/or enhancers (Merika, 1995).

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serpent: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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