stripe

Egr-1 and Egr-2 regulation of miscellaneous genes

The early growth response-1 (EGR-1) protein is an anti-proliferative signal for certain tumor cells and is required for apoptosis induced by stimuli that elevate intracellular Ca2+. EGR-1 transactivates the promoter of the p53 gene and up-regulates p53 RNA and protein levels. Inhibition of p53 function with dominant-negative p53 mutants abrogates EGR-1-dependent apoptosis. These findings establish a direct functional link between EGR-1 and the p53-mediated cell death pathway and suggest that mutant forms of p53 in tumor cells may provide resistance to the anti-proliferative effects of EGR-1 (Nair, 1997).

A promoter within the proximal 250 base pairs upstream of the mouse Tenascin (See Drosophila Tenascin-major) gene contains several putative regulatory elements that are conserved among vertebrate genes. Four different DNA elements have been identified within this promoter, and they contribute in different ways to TN gene expression in cultured cells. These four elements are: a binding site for Krox proteins, one for nuclear factor1, an octamer motif that binds POU-homeodomain proteins, and a novel TN control element. The nuclear factor 1 and TN control element have positive effects on TN promoter activity and form similar DNA-protein complexes with nuclear extracts from three cell lines. The Krox element has a negative effect on TN promoter activity in one cell line and a positive effect in another. Two DNA binding complexes, one correlated with the negative and the other with the positive activities of the Krox element are found to contain the protein Krox24. The octamer motif is required for induction of TN promoter activity by the POU-homeodomain protein Brn2 (See Drosophila Drifter) in one cell line but is inactive in another. These results provide a striking example of the diversity of regulatory mechanisms that can be called forth by combining different promoter motifs with transcriptional activators or repressors (Copertino, 1997).

The expression of Id1 (Drosophila homolog: extramachrochaete), a helix-loop-helix protein that inhibits the activity of basic helix-loop-helix transcription factors, is down-regulated during cellular differentiation and cell cycle withdrawal both in tissue culture models and in mouse embryos. In order to study the mechanism of control of Idl expression, a 210-bp enhancer element was isolated from the upstream region of the Id1 gene. Activity of this element recapitulates Id1 expression in C2C12 muscle cells and C3H10T1/2 fibroblasts: i.e., this element is active in proliferating cells in the presence of serum and completely inactivated upon mitogen depletion, cell cycle withdrawal, and (in the case of C2C12) induced myoblast differentiation. Using linker-scanning mutations and site-directed mutagenesis in transient transfection experiments, two functional elements were identified within the 210-bp enhancer that are required for proper serum responsiveness. One element (A) contains a consensus Egr-1 binding site and additional flanking sequences required for optimal activity, and the other element (B) fits no known consensus. Gel shift experiments demonstrate that the protein complex binding to the A site contains Egr-1 and other proteins. This complex as well as a protein complex that binds to the B site is lost within 24 h of serum depletion, correlating with the down-regulation of Id1 expression. On the basis of these findings, it is propose that the regulation of the Id1 response to serum is mediated in part by the early response gene Egr-1 and as such provides a signaling link between the early-growth-response transcription factors and dominant-negative helix-loop-helix proteins (Tournay, 1997).

The intercellular adhesion molecule (ICAM) 1/CD54 plays an important role in T cell dependent B cell activation and in the function of B lymphocytes as antigen-presenting cells. ICAM-1 expression is upregulated as a consequence of B lymphocyte antigen receptor (BCR) signaling, thereby serving to render antigen-stimulated B cells more receptive to T cell-mediated costimulatory signals. Icam-1 is dependent on BCR-induced expression of the transcription factor EGR1. Icam-1 transcription, induced by BCR cross-linking or bypassing the BCR with phorbol ester, is absent in a B cell line in which the EGR1-encoding gene (egr-1) is methylated and not expressed. A potential EGR1-binding site is located at -701 bp upstream of the murine Icam-1 gene transcription start site and this binding site binds to murine EGR1. Mutation of the site significantly abrogates transcriptional induction by phorbol ester and anti-mu stimulation in primary B cells. A direct effect of EGR1 on the Icam-1 promoter is suggested by the ability of EGR1 expressed from a expression vector to transactivate the wild-type Icam-1 promoter, whereas mutation of the EGR1 mutation of the EGR1 binding motif at -701 bp markedly compromises this induction (Maltzman, 1997).

A number of pathophysiologically relevant genes, including platelet-derived growth factor B-chain (PDGF-B), are induced in the vasculature after acute mechanical injury. In rat aorta, the activated expression of these genes was preceded by a marked increase in the amount of the early-growth-response gene product Egr-1 at the endothelial wound edge. Egr-1 interacts with a novel element in the proximal PDGF-B promoter, as well as with consensus elements in the promoters of other genes induced by endothelial injury. This interaction is crucial for injury-induced PDGF-B promoter-dependent expression. Sp1, whose binding site in the PDGF-B promoter overlaps that of Egr-1, occupies this element in unstimulated cells and is displaced by increasing amounts of Egr-1. These findings implicate Egr-1 in the up-regulated expression of PDGF-B and other potent mediators in mechanically injured arterial endothelial cells (Khachigian, 1996).

Tissue factor (TF) is induced in THP-1 cells stimulated with lipopolysaccharide (LPS). DNase I footprinting identifies six sites of protein-DNA interaction between -383 and the cap site that varies between control and induced extracts. Four footprints show qualitative differences in nuclease sensitivity. Footprints I (-85 to -52) and V (-197 to -175) are induction-specific and localize to regions of the promoter that mediate serum, phorbol ester, partial LPS response (-111 to +14), and the major LPS-inducible element (-231 to -172). Electrophoretic mobility shift assays with the -231 to -172 probe demonstrate JunD and Fos binding in both control and induced nuclear extracts; however, binding of c-Jun is only detected following LPS stimulation. Antibody inhibition studies implicate binding of Ets-1 or Ets-2 to the consensus site between -192 and -177, a region that contains an induction-specific footprint. The proximal region (-85 to -52), containing the second inducible footprint, binds Egr-1 following induction. These data suggest that LPS stimulation of THP-1 cells activate binding of c-Jun, Ets, and Egr-1 to the TF promoter and implicates these factors in the transcriptional activation of TF mRNA synthesis (Groupp, 1997).

Adrenomedullary chromaffin cells express at least two subtypes of acetylcholine nicotinic receptors, which differ in their sensitivity to the snake toxin alpha-bungarotoxin. One subtype is involved in the activation step of the catecholamine secretion process and is not blocked by the toxin. The other is alpha-bungarotoxin-sensitive, and its functional role has not yet been defined. The alpha7 subunit is a component of this subtype. Autoradiography of bovine adrenal gland slices with alpha-bungarotoxin indicates that these receptors are restricted to medullary areas adjacent to the adrenal cortex and colocalize with the enzyme phenylethanolamine N-methyl transferase (PNMT), which confers the adrenergic phenotype to chromaffin cells. Transcripts corresponding to the alpha7 subunit also are localized exclusively to adrenergic cells. To identify possible transcriptional regulatory elements of the alpha7 subunit gene involved in the restricted expression of nicotinic receptors, its 5' flanking region was isolated and characterized, revealing putative binding sites for the immediate early gene transcription factor Egr-1, which is known to activate PNMT expression. In reporter gene transfection experiments, Egr-1 increases alpha7 promoter activity by up to sevenfold. Activation is abolished when the most promoter-proximal of the Egr-1 sites is mutated, whereas modification of a close upstream site produces a partial decrease of the Egr-1 response. Because Egr-1 is found to be expressed exclusively in adrenergic cells, it is suggested that this transcription factor may be part of a common mechanism involved in the induction of the adrenergic phenotype and the differential expression of alpha-bungarotoxin-sensitive nicotinic receptors in the adrenal gland (Criado, 1997).

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