Egr-1 and Egr-2 corepressors

Previous work has identified a corepressor, NAB1, which represses transcriptional activation mediatedby NGFI-A (also known as Egr-1, zif268, and Krox24) and Krox20. These zinc finger transcriptionfactors are encoded by immediate-early genes and have been implicated in a wide variety ofproliferative and differentiative processes. Another corepressor,NAB2, is highly related to NAB1 within two discrete domains. The first conserved domain ofNAB2 mediates an interaction with the R1 domain of NGFI-A. NAB2 represses the activity of bothNGFI-A and Krox20; its expression is regulated by some of the same stimuli that induce NGFI-Aexpression, including serum stimulation of fibroblasts and nerve growth factor stimulation of PC12cells. The human NAB2 gene has been localized to chromosome 12ql3.3-14.1, a region that isrearranged in several solid tumors, lipomas, uterine leiomyomata, and liposarcomas. Sequencing of theC. elegans genome has identified a gene that bears high homology to both NAB1 andNAB2, suggesting that NAB molecules fulfill an evolutionarily conserved role (Svaren, 1996).

The two Nab genes, coding for transcriptional corepressors of NGFI-A (Egr-1, Krox24, zif268) andKrox20, have been localized to two regions of the genome, each of which contains at least twomembers of the Stat gene family. The association of the two Nab genes with the Stat clusters onmouse chromosomes 1 and 10 (human chromosomes 2 and 12) suggest that a Nab gene was involvedin at least one of the duplication events that resulted in dispersion of the primordial Stat gene pair tothree different mouse chromosomes. Sequencing of the Nab2 genomic locus reveals that it is situatedvery close to the Stat6 gene. The transcripts of the two genes converge, such that the 3' ends of theStat6 and Nab2 mRNAs overlap by 58 bp. Both transcripts terminate within a 78-bp region that isabsolutely conserved between mouse and human. Analysis of Nab2 cDNA reveals that there is analternatively spliced form of the Nab2 transcript (lacking exon 3) that produces a protein that lacks theability to repress transcription by NGFI-A and Krox20 (Svaren, 1997).

Nab proteins constitute an evolutionarily conserved family of corepressors that specifically interactwith and repress transcription mediated by three members (Egr-1, Krox24, zif/268) of the NGFI-A family of immediate-early gene transcription factors, which includes NGFI-C, Krox20, and Egr3. The mechanism of Nab1 repression was explored and structural domains were identified that are required for Nab1function. Nab1 does not act by blocking DNA binding or nuclear localization of NGFI-A. In fact, Nab1repression is not unique to NGFI-A because multiple types of non-NGFI-A activation domains arerepressed, as was a heterologous transcription factor carrying the NGFI-A R1 domain, which isrequired for Nab1 interaction. Additionally, Nab1 tethered directly to DNA represses constitutivelyactive promoters. Tethered repression is not dependent on the identity of the basal promoterelements, the presence of a distal enhancer, or the distance separating the binding sites from thepromoter. These results suggest that Nab1 repression is not specific to particular activators and thatNab1 is an active repressor that works by a direct mechanism. A bipartite-like nuclearlocalization sequence was identified and the repression function was localized to the Nab conserved domain 2 (NCD2), aregion found in the carboxy-terminal half of all Nab proteins. Three small regions of homology betweenNab1 and previously characterized corepressors, Dr1 and E1b 55-kDa protein, were identified withinNCD2. Replacement mutagenesis of residues conserved between these proteins interfers with Nab1repression, although Nab1 does not function by the same mechanism as Dr1. The human NAB1genomic locus was mapped to chromosome 2q32.3-33 (Swirnoff, 1998).

Egr-1 and Egr-2 and growth repression

The early growth response 1 (EGR-1) gene product is a transcription factor with roles in differentiationand growth. Expression of exogenous EGR-1 in various human tumorcells unexpectedly and markedly reduces growth and tumorigenicity and, conversely, suppressionof endogenous Egr-1 expression by antisense RNA eliminates protein expression, enhances growth,and promotes phenotypic transformation. However, the mechanism of these effects remainedunknown. The promoter of human transforming growth factor beta 1 (TGF-beta 1) contains twoGC-rich EGR-1 binding sites. Expression of EGR-1 in human HT-1080 fibrosarcomacells exhibits increased secretion of biologically active TGF-beta 1 in direct proportion to the amount of EGR-1 expressed and addition of recombinant human TGF-beta 1 is stronglygrowth-suppressive for these cells. Addition of monoclonal anti-TGF-beta 1 antibodies toEGR-1-expressing HT-1080 cells completely reverses the growth inhibitory effects of EGR-1.Reporter constructs bearing the EGR-1 binding segment of the TGF-beta 1 promoter are activated 4-to 6-fold relative to a control reporter in either HT-1080 cells that stably express or parental cellscotransfected with an EGR-1 expression vector. Expression of delta EGR-1, a mutant that cannotinteract with the corepressors (nerve growth factor-activated factor binding proteins NAB1 and NAB2)due to deletion of the repressor domain, exhibits enhanced transactivation of 2- to 3.5-fold over that ofwild-type EGR-1, showing that the reporter construct reflects the appropriate in vivo regulatorycontext. The EGR-1-stimulated transactivation is inhibited by expression of the Wilms tumorsuppressor, a known specific DNA-binding competitor. These results indicate that EGR-1 suppressesgrowth of human HT-1080 fibrosarcoma cells by induction of TGF-beta 1 (Liu, 1996).

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 andis 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 deathpathway and suggest that mutant forms of p53 in tumor cells may provide resistance to theanti-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 ofregulatory mechanisms that can be called forth by combining different promoter motifs withtranscriptional 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-helixtranscription factors, is down-regulated during cellular differentiation and cell cycle withdrawal both intissue culture models and in mouse embryos. In order to study the mechanism of control of Idlexpression, 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., thiselement is active in proliferating cells in the presence of serum and completely inactivated uponmitogen 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 properserum responsiveness. One element (A) contains a consensus Egr-1 binding site and additional flankingsequences required for optimal activity, and the other element (B) fits no known consensus. Gel shiftexperiments demonstrate that the protein complex binding to the A site contains Egr-1 and otherproteins. This complex as well as a protein complex that binds to the B site is lost within 24 h of serumdepletion, correlating with the down-regulation of Id1 expression. On the basis of these findings, it ispropose that the regulation of the Id1 response to serum is mediated in part by the early response geneEgr-1 and as such provides a signaling link between the early-growth-response transcription factorsand dominant-negative helix-loop-helix proteins (Tournay, 1997).

The intercellular adhesion molecule (ICAM) 1/CD54 plays an important role in T cell dependent B cellactivation and in the function of B lymphocytes as antigen-presenting cells. ICAM-1 expression isupregulated as a consequence of B lymphocyte antigen receptor (BCR) signaling, thereby serving torender 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 potentialEGR1-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 activatedexpression 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 novelelement in the proximal PDGF-B promoter, as well as with consensus elements in the promoters ofother genes induced by endothelial injury. This interaction is crucial for injury-induced PDGF-Bpromoter-dependent expression. Sp1, whose binding site in the PDGF-B promoter overlaps that ofEgr-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 inmechanically injured arterial endothelial cells (Khachigian, 1996).

Tissue factor (TF) is induced in THP-1cells stimulated with lipopolysaccharide (LPS). DNase I footprinting identifies six sites of protein-DNAinteraction between -383 and the cap site that varies between control and induced extracts. Fourfootprints 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 inboth control and induced nuclear extracts; however, binding of c-Jun is only detected following LPSstimulation. 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 LPSstimulation of THP-1 cells activate binding of c-Jun, Ets, and Egr-1 to the TF promoter and implicatesthese 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 theactivation step of the catecholamine secretion process and is not blocked by the toxin. The other isalpha-bungarotoxin-sensitive, and its functional role has not yet been defined. The alpha7 subunit is acomponent of this subtype. Autoradiography of bovine adrenal gland slices with alpha-bungarotoxinindicates that these receptors are restricted to medullary areas adjacent to the adrenal cortex andcolocalize with the enzyme phenylethanolamine N-methyl transferase (PNMT), which confers theadrenergic phenotype to chromaffin cells. Transcripts corresponding to the alpha7 subunit also arelocalized exclusively to adrenergic cells. To identify possible transcriptional regulatory elements of thealpha7 subunit gene involved in the restricted expression of nicotinic receptors, its 5' flanking region was isolated andcharacterized, revealing putative binding sites for the immediate early genetranscription factor Egr-1, which is known to activate PNMT expression. In reporter gene transfectionexperiments, Egr-1 increases alpha7 promoter activity by up to sevenfold. Activation is abolishedwhen the most promoter-proximal of the Egr-1 sites is mutated, whereas modification of a closeupstream site produces a partial decrease of the Egr-1 response. Because Egr-1 is found to beexpressed exclusively in adrenergic cells, it is suggested that this transcription factor may be part of acommon mechanism involved in the induction of the adrenergic phenotype and the differentialexpression of alpha-bungarotoxin-sensitive nicotinic receptors in the adrenal gland (Criado, 1997).

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