GABP Alpha Research Articles

GABP Transcription Factor Is Required for B Lymphocyte Maturation and Plasma Cell Development

B lymphocytes develop in the bone marrow and later encounter antigen in lymph nodes, where they complete their development as plasma cells or B memory cells. Several key transcription factors have been identified that are required for B cell development, including Pax5, BCL6, C-MYC, and others. GABP is a tetrameric ets transcription factor that includes the DNA-binding GABP alpha protein, and the unrelated protein, GABP beta, which contains multimerization and transcriptional activation domains. GABP plays key roles in cell cycle control and mitochondrial biogenesis. It is also required for lineage specific gene expression, and it was previously shown to control gene expression of the IL-7 receptor and Pax5, both of which are required for lymphocyte development. Disruption of mouse Gabpα caused cell cycle arrest in hematopoietic stem cells (HSC), profound loss of progenitor cells, and aberrant myeloid differentiation. We created a conditional knockout model of Gabpα in B lymphocytes by breeding mice with lox-P flanked Gabpa to mice that bear Cre recombinase knocked into the B-cell specific CD19 locus; the mice also carry the Rosa 26 lox-STOP-lox YFP transgene, which permits identification and isolation of individual Gabpα null cells, based on expression of YFP. Loss of Gabpα was highly lineage specific for B lymphocytes. Gabpa null mice were healthy and vigorous through young adulthood, but some developed rectal prolapse by nine months of age, and necropsy demonstrated thinning of the intestinal wall and loss of Peyer's Patches and other lymphoid tissue. We immunologically characterized mice between 6 and 8 weeks of age, in order to minimize secondary effects of the inflammatory process associated with rectal prolapse. There was no deletion of Gabpα in T lymphocytes, and no discernable effect on T-cell subpopulations. We observed a significant reduction in Gabpα null (YFP+) B cells, in comparison with the Gabpα replete (YFP-) B cells in bone marrow and spleen. Gabpα null cells contributed to the pro-B cell population, but there was a progressively reduced contribution of Gabpα null cells to later stages of B cell maturation. We detected no Gabpα null cells among mature naive IgD+/IgM+ B cells, indicating a profound block in B cell maturation in cells that lack Gabpα. Importantly, no YFP+ CD138+ cells were detected, indicating that Gabpα null cells could not contribute to plasma cell development. We conclude that Gabp is required for full B cell maturation and plasma cell development in mice, and that its deletion is associated with loss of Peyer's Patches and rectal prolapse. GABP was previously shown to regulate expression of IL-7R and Pax5, which are expressed in lymphoid progenitor cells long before activation of CD19 expression. Thus, failure of B cell development and plasma cell formation in this CD19-Cre Gabpα null model is independent of the effect of GABP on those other B cell factors, and indicates a new, critical role for GABP in later stages of B cell and plasma cell development. Although rectal prolapse has been observed in mice with T cell defects, this represents the first demonstration that B cell defects cause such a phenotype. DisclosuresGerstein:Vertex Pharmaceuticals: Other: employer of spouse.

Characterization of a negative transcriptional element in the BRCA1 promoter

IntroductionDecreased transcription of the BRCA1 gene has previously been observed to occur in sporadic breast tumours, making elucidation of the mechanisms regulating the expression of this gene important for our understanding of the etiology of the disease.MethodsTranscriptional elements involved in the regulation of the BRCA1 promoter were analysed by co-transfection experiments into the human MCF-7 and T-47D breast cancer cell lines.ResultsWe have identified a repressor element, referred to as the UP site, within the proximal BRCA1 promoter whose inactivation results in increased promoter activity. An E2F recognition element, previously suggested to mediate repression via E2F-6, is adjacent to the UP site and its inactivation also leads to increased BRCA1 expression. These two elements appear to form a composite repressor element whose combined effect is additive. The UP element is composed of two sequences, one of which binds the ubiquitously expressed ets family transcription factor GABP alpha/beta. This site is distinct from a previously identified GABP alpha/beta site, the RIBS element, though the RIBS site appears to be necessary for derepression of the promoter via mutations in the UP site. Knockdown of GABP alpha using an shRNA vector confirms that this protein is important for the function of both the RIBS and UP sites.ConclusionThe identification of a repressor element in the BRCA1 promoter brings a new level of complexity to the regulation of BRCA1 expression. The elements characterized here may play a normal role in the integration of a variety of signals, including two different growth related pathways, and it is possible that loss of the ability to derepress the BRCA1 promoter during critical periods may contribute to breast transformation.

GA Binding Protein (GABP) Is Required for Development and Maturation of Myeloid Cells.

GABP is an ets transcription factor that regulates transcription of key myeloid genes, including CD18 (beta2 leukocyte integrin), neutrophil elastase, lysozyme, and other key mediators of the inflammatory response; it is also known to regulate important cell cycle control genes. GABP consists of two distinct and unrelated proteins that, together, form a functional transcription factor complex. GABPalpha (GABPa) is an ets protein that binds to DNA; it forms a tetrameric complex by recruiting its partner, GABPbeta (GABPb), which contains the transactivation domain. GABPa is a single copy gene in both the human and murine genomes and it is the only protein that can recruit GABPb to DNA. We cloned GABPa from a murine genomic BAC library and prepared a targeting vector in which exon 9 (which encodes the GABPa ets domain) was flanked by loxP (floxed) recombination sites. The targeting construct was electroporated into embryonic stem cells, homologous recombinants were implanted into pseudopregnant mice, heterozygous floxed GABPa mice were identified, and intercrossing yielded expected Mendelian ratios of wild type, heterozygous, and homozygous floxed GABPa mice. Breeding of heterozygous floxed GABPa mice to CMV-Cre mice (which express Cre recombinase in all tissues) yielded expected numbers of hemizygous mice (only one intact GABPa allele), but no nullizygous (GABPa−/−) mice among 64 pups; we conclude that homozygous deletion of GABPa causes an embryonic lethal defect. To determine the effect of GABPa deletion on myeloid cell development, we bred heterozygous and homozygous floxed mice to LysMCre mice, which express Cre only in myeloid cells. These mice had a normal complement of myeloid cells but, unexpectedly, PCR indicated that their Gr1+ myeloid cells retained an intact (undeleted) floxed GABPa allele. We detected similar numbers of in vitro myeloid colonies from bone marrow of wild type, heterozygous floxed, and homozygous floxed progeny of LysMCre matings. However, PCR of twenty individual in vitro colonies from homozygous floxed mice indicated that they all retained an intact floxed allele. Breeding of floxed GABPa/LysMCre mice with hemizygous mice indicated that retention of a floxed allele was not due to incomplete deletion by LysMCre; rather, it appears that only myeloid cells that retain an intact GABPa allele can survive to mature in vitro or in vivo. We prepared murine embryonic fibroblasts from homozygous floxed mice and efficiently deleted GABPa in vitro. We found striking abnormalities in proliferation and G1/S phase arrest. We used quantitative RT-PCR to identify mechanisms that account for the altered growth of GABPa null cells. We found dramatically reduced expression of known GABP target genes that regulate DNA synthesis and cell cycle that appear to account for the proliferative defect. We conclude that GABPa is required for growth and maturation of myeloid cells and we identified downstream targets that may account for their failure to proliferate and mature in vitro and in vivo.

Identification of a GABP alpha/beta binding site involved in the induction of oxytocin receptor gene expression in human breast cells, potentiation by c-Fos/c-Jun.

Oxytocin (OT) receptors (OTRs) mediate reproductive functions, including the initiation of labor and milk ejection. OTR messenger RNA levels are highly regulated, reaching the greatest concentration in the uterus at the end of gestation, and in the mammary gland during lactation. Factors directly effecting changes in OTR gene expression in the mammary gland are not known, so the present studies were done to elucidate possible regulators by characterizing the human OTR gene promoter and 5'-flanking sequence. By analyzing expression of promoter-luciferase constructs, we localized a region between -85 and -65 that was required for both basal and serum-induced expression in a mammary tumor cell line (Hs578T) that expresses inducible, endogenous OTRs. This DNA region contains an ets family target sequence (5'-GGA-3'), and a CRE/AP-1-like motif. The specific Ets factor binding to the OTR promoter was identified, by electrophoretic mobility immunoshift assays, to be GABP alpha/beta. Co-transfection of a -85 OTR/luciferase construct with vectors expressing GABP alpha and GABP beta1 had only a modest effect on expression, but cotransfection with GABP alpha/beta- with c-Fos/c-Jun-expressing plasmids resulted in an increase of almost 10-fold in luciferase activity. Mutation of either the GABP- or CRE-like binding sites obliterated the induction. These findings are consistent with the involvement of protein kinase C activity in serum induction of the endogenous gene in Hs578T cells. We showed the requirement for GABP alpha/beta and c-Fos/c-Jun in endogenous OTR gene expression, using oligonucleotide GABP and AP-1 binding decoys to inhibit serum-induced increases in 125I-labeled OT antagonist binding to Hs578T cells. Our work is the first characterization of the proximal promoter region of the human OTR gene, and it sets the stage for studying regulation of OTR expression in breast cells.

Elevated expression of ETS-1 gene in a metastatic, tumorigenic human prostate epithelial cell line transformed by the v-Ki-ras oncogene

A suitable in vitro model system to investigate mechanisms of human prostate carcinogenesis is much needed. We have previously demonstrated that an immortal, but non-tumorigenic, human prostate epithelial cell line (267B(1)) can be malignantly transformed by the v-Ki-ras oncogene, and it can serve as a useful model for investigation of the progression steps of prostate carcinogenesis. In this study, we report for the first time the invasive/metastatic phenotype of the v-Ki-ras transformed 267B, cells (267B(1)/Ki-ras). In addition, comparing non-tumorigenic 267B, and metastatic tumorigenic 267B(1)/Ki-ras human prostate epithelial cell lines, we have found that expression of ETS-1 and ERGB mRNA was elevated to 2-5 fold in the metastatic and tumorigenic 267B(1)/Ki-ras cell line. A specific ETS-1 monoclonal antibody E44 also revealed that the expression of ETS-1 protein level in 267B(1)/Ki-ras cell line was higher than those in 267B, cell line. However, other members of the ETS gene family such as ETS-2, GABP alpha and their mRNA expression levels were similar in both cell lines. The activation of MAP kinase, a downstream target for Ki-ras, was also shown. The expression of urokinase plasminogen activator (u-PA) was also increased in the metastatic 267B(1)/Ki-ras line. An obvious capability of invasion was observed in the 267B(1)/Ki-ras cell line, but not in the 267B(1) line using BioCoat Matrigel invasion chamber assay system. The present study has provided evidence that the v-Ki-ras oncogene may activate the nuclear target gene, ETS-1 gene, to mediate tumorigenic and metastatic capacity of the v-Ki-ras transformed prostate epithelial cells.

GABP factors bind to a distal interleukin 2 (IL-2) enhancer and contribute to c-Raf-mediated increase in IL-2 induction.

Triggering of the T-cell receptor-CD3 complex activates two major signal cascades in T lymphocytes, (i) Ca2+-dependent signal cascades and (ii) protein kinase cascades. Both signal cascades contribute to the induction of the interleukin 2 (IL-2) gene during T-cell activation. Prominent protein kinase cascades are those that activate mitogen-activated protein (MAP) kinases. We show here that c-Raf, which is at the helm of the classic MAP-Erk cascade, contributes to IL-2 induction through a distal enhancer element spanning the nucleotides from positions -502 to -413 in front of the transcriptional start site of the IL-2 gene. Induction of this distal IL-2 enhancer differs from induction of the proximal IL-2 promoter-enhancer, since it is induced by phorbol esters alone and independent from Ca2+ signals. In DNA-protein binding studies, we detected the binding of transcription factors GABP alpha and -beta to a dyad symmetry element (DSE) of the distal enhancer, which is formed by palindromic binding sites of Ets-like factors. Introduction of point mutations suppressing GABP binding to the DSE interfered with the induction of the distal enhancer and the entire IL-2 promoter-enhancer, while overexpression of both GABP factors enhanced the IL-2 promoter-enhancer induction. Overexpression of BXB, a constitutive active version of c-Raf, and of further members of the Ras-Raf-Erk signal cascade exerted an increase of GABP-mediated promoter-enhancer induction. In conjunction with previously published data on c-Raf-induced phosphorylation of GABP factors (E. Flory, A. Hoffmeyer, U. Smola, U. R. Rapp, and J. T. Bruder, J. Virol. 70:2260-2268, 1996), these results indicate a contribution of GABP factors to the Raf-mediated enhancement of IL-2 induction during T-cell activation.

GA-binding protein is involved in altered expression of ribosomal protein L32 gene

Differentiation of BC3H1 myoblasts to myocytes is accompanied by a 67% drop in the rate of rpL32 gene transcription. Addition of high concentrations of serum to resting myocyte populations stimulates cell growth and subsequent dedifferentiation to proliferating myoblasts with a return to the normal rate of rpL32 gene transcription. During these growth rate changes the binding activities of previously identified factors (beta, gamma, delta) which interact with the rpL32 gene promoter were examined by mobility shift assays. Binding of the beta factor (an Ets related protein) to an oligonucleotide containing the beta element was reduced significantly in myocyte nuclear extracts, but subsequent dedifferentiation increased binding within 30 min in either the presence or absence of the cycloheximide. Binding of the gamma and delta factors to their respective elements changed only slightly during these processes. Dephosphorylation of either myoblast or myocyte extracts resulted in increased binding of the beta factor suggesting that binding activity of the beta factor is modulated by phosphorylation during the changes in BC3H1 myoblasts growth rate. In addition, mobility shift assays with recombinant GABP alpha and beta proteins and their specific antibodies revealed that GABP proteins bind to the rpL32 gene promoter in a sequence dependent manner, and that similar proteins are present in BC3H1 myoblast/myocyte extracts. These results support the premise that the GABP heterodimer is the rpL32 beta factor. Furthermore, during BC3H1 myoblast differentiation and dedifferentiation neither the levels of the GABP alpha and beta proteins nor their respective mRNAs change. These results suggest that GABP is a constitutively expressed protein and is involved in regulating rpL32 gene by post-transcriptional modifications.

A domain of TEL conserved in a subset of ETS proteins defines a specific oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein.

TEL is a novel member of the ETS family of transcriptional regulators which is frequently involved in human leukemias as the result of specific chromosomal translocations. We show here by co-immunoprecipitation and GST chromatography analyses that TEL and TEL-derived fusion proteins form homotypic oligomers in vitro and in vivo. Deletion mutagenesis identifies the TEL oligomerization domain as a 65 amino acid region which is conserved in a subset of the ETS proteins including ETS-1, ETS-2, FLI-1, ERG-2 and GABP alpha in vertebrates and PNTP2, YAN and ELG in Drosophila. TEL-induced oligomerization is shown to be essential for the constitutive activation of the protein kinase activity and mitogenic properties of TEL-platelet derived growth factor receptor beta (PDGFR beta), a fusion oncoprotein characteristic of the leukemic cells of chronic myelomonocytic leukemia harboring a t(5;12) chromosomal translocation. Swapping experiments in which the TEL oligomerization domain was exchanged by the homologous domains of representative vertebrate ETS proteins including ETS-1, ERG-2 and GABP alpha show that oligomerization is a specific property of the TEL amino-terminal conserved domain. These results indicate that the amino-terminal domain conserved in a subset of the ETS proteins has evolved to generate a specialized protein-protein interaction interface which is likely to be an important determinant of their specificity as transcriptional regulators.

GABP Mediates Insulin-increased Prolactin Gene Transcription

The insulin-response element from the prolactin gene is identical to the Ets-binding site, and dominant-negative Ets protein inhibits insulin-increased prolactin gene expression. Immunoblotting identified the Ets-related transcription factor GABP in nuclear extracts from GH cells. Expression of GABP alpha and GABP beta 1 squelches insulin-increased prolactin gene expression. GABP alpha and GABP beta 1 bind the insulin-response element of the prolactin promoter, and anti-GABP alpha and anti-GABP beta 1 antibodies supershift a species seen with nuclear extracts from GH cells. GABP alpha immunoprecipitated from insulin-treated, 32P-labeled GH cells was phosphorylated 3-fold more than GABP alpha from control cells. There was no increase in phosphorylation of GABP beta in response to insulin. Mitogen-activated protein (MAP) kinase activity is increased 10-fold in insulin-treated GH4 cells. MAP kinase immunoprecipitated from control cells does not phosphorylate GABP alpha while MAP kinase immunoprecipitated from insulin-treated cells shows substantial phosphorylation of GABP alpha. These studies suggest that GABP mediates insulin-increased transcription of the prolactin gene. GABP may be regulated by MAP kinase phosphorylation.

Raf-1 kinase targets GA-binding protein in transcriptional regulation of the human immunodeficiency virus type 1 promoter.

The serine/threonine protein kinase Raf-1 is a component of a conserved intracellular signaling cascade that controls responses to various extracellular stimuli. Transcription from several promoters, including the oncogene-responsive element in the polyomavirus enhancer, the c-fos promoter, as well as other AP-1- and Ets-dependent promoters, can be induced by Raf-1 kinase. Previously, we have shown that activated Raf-1 kinase transactivates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat and have identified the NF-kappaB binding motif as a Raf-1-responsive element (RafRE). We now report that Raf-1 kinase-induced transactivation from the HIV RafRE involves the purine-rich-repeat-binding protein (GABP), which is composed of two distinct subunits (alpha and beta). GABP alpha is an Ets oncogene-related DNA-binding protein, and GABP beta contains four ankyrin-like repeats that have been shown to be essential in protein-protein interactions. In electrophoretic mobility shift assays using nuclear extracts from human Jurkat T cells, a protein-DNA complex which was supershifted with antiserum against GABP alpha and GABP beta was observed. Purified recombinant GABP alpha and beta interact with the HIV RafRE as judged from DNA binding assays. Cotransfection experiments with GABP alpha and beta and Raf-1 kinase demonstrate synergistic transactivation of the HIV-1 promoter. Point mutations in the HIV RafRE abolished the Raf-1 kinase as well as GABP alpha- and beta-induced transactivation. The observed Raf-1-GABP synergism presumably involves phosphorylation of GABP subunits, as treatment of cells with Raf-1 kinase activators serum and 12-O-tetradecanoylphorbol-13-acetate increases phosphorylation of GABP in vivo. However, GABP is not a target of Raf-1 kinase; instead, it is a substrate of mitogen-activated protein kinase (MAPK/ERK), since in vitro phosphorylation of GABP alpha and beta was achieved by the reconstituted protein kinase cascade but not with purified Raf-1 or MEK. These results suggest that Raf-1 kinase- induced activation of the HIV-1 promoter is mediated by the classical cytoplasmic cascade resulting in MAPK/ERK-mediated phosphorylation of GABP alpha and beta. Because the HIV RafRE corresponds to a region within the promoter which is essential for regulation of HIV-1 expression, the data indicate that in addition to NK-kappaB, GABP transcription factors are important for induced expression of HIV.

A DNA Methylation Site in the Male-Specific P450 (Cyp 2d-9) Promoter and Binding of the Heteromeric Transcription Factor GABP

The Cyp 2d-9 gene encodes the male-specific steroid 16 alpha-hydroxylase in mouse liver and shares a conserved regulatory element (-100TTCCGGGC-93) with another male-specific Slp promoter. As shown with the Slp promoter (N. Yokomori, R. Moore, and M. Negishi, Proc. Natl. Acad. Sci. USA 92:1302-1306, 1995), the male-preferential demethylation also occurs at CpG/-97 in the Cyp 2d-9 promoter. The transcription factor which specifically binds to the demethylated element has been purified. The peptide sequences reveal that the factor consists of GABP alpha and GABP beta 1 with Ets and Notch motifs, respectively. Both DNase I footprinting and gel shift assays indicate that the bacterially expressed glutathione S-transferase-GABP fusion proteins bind to the regulatory element only when CpG/-97 is demethylated. Moreover, Cyp 2d-9 promoter is trans-activated by coexpression of GABP proteins in HepG2 cells. Given the additional results that CpG/-50 of the female-specific steroid 15 alpha-hydroxylase (Cyp 2a-4) promoter is preferentially demethylated in the females, the sex-specific expressions of the P450 genes correlate very well with DNA demethylation. We also conclude that GABP is a methylation-sensitive transcription factor and is a potential transcription activator of the male-specific Cyp 2d-9 promoter.

Molecular and genetic characterization of GABP beta.

This report outlines three observations relating to GABP beta, a polypeptide constituent of the heterotetrameric transcription factor GABP. Evidence is presented showing that the mouse genome encodes two highly related GABP beta polypeptides, designated GABP beta 1-1 and GABP beta 2-1. Genomic and cDNA copies of the newly defined Gabpb2 gene were cloned and characterized, providing the conceptually translated amino acid sequence of GABP beta 2-1. The genes encoding these two proteins, as well as GABP alpha, were mapped to three unlinked chromosomal loci. Although physically unlinked, the patterns of expression of the three genes were strikingly concordant. Finally, the molecular basis of GABP beta dimerization was resolved. Carboxy-terminal regions of the two GABP beta polypeptides, which mediate dimerization, bear highly related primary amino acid sequences. Both sequences are free of alpha-helix destabilizing residues and, when displayed on idealized alpha-helical projections, reveal marked amphipathy. Two observations indicate that these regions adopt an alpha-helical conformation and intertwine as coiled-coils. First, the dimer-forming region of GABP beta 2-1 can functionally replace the leucine zipper of a bZIP transcription factor. Second, a synthetic peptide corresponding to this region shows distinctive helical properties when examined by circular dichroism spectroscopy. Finally, evidence is presented showing that GABP beta 1-1 and GABP beta 2-1 can heterodimerize through this carboxy-terminal domain, but neither protein can heterodimerize via the dimer-forming region of the bZIP protein C/EBP beta.

Binding of an ETS-related protein within the DNase I hypersensitive site of the HER2/neu promoter in human breast cancer cells.

Promoter elements accounting for HER2 (c-erbB-2/neu) overexpression were searched for in several human breast cancer cell lines (MDA-453, BT-474, ZR-75-1, MCF-7) known to express constitutively a 30-fold range in HER2 transcripts per gene copy. HER2 overexpressing cells showed a single prominent DNase I hypersensitive site near a conserved and hitherto unrecognized ets response element (GAGGAA), located 38 bases down-stream from the CAAT box and directly 5' of the TATA box in the human HER2 promoter. Transient transfection of HER2 promoter constructs (0.125, 0.5, and 2.0 kilobase pairs (kb)) demonstrated that the most proximal promoter region (0.125 kb) was capable of conferring up to 30-fold enhanced activity in HER2-overexpressing cell lines relative to low HER2-expressing control lines. Site-directed mutagenesis of the ets response element (GAGGAA-->GAGAGA) caused a > or = 60% reduction in promoter activity affecting at least 0.5 kb of upstream HER2 regulatory sequence. Gel-shift assays with nuclear extracts and oligonucleotide sequences spanning the 0.125-kb promoter region detected an ETS-immunoreactive complex, present most abundantly in cells overexpressing HER2, whose high-affinity binding depended on the GAGGAA response element. Methylation interference confirmed the ETS-specific pattern of protein binding by this complex to guanine bases in the ets response element. UV cross-linking and immunoprecipitation implicate a approximately 60-kDa ETS protein, and candidate ETS genes expressed in these breast cancer cells include GABP alpha, elk-1, elf-1, and PEA3.

An intricate arrangement of binding sites for the Ets family of transcription factors regulates activity of the alpha 4 integrin gene promoter.

alpha 4 integrins mediate cell-cell and cell-extracellular matrix interactions that are critical for maturation and function of the immune system as well as differentiation of skeletal muscle. Here we examine molecular mechanisms controlling the pattern of alpha 4 expression. The activity of constructs containing 5' deletion mutants of the alpha 4 gene promoter was compared in transfection assays into cell lines that express alpha 4 and cell lines that do not. The sequence between position -42 and -76 base pairs (bp) was required for efficient transcription in cells that express alpha 4, but it showed no activity in HeLa cells, which do not express alpha 4. Three binding sites for the Ets family of transcription factors are found in this region: two adjacent sites at positions -50 and -54 bp and a more 5' site at position -67 bp. Using a series of constructs containing deletions and mutations in this region, we found that the 3'-most site alone was sufficient for binding GA-binding protein alpha (GABP alpha)/GABP beta and for a low level of transcriptional activation. When all three sites were present, a second complex "a" was detected, which contains an unknown member of the Ets family. Formation of complex a was cell-type specific and correlated with a high level of transcription. Deletion of the 5'-most Ets site had no effect on binding to GABP alpha/GABP beta, but it eliminated a. Concomitant with this loss of a, a new Ets-1-containing complex "c" appeared. Complex c substituted efficiently for complex a in transcriptional activation. We conclude that although neither of the two 5'-most Ets sites alone binds nuclear protein, they appear to act as modulators which control the pattern of Ets proteins that bind the alpha 4 gene promoter. This arrangement of Ets sites, coupled with the tissue- and developmental-specific expression of Ets members, likely play a key role in defining the pattern of alpha 4 integrin.

Characterization of the human aldose reductase gene promoter

The promoter region of the human aldose reductase gene has been identified upstream of the translation start ATG codon. The promoter contains a TATA box, a CCAAT promoter element, and three Sp1 protein binding consensus sequences upstream of the capsite. A 640-base pair insert spanning +31 to -609 directs expression of the reporter gene chloramphenicol acetyltransferase in an orientation-specific manner in transfected Hep G2 cells. The promoter activity remained constant with deletions from base pairs -609 to -186. The TATA and the CCAAT consensus sequences show significant promoter activity, whereas the three Sp1 binding consensus sequences, individually, have no significant promoter activity. A GA-rich region (-186 to -146) contains two CGGAAA/G motifs, which show promoter activity and interaction with Hep G2 nuclear extract and GA-binding proteins (GABP alpha and GABP beta 1) as shown by mobility shift assays and DNase I footprinting. Similar cis-elements in herpes simplex virus type 1 interact with rat liver GABP and the viral VP16 protein to mediate the induction of immediate early viral genes. A GC-rich region (-87 to -31) is identified by mobility shift assay, and a consensus sequence of an androgen response element is present at -396 to -382. The human aldose reductase promoter, thus, has regulatory response elements that may be important during early development and puberty. These regulatory elements may play a significant role in the development of certain diabetic complications.

Specificities of protein-protein and protein-DNA interaction of GABP alpha and two newly defined ets-related proteins.

The ets-related protein GABP alpha interacts with the four ankyrin-type (ANK) repeats of GABP beta to form a high-affinity DNA-binding complex that recognizes a site important for herpes simplex virus type I immediate early gene activation. To investigate the selectivity and specificity of the GABP complex, we have isolated two new ETS family members, termed ER81 and ER71. ER81 and GABP alpha were present in most tissues of adult mice, whereas ER71 was restricted to testis. We have compared the DNA-binding specificities of these proteins by binding site selection. GABP alpha, ER71, and ER81 recognized the common pentanucleotide DNA sequence 5'-CGGAA/T-3'. Although subtle differences were observed for nucleotide preferences flanking this pentanucleotide core, the overall similarity of the selected sequences was most striking. Given the observation that GABP alpha interaction with GABP beta requires its intact ETS domain, we further compared the ability of GABP beta to interact with other ETS proteins. GABP beta did not augment the DNA-binding activity of the highly similar ETS domains of ER81, ER71, or Ets-1. Moreover, probing of total tissue extracts with radiolabeled GABP beta demonstrated its exceedingly stringent specificity for GABP alpha. Given that the DNA-binding specificities of these ETS proteins are similar and that the protein-protein interactions between GABP beta and GABP alpha are highly specific, we conclude that the protein interactions determine the target site selection by GABP alpha.

Evolutionarily conserved Ets family members display distinct DNA binding specificities.

Members of the Ets family of proto-oncogenes encode sequence-specific transcription factors that bind to a purine-rich motif centered around a conserved GGA trinucleotide. Ets binding sites have been identified in the transcriptional regulatory regions of multiple T cell genes including the T cell receptor alpha and beta (TCR-alpha and -beta) enhancers and the IL-2 enhancer, as well as in the enhancers of several T cell-trophic viruses including Maloney sarcoma virus, human leukemia virus type 1, and human immunodeficiency virus-2. T cells express multiple members of the Ets gene family including Ets-1, Ets-2, GABP alpha, Elf-1, and Fli-1. The different patterns of expression and protein-protein interactions of these different Ets family members undoubtedly contribute to their ability to specifically regulate distinct sets of T cell genes. However, previous studies have suggested that different Ets family members might also display distinct DNA binding specificities. In this report, we have examined the DNA binding characteristics of two Ets family members, Ets-1 and Elf-1, that are highly expressed in T cells. The results demonstrate that the minimal DNA binding domain of these proteins consists of adjacent basic and putative alpha-helical regions that are conserved in all of the known Ets family members. Both regions are required for DNA binding activity. In vitro binding studies demonstrated that Ets-1 and Elf-1 display distinct DNA binding specificities, and, thereby interact preferentially with different naturally occurring Ets binding sites. A comparison of known Ets binding sites identified three nucleotides at the 3' end of these sequences that control the differential binding of the Ets-1 and Elf-1 proteins. These results are consistent with a model in which different Ets family members regulate the expression of different T cell genes by binding preferentially to purine-rich sequences that share a GGA core motif, but contain distinct flanking sequences.

Different binding site requirements for binding and activation for the bipartite enhancer factor EF-1A.

The human transcription factor EF-1A binds to the purine-rich E1A core enhancer sequence in the adenovirus E1A and E4 and polyomavirus enhancer regions. The consensus binding site for EF-1A resembles that of members of the ets domain protein family. EF-1A activation of transcription requires a dimeric binding site. Analysis of binding sites containing point mutations revealed that EF-1A binding is determined by the core nucleotides of the binding site, while transcriptional activation is determined both by the core and some peripheral nucleotides that do not affect binding. We have purified EF-1A and analyzed its two constituent subunits, EF-1A alpha and EF-1A beta. EF-1A alpha (MW approximately 60kD) makes the primary DNA contacts. EF-1A beta (MW approximately 50 kD) forms a heteromultimeric complex with EF-1A alpha both in solution and on a dimeric binding site. Binding of both EF-1A subunits is necessary, but not sufficient, for transcriptional activation. We present immunochemical and functional evidence that EF-1A alpha is related to the murine ets-related protein GABP alpha and that EF-1A beta is related to the murine protein GABP beta.

Convergence of Ets- and Notch-Related Structural Motifs in a Heteromeric DNA Binding Complex

Analysis of the heteromeric DNA binding protein GABP has revealed the interaction of two distinct peptide sequence motifs normally associated with proteins located in different cellular compartments. The alpha subunit of GABP contains an 85-amino acid segment related to the Ets family of DNA binding proteins. The ETS domain of GABP alpha facilitates weak binding to DNA and, together with an adjacent segment of 37 amino acids, mediates stable interaction with GABP beta. The beta subunit of GABP contains four imperfect repeats of a sequence present in several transmembrane proteins including the product of the Notch gene of Drosophila melanogaster. These amino-terminal repeats of GABP beta mediate stable interaction with GABP alpha and, when complexed with GABP alpha, directly contact DNA. These observations provide evidence for a distinct biochemical role for the 33-amino acid repeats, and suggest that they may serve as a module for the generation of specific dimerization interfaces.