Ets Family Proteins Research Articles

Characterization of a novel protein kinase C response element in the glucagon gene.

To maintain glucose levels in blood within narrow limits, the synthesis and secretion of pancreatic islet hormones are controlled by a variety of neural, hormonal, and metabolic messengers that act through multiple signal transduction pathways. Glucagon gene transcription is stimulated by cyclic AMP and depolarization-induced calcium influx. In this study, the effect of protein kinase C on glucagon gene transcription was investigated. After transient transfection of a glucagon-reporter fusion gene into the glucagon-producing islet cell line alphaTC2, activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated glucagon gene transcription. By 5' deletions, 3' deletions, internal deletion, and oligonucleotide cassette insertion, the TPA-responsive element was mapped to the G2 element (from -165 to -200). Like TPA, overexpression of oncogenic Ras (V-12 Ras) stimulated G2-mediated transcription whereas overexpression of a dominant negative Ras mutant (N-17 Ras) blocked the effect of TPA. A mutational analysis of G2 function and nuclear protein binding indicated that protein kinase C and Ras responsiveness is conferred to the glucagon gene by HNF-3beta functionally interacting with a protein that binds to a closely associated site with sequence similarity to binding sites of Ets family proteins. HNF-3beta belongs to the winged-helix family of transcription factors and has been implicated in the control of cell-specific and developmental gene expression. The results of the present study show that the cell lineage-specific transcription factor HNF-3beta is an essential component of a novel protein kinase C response element in the glucagon gene.

Characterization of the Human Thrombopoietin Gene Promoter: A POSSIBLE ROLE OF AN Ets TRANSCRIPTION FACTOR, E4TF1

Thrombopoietin (TPO), the ligand for c-Mpl, is a cytokine that regulates megakaryocyte growth and development. We have cloned the 5'-flanking region of the human TPO gene and analyzed its promoter activity. The human TPO gene promoter lacks a TATA box and directs transcription initiation at multiple sites over a 50-nucleotide region. Transient expression in a human liver cell line (PLC) of promoter fragment-luciferase reporter gene constructs containing a series of 5'-truncated sequences or site-directed mutations identified a sequence 5'-ACTTCCG-3' from -69 to -63 as a positive cis-acting element for high level expression of TPO gene. This sequence contains a core motif (C/A)GGA(A/T) for Ets family proteins in the noncoding strand. Gel mobility shift assays performed with nuclear protein from PLC cells identified a DNA binding protein(s) specific for the element. Anti-E4TF1-60(GABPalpha) or anti-E4TF1-53/47(GABPbeta) antibodies supershifted the complex in gel shift assay. Furthermore, co-expression of E4TF1-60 and E4TF1-53/47 squelched TPO gene expression in PLC and HepG2 cells. It is concluded that Ets family transcription factor E4TF1(GABPalpha/beta), an ubiquitously expressed protein, is required for high level expression of the TPO gene in liver.

Specific binding of the ETS-domain protein to the interferon-stimulated response element.

Interferon (IFN) activation of genes bearing an IFN-stimulated response element (ISRE) is regulated through binding of IFN-stimulated gene factors (ISGF) to the ISRE found in many IFN-stimulated genes. Using a multimerized human 2-5A synthetase ISRE as probe, we screened lambda gt11 expression libraries for cDNA encoding ISRE-binding activity and isolated a clone for murine proto-oncogene ets-1. The Ets-1 protein binds to the 2-5A synthetase ISRE at a site that also binds ISGF3, a multicomponent factor whose ISRE binding correlates with IFN-induced activation of transcription from ISRE-containing promoters. IFN-induced ISGF3 complex formation on the ISRE can be inhibited by specific Ets-1 antibody. Coexpression of Ets-1 represses ISRE-dependent reporter activity, suggesting that one or more members of the Ets protein family may negatively regulate transcriptional activity mediated by the 2-5A synthetase ISRE.

A cAMP Response Element and an Ets Motif Are Involved in the Transcriptional Regulation of flt-1 Tyrosine Kinase (Vascular Endothelial Growth Factor Receptor 1) Gene

The flt-1 gene encodes a transmembrane tyrosine kinase, Flt-1, a receptor for vascular endothelial growth factor. The expression of flt-1 gene is restricted to endothelial cells in vivo. To understand the molecular mechanism underlying endothelial-specific expression of this gene, we studied the functional significance of transcriptional motifs in the 200-base pair region of the human flt-1 gene promoter, which has been identified to confer cell type specificity. By point mutation analysis using chloramphenicol acetyltransferase plasmids in 293E1 cells, which express significant levels of flt-1 mRNA, we found that an Ets motif, E4, at -54 to -51 and a cAMP response element (CRE) at -83 to -76 are involved in the transcriptional regulation of this gene. Disruption of either this CRE or E4 within the promoter sequence of 90 base pairs resulted in a decrease in chloramphenicol acetyltransferase activity of 90%, indicating that co-existence of both of CRE and Ets motif E4 is necessary for transcription of the flt-1 gene. Co-transfection of an expression vector containing c-ets-1, c-ets-2, or c-erg cDNA with this 90-base pair sequence yielded a 5-8-fold elevation of chloramphenicol acetyltransferase activity, further supporting the idea that Ets family protein(s) participates in the regulation of the flt-1 gene. Gel shift assays using nuclear extracts of 293E1 and endothelial cells demonstrated the existence of protein factor(s) that specifically binds to CRE and Ets motif E4, respectively. Taken together, our results strongly suggest cooperation of a CRE and an Ets motif for the function of the flt-1 gene promoter.

An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein.

Expression of the human interleukin-2 (IL-2) receptor alpha chain gene is potently upregulated by its own ligand, IL-2. In this study, we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs, two putative Ets binding sites (EBS), one of which overlaps the consensus GAS motif, and a GATA motif, which overlaps the non-consensus GAS motif. We demonstrate that although the individual components of this element do not respond to IL-2, together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter. Multiple factors including Stat5, Elf-1, HMG-I(Y) and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element. An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter. Thus, IL-2-induced IL-2R alpha promoter activity requires a complex upstream element, which appears to contain binding sites for both positive and negative regulatory factors.

Tissue-specific Activity of the γc Chain Gene Promoter Depends upon an Ets Binding Site and Is Regulated by GA-binding Protein

The gammac chain is a subunit of multiple cytokine receptors (interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15), the expression of which is restricted to hematopoietic lineages. A defect in gammac leads to the X-linked severe combined immunodeficiency characterized by a block in T cell differentiation. In order to better characterize the human gammac promoter and define the minimal tissue-specific promoter region, progressive 5'-deletion constructs of a segment extending 1053 base pairs upstream of the major transcription start site were generated and tested for promoter activity in various hematopoietic and nonhematopoietic cell types. The -1053/+34 construct allowed promoter activity only in cells of hematopoietic origin, and tissue specificity was conserved in all other constructs tested. The region downstream of -90 appeared critical for basal promoter activity. It contains two potential Ets binding sites conserved in the murine gammac promoter gene, one of which was found essential for functional promoter activity as determined by mutational analysis. The functional Ets binding site was found to bind Ets family proteins, principally GA-binding protein and Elf-1 and could be transactivated by GABPalpha and -beta synergistically. These results indicate that, as already reported for the IL2Rbeta promoter, GA-binding protein is an essential component of gammac basal promoter activity. Although GABP expression is not restricted to the hematopoietic lineage, its interaction with other specific factors may contribute to the tissue-specific expression of the gammac gene.

SRE elements are binding sites for the fusion protein EWS-FLI-1

EWS-FLI-1 is a chimeric protein produced in most Ewing's sarcomas. It results from the fusion of the N-terminal-encoding region of the EWS gene to the C-terminal DNA-binding domain (the ETS domain) encoded by the FLI-1 ets family gene. Both EWS-FLI-1 and FLI-1 proteins function as transcription factors that bind specifically to ets sequences (the ets boxes) present in promoter elements. EWS- FLI-1 is a powerful transforming protein, whereas FLI-1 is not. In a search for potential DNA binding sites for these two proteins, we have tested their ability to recognize the serum responsive element (SRE) in the c-fos promoter. This cis element contains an ets box which can be occupied by members of the ETS protein family which do not bind DNA autonomously but form a ternary complex with a second protein, p67SRF (serum responsive factor). We demonstrate here that EWS-FLI-1, but not FLI-1, is able to form a ternary complex on the c-fos SRE. Using a GST pull-down assay, we show that both FLI-1 and EWS-FLI-1 interact in vitro with SRF in the absence of DNA. In electromobility shift assays, EWS-FLI-1 binding to the SRE is detectable in the absence of SRF whereas the binding of FLI-1 is not, suggesting that the interaction with DNA is the step which limits ternary complex formation by FLI-1. Deletion of the N-terminal portion of FLI-1 resulted in a protein which behaved as EWS-FLI-1, suggesting the existence of an N- terminal inhibitory domain in the normal protein. Taken together, our data indicate that there are intrinsic differences in the binding of EWS-FLI-1 and FLI-1 proteins to distinct ets sequences.

Importance of low affinity Elf-1 sites in the regulation of lymphoid-specific inducible gene expression.

Elf-1 is an Ets family transcription factor that regulates a number of inducible lymphoid-specific genes, including those encoding interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), and the IL-2 receptor (IL-2R) alpha chain. A minimal oligonucleotide spanning the IL-2R alpha Elf-1 site (-97/-84) bound Elf-1 poorly, but binding activity markedly increased when this oligonucleotide was multimerized or flanking sequences were added. This result is consistent with the requirement of accessory proteins for efficient Elf-1 binding, as has been demonstrated for the GM-CSF and IL-3 promoters. A binding site selection analysis revealed the optimal Elf-1 consensus motif to be A(A/t)(C/a)CCGGAAGT(A/S), which is similar to the consensus motif for the related Drosophila E74 protein. This minimal high affinity site could bind Elf-1 and functioned as a stronger transcription element than the -97/-84 IL-2R alpha oligonucleotide when cloned upstream of a heterologous promoter. In contrast, in the context of the IL-2R alpha promoter, conversion of the naturally occurring low affinity Elf-1 site to an optimal site decreased inducible activation of a reporter construct in Jurkat cells. This finding may be explained by the observation that another Ets family protein, ER GB/Fli-1, can efficiently bind only to the optimal site, and in this context, interferes with Elf-1 binding. Therefore, high affinity Elf-1 sites may lack sufficient binding specificity, whereas naturally occurring low affinity sites presumably favor the association of Elf-1 in the context of accessory proteins. These findings offer an explanation for the lack of optimal sites in any of the known Elf-1-regulated genes.

Activation of transcription by PU.1 requires both acidic and glutamine domains.

The B-lymphocyte- and macrophage-specific transcription factor PU.1 is a member of the ets family of proteins. To understand how PU.1 functions as a transcription factor, we initiated a series of experiments to define its activation domain. Using deletion analysis, we showed that the activation domain of PU.1 is located in the amino-terminal half of the protein. Within this region, we identified three acidic subdomains and one glutamine-rich subdomain. The deletion of any of these subdomains resulted in a significant loss in the ability of PU.1 to transactivate in cotransfection studies. Amino acid substitution analysis showed that the activation of transcription by PU.1 requires acidic residues between amino acids 7 and 74 and a group of glutamine residues between amino acids 75 and 84. These data show that PU.1 contains two types of known activation domains and that both are required for maximal transactivation.

Generation and characterization of monoclonal antibodies against the ERGB/FLI-1 transcription factor.

Five monoclonal antibodies were produced from mice immunized with recombinant full length human ERGB protein. Among these monoclonal antibodies, four clones did not cross react with other ets family proteins and thus are specific for the ERGB protein; however, one clone did react with the ERG protein, which has high amino acid identity with the ERGB protein. The epitope location of these antibodies was studied using bacterially expressed fragments of the human, ERGB protein. These monoclonal antibodies recognized 51 kDa (p51) and 48 kDa (p48), two ERGB gene-encoded proteins, from human, mouse, and rat cell lines. These results suggest that the monoclonal antibodies can be used in human, mouse, or rat cell lines and will be useful for the biochemical and functional analysis of the ERGB protein.

A Composite Ets/Pit-1 Binding Site in the Prolactin Gene Can Mediate Transcriptional Responses to Multiple Signal Transduction Pathways

Binding sites for the tissue-specific transcription factor, Pit-1, are required for basal and hormonally induced prolactin gene transcription. Although Pit-1 is phosphorylated in response to several signaling pathways, the mechanism by which Pit-1 contributes to hormonal induction of gene transcription has not been defined. Recent reports suggest that phosphorylation of Pit-1 may not be required for hormonal regulation of the prolactin promoter. Analysis of the contribution of individual Pit-1 binding sites has been complicated due to the fact that some of the elements appear to be redundant. To better understand the role of Pit-1 sites in mediating hormonal regulation of the prolactin gene, we have performed enhancer tests using the three most proximal Pit-1 binding sites of the rat prolactin gene which are designated the 1P, 2P, and 3P sites. The results demonstrate that multimers of the 3P Pit-1 binding site are much more responsive to several hormonal and intracellular signaling pathways than multimers of the 1P or 2P sites. The 3P DNA element was found to contain a consensus binding site for the Ets family of proteins. Mutation of the Ets binding site greatly decreased the ability of epidermal growth factor, phorbol esters, Ras, or the Raf kinase to induce reporter gene activity. Mutation of the Ets site had little effect on basal enhancer activity. In contrast, mutation of the consensus Pit-1 binding site in the 3P element essentially abolished all basal enhancer activity. Overexpression of Ets-1 in GH3 pituitary cells enhanced both basal and Ras induced activity from the 3P enhancer. These data describe a composite element in the prolactin gene containing binding sites for two different factors and the studies suggest a mechanism by which Ets proteins and Pit-1 functionally cooperate to permit transcriptional regulation by different signaling pathways.

π1 Binding Sites Are Negative Regulators of bcl-2 Expression in Pre-B Cells

The bcl-2 gene is differentially regulated during B-cell development, with low-level expression in pre-B cells and higher-level expression in mature B cells. These changes correlate with susceptibility to cell death by apoptosis and suggest that the Bcl-2 protein may play a role in the control of cell death during B-cell development. We have identified two negative regulatory regions in the human bcl-2 5' flanking and 5' untranslated regions in pre-B cells; these regions have no significant function in mature B cells. Further investigation of these regions revealed two pre-B-cell-specific enhancer elements (pi 1 sites) in the 5' negative regulatory region and one in the 3' negative regulatory region. Mutational analysis confirmed that these three sites functioned as negative regulators of the bcl-2 promoter in the pre-B-cell line Nalm-6. Electrophoretic mobility shift assays with each of the three sites demonstrated a complex of identical mobility to that formed with the immunoglobulin heavy-chain enhancer pi 1 site. UV cross-linking experiments revealed that a protein with a molecular mass of 58 kDa bound to the three bcl-2 sites and to the immunoglobulin enhancer site. This protein reacted with an antibody against Ets family proteins. Constructs with the isolated pi 1 sites linked to the simian virus 40 promoter were used in transient transfection experiments in the pre-B-cell line. The bcl-2 sites decreased expression of the simian virus 40 promoter, while the immunoglobulin enhancer site increased its expression. The pi 1 sites in the bcl-2 gene may play a role in the developmental regulation of bcl-2 expression during B-cell differentiation.

Characterization of a gp91-phox Promoter Element That Is Required for Interferon γ-induced Transcription

The cytochrome b558 heavy chain (gp91-phox) is expressed nearly exclusively in terminally differentiating myelomonocytic cells, thereby providing a model to study the events of late myeloid differentiation. We describe a tissue culture assay for studying interferon gamma induction of gp91-phox expression and a cis-element in the gp91-phox promoter that is necessary but not sufficient for this activity. In vitro assays reveal two DNA-binding proteins that interact with this cis-element. One factor is restricted to hematopoietic cells, is required for an interferon gamma response, and binds to an element similar to the Ets protein family consensus, although it does not correspond to known family members. The second factor is the ubiquitous CCAAT-binding protein CP1, which is dispensable for an interferon gamma response. Single base pair mutations in the gp91-phox promoter that specifically abolish the binding of the hematopoietic-associated factor have previously been identified in chronic granulomatous disease patients (Newburger, P. E., Skalnik, D. G., Hopkins, P. J., Eklund, E. A., and Curnutte, J. T. (1994) J. Clin. Invest. 94, 1205-1211). The data reported here directly demonstrate the functional significance of the hematopoietic-associated factor for gp91-phox promoter activity and reveal the binding properties and tissue distribution of this novel DNA-binding protein.

Transcriptional activation of the immediate early gene pip92 by serum growth factors requires both Ets and CArG-like elements.

pip92 is an immediate early gene that is transcriptionally activated in mouse 3T3 fibroblasts upon treatment with serum growth factors or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Here we show that a 73-base pair pip92 promoter fragment located between base pairs 1231 and 1158 upstream of the transcription start site is sufficient to mediate transcriptional activation. This promoter fragment contains two binding sites for transcription factors of the Ets family and a low affinity binding site for the serum response factor. The minimal sequence that mediates serum induction includes at least one copy of the Ets-binding site and a low affinity binding site for the serum response factor. This sequence can interact with at least two proteins in a fibroblast nuclear extract that have binding characteristics of an Ets family protein and a serum response factor-like protein. These proteins can bind the pip92-inducible element simultaneously, thus forming a ternary complex. Furthermore, the same element interacts with recombinant serum response factor and Elk1 proteins individually as well as simultaneously to form a ternary complex. This mode of ternary complex formation is in contrast to the one seen in the promoter of the c-fos protooncogene, where formation of the ternary complex is dependent on the prior assembly of the serum response factor-DNA binary complex. The activation of a number of immediate early genes appears to be mediated through a ternary complex involving members of the Ets family of transcription factors and the serum response factor. We propose that different mechanisms can lead to the formation of such ternary complexes.

Multiple transcription factors including members of the ets protein family interact with the human granzyme B promoter to control specific transcription in activated T cells

Multiple transcription factors including members of the ets protein family interact with the human granzyme B promoter to control specific transcription in activated T cells

The Ets family of transcription factors.

Interest in the Ets proteins has grown enormously over the last decade. The v-ets oncogene was originally discovered as part of a fusion protein expressed by a transforming retrovirus (avian E26), and later shown to be transduced from a cellular gene. About 30 related proteins have now been found in species ranging from flies to humans, that resemble the vEts protein in the so-called 'ets domain'. The ets domain has been shown to be a DNA-binding domain, that specifically interacts with sequences containing the common core trinucleotide GGA. Furthermore, it is involved in protein-protein interactions with co-factors that help determine its biological activity. Many of the Ets-related proteins have been shown to be transcription activators, like other nuclear oncoproteins and anti-oncoproteins (Jun, Fos, Myb, Myc, Rel, p53, etc.). However, Ets-like proteins may have other functions, such as in DNA replication and a general role in transcription activation. Ets proteins have been implicated in regulation of gene expression during a variety of biological processes, including growth control, transformation, T-cell activation, and developmental programs in many organisms. Signals regulating cell growth are transmitted from outside the cell to the nucleus by growth factors and their receptors. G-proteins, kinases and transcription factors. We will discuss how several Ets-related proteins fit into this scheme, and how their activity is regulated both post- and pre-translationally. Loss of normal control is often associated with conversion to an oncoprotein. vEts has been shown to have different properties from its progenitor, which might explain how it has become oncogenic. Oncogene-related products have been implicated in the control of various developmental processes. Evidence is accumulating for a role for Ets family members in Drosophila development, Xenopus oocyte maturation, lymphocyte differentiation, and viral infectious cycles. An ultimate hope in studying transformation by oncoproteins is to understand how cells become cancerous in humans, which would lead to more effective treatments. vEts induces erythroblastosis in chicken. Cellular Ets-family proteins can be activated by proviral insertion in mice and, most interestingly, by chromosome translocation in humans. We are at the beginning of understanding the multiple facets of regulation of Ets activity. Future work on the Ets family promises to provide important insights into both normal control of growth and differentiation, and deregulation in illness.

Identification of a Killer Cell-Specific Regulatory Element of the Mouse Perforin Gene: an Ets-Binding Site-Homologous Motif That Interacts with Ets-Related Proteins

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins.

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

Regulatory Elements Involved in Tax-Mediated Transactivation of the HTLV-I LTR

HTLV-I is the etiologic agent of adult T-cell leukemia. In this study, we investigated the regulatory elements and cellular transcription factors which function in modulating HTLV-I gene expression in response to the viral transactivator protein, tax. Transfection experiments into Jurkat cells of a variety of site-directed mutants in the HTLV-1 LTR indicated that each of the three motifs A, B, and C within the 21-bp repeats, the binding sites for the Ets family of proteins, and the TATA box all influenced the degree of tax -mediated activation. Tax is also able to activate gene expression of other viral and cellular promoters. Tax activation of the IL-2 receptor and the HIV-1 LTR is mediated through NF-κB motifs. Interestingly, sequences in the 21-bp repeat B and C motifs contain significant homology with NF-κB regulatory elements. We demonstrated that an NF-κB binding protein, PRDII-BF1, but not the rel protein, bound to the B and C motifs in the 21-bp repeat. PRDII-BF 1 was also able to stimulate activation of HTLV-I gene expression by tax. The role of the Ets proteins on modulating tax activation was also studied. Ets 1 but not Ets 2 was capable of increasing the degree of tax activation of the HTLV-I LTR. These results suggest that tax activates gene expression by either direct or indirect interaction with several cellular transcription factors that bind to the HTLV-I LTR.

Characterization of the Human Interleukin-2 Receptor β-Chain Gene Promoter: Regulation of Promoter Activity by ets Gene Products

The interleukin-2 receptor (IL-2R) beta chain (IL-2R beta) is an essential signaling component of high- and intermediate-affinity IL-2Rs. Our laboratory previously reported that a DNA fragment containing 857 bp of 5'-flanking sequence of the human IL-2R beta gene exhibited promoter activity. We have now further characterized the promoter and delineated cis-acting regulatory regions. The region downstream of -363 is critical for basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and contains at least three enhancer-like regions. Among them, the -56 to -34 enhancer was the most potent and had high-level activity in two T-cell lines but not in nonlymphoid HeLaS3 and MG63 cells. This enhancer contains a GGAA Ets binding site which bound two Ets family proteins, Ets-1 and GA-binding protein in vitro. Mutation of the Ets motif strongly diminished both promoter and enhancer activities. We conclude that this Ets binding site plays a key role in regulating basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and may also contribute to tissue-specific expression of the IL-2R beta gene.