Ets Element Research Articles

An Ets element regulates the transcription of the human 2B4 gene in natural killer cells

2B4 (CD244) acts as an activation receptor on human NK cells, whereas it sends inhibitory signals in murine NK cells. A previous study indicated a prominent role for AP-1 in the transcription of 2B4 gene. To further understand the transcriptional regulation we analyzed the upstream positive regulatory region (−1151 to −704) of the 2B4 promoter. We have identified an Ets element that regulates the 2B4 gene transcription in an AP1 dependent manner.

Characterization of the survival motor neuron (SMN) promoter provides evidence for complex combinatorial regulation in undifferentiated and differentiated P19 cells.

There exist two SMN (survival motor neuron) genes in humans, the result of a 500 kb duplication in chromosome 5q13. Deletions/mutations in the SMN1 gene are responsible for childhood spinal muscular atrophy, an autosomal recessive neurodegenerative disorder. While the SMN1 and SMN2 genes are not functionally equivalent, up-regulation of the SMN2 gene represents an important therapeutic target. Consequently, we exploited in silico, in vitro and in vivo approaches to characterize the core human and mouse promoters in undifferentiated and differentiated P19 cells. Phylogenetic comparison revealed four highly conserved regions that contained a number of cis-elements, only some of which were shown to activate/repress SMN promoter activity. Interestingly, the effect of two Sp1 cis-elements varied depending on the state of P19 cells and was only observed in combination with a neighbouring Ets cis-element. Electrophoretic mobility-shift assay and in vivo DNA footprinting provided evidence for DNA-protein interactions involving Sp, NF-IL6 and Ets cis-elements, whereas transient transfection experiments revealed complex interactions involving these recognition sites. SMN promoter activity was strongly regulated by an NF-IL6 response element and this regulation was potentiated by a downstream Ets element. In vivo results suggested that the NF-IL6 response must function either via a protein-tethered transactivation mechanism or a transcription factor binding an upstream element. Our results provide strong evidence for complex combinatorial regulation and suggest that the composition or state of the basal transcription complex binding to the SMN promoter is different between undifferentiated and differentiated P19 cells.

Alternative initiation of transcription of the human presenilin 1 gene in SH‐SY5Y and SK‐N‐SH cells

We have identified DNA sequences required for the expression of the presenilin 1 (PS1) gene. A promoter region has been mapped in SK-N-SH cells and includes sequences between -118 and +178 flanking the major initiation site (+1). The PS1 gene is also efficiently transcribed in the SH-SY5Y subclone of SK-N-SH cells. However the promoter appears to be utilized in alternative ways in both cell types. Sequences both upstream as well as downstream from the initiation site mapped in SK-N-SH cells were shown by 5'- and 3'-deletion analysis to play a crucial role in both cell lines. However, in SH-SY5Y cells either upstream or downstream sequences are sufficient to direct transcription, whereas in SK-N-SH cells 5'-deletions past the +1 site eliminate over 95% of transcription. Several Ets motifs (GGAA) as well as Sp1 motifs [(G/T)GGCGGRRY] are juxtaposed both upstream and downstream from +1. To understand how the promoter may be utilized alternatively in different cell types we have examined the effect of point mutations in these elements. Altering an Ets motif at -10 eliminates 80% of transcription in SK-N-SH cells whereas the same mutation has only a minor effect in SH-SY5Y cells. Conversely, mutation of the Ets element at +90, which eliminates 70% of transcription in SH-SY5Y cells, has a lesser effect in SK-N-SH cells. In both cell types a promoter including mutations at both -10 and +90 sites loses over 90% transcription activity indicating the crucial importance of these two Ets motifs. The effect of Sp1 mutations appears to be similar in both cell types. Hence the differential expression in each cell type may be at least partially determined by Ets factors and the -10/+90 sites. We have identified several Ets factors that recognize specifically the -10 Ets motif by the yeast one-hybrid selection including avian erythroblastosis virus E26 oncogene homologue 2, Ets-like gene 1, Ets translocation variant 1 and Ets related molecule (ERM). We show here that ERM specifically recognizes Ets motifs on the PS1 promoter located at -10 as well as downstream at +90, +129 and +165 and activates PS1 transcription with promoter fragments containing or not the -10 Ets site.

An abbreviated procedure for the cloning and identification of Ets transcription factors regulating the expression of the human presenilin 1 gene

We have previously defined a crucial DNA element controlling 90% of the expression of the presenilin 1 gene at (−35 to +6). This region contains an Ets transcription factor binding motif, and a two-basepair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by over 90%. We have shown that Ets1/2 transcription factors bind specifically to the −10 Ets element and activate PS1 transcription. The identification of other transcription factors recognizing specifically this promoter area should provide insights into the regulation of PS1. We have used the −10 Ets element as a bait in yeast one hybrid screening of a human brain cDNA library using a His3 reporter construct. We describe an abbreviated one-hybrid protocol to screen cDNA libraries. This assay selected four factors from the Ets family: Ets2, ER81, ERM and Elk1. We have also shown that specific DNA binding activity to the −10 Ets element of PS1 could easily be detected in yeast clones by EMSAs including protein extracts from yeast cells, thus confirming specific DNA binding activity without further sequencing and subcloning into suitable expression vectors. Ultimately the identity of putative clones was confirmed by DNA sequencing. We also confirmed the specific DNA binding properties of the factors identified by showing that the proteins produced by in vitro translation of the entire cDNAs from Elk1 and ER81 indeed binds specifically to the −10 region of the PS1 promoter.

Ets-1 Protects Vascular Smooth Muscle Cells from Undergoing Apoptosis by Activating p21WAF1/Cip1: ETS-1 REGULATES BASAL AND INDUCIBLE p21WAF1/Cip1 TRANSCRIPTION VIA DISTINCT CIS-ACTING ELEMENTS IN THE p21WAF1/Cip1 PROMOTER

The cyclin-dependent kinase inhibitor (CKI) p21WAF1/Cip1 is regulated at the level of transcription by nuclear factors such as the co-activator p300. It is presently unknown whether the Ets family of transcription factors control p21WAF1/Cip1 gene expression. Ets-1 inhibits apoptosis in vascular smooth muscle cells as determined by both fluorescein isothiocyanate-linked annexin V/propidium iodide staining of cells and fluorescence-activated cell sorting analysis and quantitative cytoplasmic histone-associated internucleosomal DNA fragmentation. p21WAF1/Cip1 can play a mitogenic and anti-apoptotic role in smooth muscle cells. Using transient transfection and Western blot analysis, we determined that Ets-1 activates p21WAF1/Cip1 transcription and protein expression. Electrophoretic mobility shift assays revealed that Ets-1 interacts selectively with the -1350GGAA-1347 Ets element in the p21WAF1/Cip1 promoter. Mutation of this element reduced basal and Ets1-inducible p21WAF1/Cip1 promoter-dependent expression. In contrast, the -1577GGAT-1574 motif mediates basal but not Ets-1 activation of the p21WAF1/Cip1 promoter. Co-immunoprecipitation and co-transfection analysis showed that Ets-1 binds p300 and cooperatively activates p21WAF1/Cip1 transcription. The phenotypic importance of Ets-1 regulation of p21WAF1/Cip1 was demonstrated by the capacity of antisense p21WAF1/Cip1 strategies to block Ets-1-inhibition of apoptosis and inhibit Ets-1-induction of proliferation.

Modulation of the folate receptor type β gene by coordinate actions of retinoic acid receptors at activator Sp1/ets and repressor AP-1 sites

AbstractFolate receptor (FR) type β is a promising target for therapeutic intervention in acute myelogenous leukemia (AML) owing particularly to its specific up-regulation in AML cells by all-trans retinoic acid (ATRA). Here we identify functional elements in the FR-β gene and examine the molecular mechanism of transcriptional induction of FR-β by ATRA. The basal promoter activity of FR-β resulted from synergistic interaction between Sp1 and ets binding sites (EBSs) and repression by upstream AP-1–like elements, whose action required EBSs. A minimal promoter containing the Sp1 and ets elements was ATRA-responsive. The repressor elements bound Fos family proteins; association of the proteins with the repressor elements correlated negatively with FR-β expression in peripheral blood neutrophils and monocytes and also in KG-1 (AML) cells grown in the absence or in the presence of ATRA. Furthermore, down-regulation of FR-β in KG-1 cells treated with O-tetradecanoylphorbol 13-acetate (TPA) was accompanied by increased AP-1 binding to the repressor elements. From chromatin immunoprecipitation (ChIP) assays, the nuclear retinoic acid receptor α (RARα) associated with the Sp1 region, and RARs β and γ associated with the AP-1 and Sp1 regions; treatment of KG-1 cells with ATRA did not alter Sp1 binding but increased the association of RARα and decreased the association of RARs β and γ. ATRA also decreased RAR expression levels. The results suggest that the FR-β gene is a target for multiple coordinate actions of nuclear receptors for ATRA directly and indirectly acting on a transcriptional complex containing activating Sp1/ets and inhibitory AP-1 proteins. The multiple mechanisms favor the prediction that ATRA will induce FR-β expression in a broad spectrum of AML cells. Further, optimal FR-β induction may be expected when all 3 RAR subtypes bind agonist.

Ets transcription factors ER81 and Elk1 regulate the transcription of the human presenilin 1 gene promoter

We have previously defined a crucial DNA element controlling 90% of the expression of the presenilin 1 gene at (−35 to +6). This region contains an Ets transcription factor binding motif, and a 2-base pair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by over 90%. We have shown that Ets1/2 transcription factors bind specifically to the −10 Ets element and activate PS1 transcription. The identification of other transcription factors recognizing specifically this promoter area should provide insights into the regulation of PS1. We have used the −10 Ets element as a bait in yeast one hybrid screening of a human brain cDNA library. This assay selected three factors from the Ets family: Ets2, ER81 and Elk1. We show that in vitro translated ER81 indeed binds specifically to the −10 region of the PS1 promoter and that ER81 activates by two- to threefold the basal transcription of a presenilin-1 promoter-chloramphenicol acetyltransferase reporter synthetic gene (−119, +178)PS1CAT in transient infection assays in neuroblastoma cells (SK-N-SH). GABPα, a member of the Ets family closely related to Ets2 and also containing a pointed domain, only increased PS1 transcription by about twofold. Cotransfection of GABPβ together with GABPα did not increase PS1 transcription. However, GABPβ alone activated PS1 transcription by two- to threefold. In contrast, the more distantly related Ets factor Elk1 repressed PS1 transcription very effectively.

Dual regulation of ets-activated gene expression by SP1

The human folate receptor (hFR) type γ gene is driven by a TATA-less promoter that uses a canonical Sp1 element for basal transcription. Using nuclear extract from 293 (human embryonic) cells, we mapped a second (non-canonical) Sp1 element to which Sp1 bound with a comparable affinity and which overlaps a functional ets binding site (EBS). Mutagenesis experiments revealed that the binding of ets to the EBS activates the promoter synergistically with Sp1 bound at the downstream site; however, binding of Sp1 to the EBS does not contribute to promoter activity. A further increase in Sp1 by inducible expression in recombinant 293 cells resulted in a small but significant decrease in the hFR-γ promoter activity, but the decrease was abolished when the EBS was deleted from the promoter. In 293 cells, which do not express hFR-γ, the Sp1 level was relatively high whereas in the hFR-γ-positive HL60 leukemia cells, the Sp1 level was low and the EBS predominantly bound an ets protein. To account for the above observations, we propose a model in which when the Sp1 level is low, ets out competes Sp1 for binding to the EBS and synergistically enhances the hFR-γ promoter activity by interacting with Sp1 bound at the canonical site whereas at higher levels, Sp1 represses the promoter by competitively inhibiting the binding of ets. As a partial extension of this model to the regulation of other ets activated genes, we show that Sp1 can predictably bind to a variety of ets elements including those responsive to Ets1 and Spi.1/Pu.1. A dual concentration-dependent action of Sp1 as an activator or a repressor offers a potential mechanism contributing to tissue-specific regulation of ets-dependent genes by Sp1.

Cooperative Interaction of Hypoxia-inducible Factor-2α (HIF-2α) and Ets-1 in the Transcriptional Activation of Vascular Endothelial Growth Factor Receptor-2 (Flk-1)

Interactions between Ets family members and a variety of other transcription factors serve important functions during development and differentiation processes, e.g. in the hematopoietic system. Here we show that the endothelial basic helix-loop-helix PAS domain transcription factor, hypoxia-inducible factor-2alpha (HIF-2alpha) (but not its close relative HIF-1alpha), cooperates with Ets-1 in activating transcription of the vascular endothelial growth factor receptor-2 (VEGF-2) gene (Flk-1). The receptor tyrosine kinase Flk-1 is indispensable for angiogenesis, and its expression is closely regulated during development. Consistent with the hypothesis that HIF-2alpha controls the expression of Flk-1 in vivo, we show here that HIF-2alpha and Flk-1 are co-regulated in postnatal mouse brain capillaries. A tandem HIF-2alpha/Ets binding site was identified within the Flk-1 promoter that acted as a strong enhancer element. Based on the analysis of transgenic mouse embryos, these motifs are essential for endothelial cell-specific reporter gene expression. A single HIF-2alpha/Ets element conferred strong cooperative induction by HIF-2alpha and Ets-1 when fused to a heterologous promoter and was most active in endothelial cells. The physical interaction of HIF-2alpha with Ets-1 was demonstrated and localized to the HIF-2alpha carboxyl terminus and the autoinhibitory exon VII domain of Ets-1, respectively. The deletion of the DNA binding and carboxyl-terminal transactivation domains of HIF-2alpha, respectively, created dominant negative mutants that suppressed transactivation by the wild type protein and failed to synergize with Ets-1. These results suggest that the interaction between HIF-2alpha and endothelial Ets factors is required for the full transcriptional activation of Flk-1 in endothelial cells and may therefore represent a future target for the manipulation of angiogenesis.

Activation of transcription of the human presenilin 1 gene by 12-O-tetradecanoylphorbol 13-acetate.

We have recently identified an Ets element controlling over 90% of the basal expression of the human presenilin 1 (PS1) gene. We have also shown that Ets1 and Ets2 act as transactivators of the PS1 gene by cotransfection experiments in SK-N-SH neuronal cells. The PS1 gene is widely but differentially expressed across tissues and the expression in brain appears to be restricted to neurons. To gain further insight into the regulation of the gene we have examined the regulation of PS1 by 12-O-tetradecanoylphorbol 13-acetate (TPA). SK-N-SH neuronal cells were treated with 0.2 micro m TPA for 30 min to 24 h and the level of expression of the endogenous PS1 gene was measured by Northern blot analysis. A two- to threefold increase in the level of PS1 mRNA appeared 4-8 h after the addition of TPA. A similar increase in transcription activity was observed in nuclear run off experiments, indicating that the increased mRNA level results from an activation in the initiation of transcription of PS1. Consistently, TPA also increased the level of PS1 protein. No activation of the PS1 endogenous gene by TPA was observed in hepatoma HepG2 cells. Next we tested the effect of TPA on the expression of the PS1 promoter by transfecting fusion genes including various fragments of the PS1 promoter linked to a CAT reporter into SK-N-SH cells. TPA also stimulated the expression of the PS1CAT constructs. Generally wild type constructs -687/+178, -118/+178, -22/+178 including the short -35/+6 fragment showed a minor two- to threefold activation by TPA. Point mutations eliminating the -10 Ets motif or the -6 CREB/AP1 motif did not decrease the stimulation by TPA. Thus TPA appears to activate the transcription of the PS1 gene by a mechanism which does not require these elements.

Control of megakaryocyte-specific gene expression by GATA-1 and FOG-1: role of Ets transcription factors.

The transcription factor GATA-1 and its cofactor FOG-1 are essential for the normal development of erythroid cells and megakaryocytes. FOG-1 can stimulate or inhibit GATA-1 activity depending on cell and promoter context. How the GATA-1-FOG-1 complex controls the expression of distinct sets of gene in megakaryocytes and erythroid cells is not understood. Here, we examine the molecular basis for the megakaryocyte-restricted activation of the alphaIIb gene. FOG-1 stimulates GATA-1-dependent alphaIIb gene expression in a manner that requires their direct physical interaction. Transcriptional output by the GATA-1-FOG-1 complex is determined by the hematopoietic Ets protein Fli-1 that binds to an adjacent Ets element. Chromatin immunoprecipitation experiments show that GATA-1, FOG-1 and Fli-1 co-occupy the alphaIIb promoter in vivo. Expression of several additional megakaryocyte-specific genes that bear tandem GATA and Ets elements in their promoters also depends on the physical interaction between GATA-1 and FOG-1. Our studies define a molecular context for transcriptional activation by GATA-1 and FOG-1, and may explain the occurrence of tandem GATA and Ets elements in the promoters of numerous megakaryocyte-expressed genes.

Complex 5' genomic structure of the human prolactin receptor: multiple alternative exons 1 and promoter utilization.

Transcription of the prolactin receptor (PRLR) is under the control of multiple promoters. Following the recent demonstration of the human non-coding exon 1, hE1(N) (hE1(N1)) and the generic exon 1 hE1(3), we have identified their promoters and characterized four other novel human exons 1 (hE1(N2-5)) that are alternatively spliced to a common non-coding exon 2 in human tissues and breast cancer cells. Genomic regions containing these exons, and 5'-flanking and intronic sequences, were determined and their order was established in chromosome 5p14-13. Promoters utilized in the transcription of previously characterized PRLR exons 1 species hE1(3) (hPII) and hE1(N1) (hP(N1)) were found to employ distinct mechanisms for controlling hPRLR transcription. hPIII requires C/EBP beta and Sp1/Sp3 for basal transcriptional activity, while hP(N1) activity is conferred by domains containing an Ets element and an NR half-site. The complex promoter control system that governs transcription of the hPRLR in multiple tissues is of relevance for studies on the regulation of PRLR expression in physiological and pathological states.

Synergistic Activation of the TATA-less Mouse Thymidylate Synthase Promoter by the Ets Transcription Factor GABP and Sp1

The mouse thymidylate synthase (TS) promoter lacks a TATA box and an initiator element and directs transcriptional initiation at multiple sites over a 90-nucleotide region. The minimum sequence required for wild-type promoter activity has been mapped to a 30-nucleotide essential promoter region that partially overlaps the 5′ end of the transcriptional initiation window. The essential promoter region contains two potential binding sites for members of the Ets family of transcription factors as well as a binding site for Sp1. Promoter mutation analyses revealed that all three of these sites are important for promoter activity. Transient cotransfection assays showed that GABP, a heterodimeric Ets factor, is able to stimulate expression of reporter genes driven by the wild-type mouse TS promoter whereas several other Ets factors have no effect. Electrophoretic mobility shift assays revealed that recombinant GABP binds to both Ets elements in the essential promoter region. Stimulation of promoter activity by GABP is diminished when either Ets element is inactivated and is prevented when both Ets elements are inactivated. Transient cotransfection assays revealed that Sp1 and GABP stimulate TS promoter activity in a highly synergistic manner.

Activation of Elk-1, an Ets transcription factor, by glucose and EGF treatment of insulinoma cells.

Elk-1, a member of the ternary complex factor family of Ets domain proteins that bind serum response elements, is activated by phosphorylation in a cell-specific manner in response to growth factors and other agents. The purpose of the current study was to determine whether Elk-1 activation contributes to glucose-/depolarization-induced Ca(2+)-dependent induction of immediate early response genes in pancreatic islet beta-cells. The results of experiments in insulinoma (MIN6) cells demonstrated that Elk-1-binding sites (Ets elements) in the Egr-1 gene promoter contribute to transcriptional activation of the gene. Treatment with either epidermal growth factor (EGF), a known inducer of beta-cell hyperplasia, glucose, or KCl-induced depolarization resulted in Ser(383) phosphorylation and transcriptional activation of Elk-1 (4 +/- 0.3-, P = 0.003, 2.3 +/- 0.19-, P = 0.002, and 2.2 +/- 0.1- fold, P = 0.001 respectively). The depolarization response was inhibited by the Ca(2+) channel blocker verapamil and by the MEK inhibitor PD98059 (53 +/- 6 and 55 +/- 0.5%, respectively). EGF-induced activation of Elk-1 was also inhibited by PD98059 (60 +/- 5%). A dominant negative Ras produced partial inhibition (42%) of the depolarization-induced Elk-1 transcriptional activation. Transfection with a constitutively active Ca(2+)/calmodulin kinase IV plasmid also resulted in Elk-1 transcriptional activation. Experiments with p38, phosphatidylinositol 3-kinase, and protein kinase A inhibitors indicated that these pathways are not involved. We conclude that Elk-1 activation contributes to glucose-/depolarization-induced Ca(2+)-dependent induction of immediate early growth response genes in pancreatic islet beta-cells. Furthermore, the results demonstrated a convergence of nutrient- and growth factor-mediated signaling pathways on Elk-1 activation through induction of Ras/mitogen-activated protein kinase ERK-1 and -2. The role of these pathways in the glucose-induced proliferation of islet beta-cells can now be assessed.

VIP Gene Transcription Is Regulated by Far Upstream Enhancer and Repressor Elements

SK-N-SH human neuroblastoma subclones differ widely in basal and second messenger induction of the gene encoding the neuropeptide vasoactive intestinal peptide (VIP). These differences were recapitulated by a chimeric gene which consisted of 5.2 kb of the human VIP gene 5′ flanking sequence fused to a reporter. Subsequent gene deletion experiments revealed several regulatory regions on the gene, including a 645-bp sequence located approximately 4.0 upstream from the transcription start site. Here we examined this upstream region in detail. Inhibitory sequences were found to be present on each end of the 645-bp fragment. When removed, basal transcription increased more than 50-fold. Subsequent deletion/mutation analysis showed that the 213-bp fragment contained at least two enhancer elements. One of these was localized to an AT-rich 42-bp sequence shown by others to bind Oct proteins in neuroblastoma cells, while the other corresponded to a composite AP-1/ets element. In addition to these enhancers, a 28-bp sequence on the 213-bp fragment with no apparent homology to known silencers inhibited transcription. The studies provide molecular details of a complex regulatory region on the VIP gene that is likely to be used to finely tune the level of gene transcription in vivo.

Sp1 and ETS family transcription factors regulate the mouse Mta2 gene expression

Dynamic changes in chromatin structure through nucleosome remodelling and core histone tail acetylation play important roles in transcriptional regulation. The purification and functional characterization of a nucleosome remodelling and histone deacetylase complex, NuRD, has suggested that nucleosome remodelling and core histone tail modification are potentially linked processes. MTA2, a component of the NuRD complex, plays an important role in regulating histone deacetylase activity of the NuRD complex. Similar to the candidate metastasis associated protein MTA1, an elevated level of MTA2 correlates with cellular proliferation. To understand the regulation of Mta2 transcription, we characterized the mouse Mta2 gene and its transcriptional regulatory elements. We found that MTA2 is encoded by 18 exons that span 10 kb. Primer extension analysis identified a major transcriptional start site locates 259 base pairs upstream of the ATG translational start codon. Transient transfection studies localized its promoter, lacking a canonical TATA box, to within 60 base pairs upstream of the transcriptional start site. Gel-mobility-shift and mutagenesis studies revealed that Sp1 and ETS elements play important roles in regulating Mta2 transcription. Information concerning the regulation of the Mta2 gene expression will be useful in understanding the regulation of NuRD histone deacetylase activity, which in turn will help in our general understanding of the transcriptional repression mechanism.

Regulation of Transcription of the Human Presenilin-1 Gene by Ets Transcription Factors and the p53 Protooncogene

The expression of the human presenilin-1 cellular gene is suppressed by the p53 protooncogene. The rapid kinetic of the down-regulation has suggested that it may result from a primary mechanism. We show here that p53 also suppresses the transcription of a presenilin-1 promoter-chloramphenicol acetyltransferase reporter synthetic gene in transient infection assays in neuroblastoma (SK-N-SH) and hepatoma (HepG2) cell lines. Only a minimum promoter including sequences from -35 to + 6 from the transcription initiation is sufficient to confer down-regulation. We have previously defined a crucial DNA element controlling 90% of the expression of the gene within the same short area, and the identification of the transcription factors involved should also provide insights into the regulation of PS1 by p53. This region contains an Ets transcription factor binding motif, and a 2-base pair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by more than 90%. We now show that Ets1 and Ets2 indeed transactivate a PS1 promoter-chloramphenicol acetyltransferase reporter including the (-35 to +6) fragment. Furthermore, in vitro translated Ets2 binds specifically to the -10 Ets motif in electrophoretic mobility shift assays. Therefore, Ets1/2 factors bind specifically to the -10 Ets element and activate PS1 transcription. We also show that the coactivator p300 enhances the activation by Ets1 and Ets2 as well as the repression by p53. p300 is known to interact with p53 as well as with Ets1 and Ets2. We show that p53 does not bind directly to the PS1 promoter. Hence the repression of PS1 transcription by p53 is likely to be mediated through protein-protein interactions.

Involvement of STAT-1 and Ets Family Members in Interferon-γ Induction of CD40 Transcription in Microglia/Macrophages

Cluster of differentiation (CD)-40 is a cell surface receptor belonging to the tumor necrosis factor receptor family that plays a critical role in the regulation of immune responses. We have previously shown that the cytokine interferon (IFN)-gamma induces CD40 expression in microglia. Herein, we have elucidated the molecular mechanisms underlying IFN-gamma induction of CD40 gene expression in microglia/macrophages. IFN-gamma up-regulates CD40 expression at the transcriptional level, and this regulation involves the STAT-1alpha transcription factor. Microglia from STAT-1alpha-deficient mice were refractive to IFN-gamma induction of CD40 expression, illustrating the importance of STAT-1alpha in this response. Functional analysis of the CD40 promoter indicates that two gamma activated sequence elements as well as two Ets elements are involved in IFN-gamma induction of CD40 promoter activity. STAT-1alpha binds to the gamma activated sequence elements, whereas PU.1 and/or Spi-B bind to the Ets elements. The expression of PU.1 and Spi-B, in conjuction with STAT-1alpha activation, correlates with IFN-gamma inducibility of CD40 expression. Collectively, our data demonstrate the involvement of STAT-1alpha, PU.1, and Spi-B in IFN-gamma induction of CD40 gene expression in cells of the macrophage lineage.

An Upstream Element Containing an ETS Binding Site Is Crucial for Transcription of the Human Presenilin-1 Gene

Deletion mapping of the human presenilin-1 (PS1) promoter delineated the most active fragment from -118 to +178 in relation to the transcription start site mapped in this study, in both human neuroblastoma SK-N-SH and hepatoma HepG2 cells. 5' deletions revealed that a crucial element controlling over 90% of the promoter activity in these cell lines is located between -22 and -6. A mutation altering only two nucleotides of the ETS consensus sequence present at -12 (GGAA to TTAA) has a similar effect. Electrophoretic mobility shift assays showed that a set of specific complexes between nuclear factors and the PS1 promoter are eliminated by this point mutation, as well as by competition with an ETS consensus oligonucleotide. Competition experiments in DNase I footprinting correlated with electrophoretic mobility shift assays and showed that only one of several footprints over the PS1 promoter is eliminated by competition with an ETS consensus oligonucleotide. It extends from -14 to -6 and surrounds the ETS motif present at -12. Thus, a crucial ETS element is present at -12 and binds a protein(s) recognizing specifically the ETS consensus motif. At least one such complex is eliminated by preincubating the nuclear extract with an antibody with broad cross-reactivity with Ets-1 and Ets-2 proteins, thus confirming that an ETS transcription factor(s) recognizes the -12 motif. Several Sp1 binding motifs at positions -70, -55, and +20 surround this ETS element. Competition DNase I footprinting showed that Sp1-like nuclear factors recognize specifically these sites in both cell lines. Furthermore, a combination of 5' and 3' deletions indicated the presence of positive promoter elements between -96 and -35 as well as between +6 and +42. Thus, transfection and footprinting assays correlate to suggest that Sp1 transcription factor(s) bind at several sites upstream and downstream from the initiation site and activate the transcription of the PS1 promoter. Sequences downstream from the transcription initiation site also contain major control elements. 3' deletions from +178 to +107 decreased promoter activity by 80%. However, further deletion to +42 increased promoter activity by 3-4-fold. Collectively, these data indicate that sequences upstream and downstream from the transcription start site each control over 80% of the promoter activity. Hence, this suggests that protein-protein interactions between factors recognizing downstream and upstream sequences are involved.

Transcriptional inhibition by interleukin-6 of the class A macrophage scavenger receptor in macrophages derived from human peripheral monocytes and the THP-1 monocytic cell line.

Expression of the class A macrophage scavenger receptor (MSR) contributes to the uptake of modified low density lipoproteins (LDL) by macrophages and transformation of these cells into lipid-laden foam cells, which characterize atherosclerosis. Many environmental factors, in particular, proinflammatory cytokines and growth factors, can exert regulatory effects on MSR expression, whereas intracellular accumulation of cholesterol itself does not influence MSR levels to any considerable extent. In the present study, by using an in vitro model, we examined whether stimulation with interleukin-6 (IL-6), an immunoregulatory, multipotential cytokine, modulates the expression and activities of the MSR in macrophages. When treated with IL-6, macrophages derived from peripheral monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 monocytic cells showed significantly reduced uptake and/or binding of the MSR ligand, acetylated LDL. This effect was paralleled by a reduction in the expression of MSR protein and mRNA. Analysis of MSR promoter activity in THP-1 cells transfected with an MSR promoter-reporter gene construct demonstrated decreased activity of the MSR promoter in IL-6-treated THP-1 macrophages. Electrophoretic mobility gel shift assay also showed a reduction in the binding of a transcription factor to the MSR promoter AP-1/ets elements in IL-6-treated cells. Thus, exposure to IL-6 may inhibit expression of the class A MSR in differentiated macrophages at transcriptional levels. This result suggests that this cytokine may modulate foam cell formation during atherogenesis.