Ets Sites Research Articles

Abstract 4968: Modes of co-factor recruitment by an oncogenic fusion protein

Abstract Ewing's sarcoma (ESFT) is a highly aggressive pediatric cancer of the bone and characterized by the chimeric ETS transcription factor EWS-FLI1 as a result of a chromosomal translocation. By whole genome gene expression and ChIP-seq analyses we obtained evidence that EWS-FLI1 directly regulates E2F target genes and possibly cooperates with E2F transcription factors. Furthermore, several E2F factors were themselves inferred to be direct targets of EWS-FLI1. To unravel the hierarchical structure of this EWS-FLI1 transcriptional network module we performed luciferase reporter gene assays in combination with DNA motif mutation analyses. The promoter regions of nine candidates for direct target genes of EWS-FLI1 and E2F factors were chosen for in-depth analysis. Corroborating the transcriptomic data, promoter activity of all 9 genes showed reduced levels upon knockdown of EWS-FLI1. The strongest modulatory effect of EWS-FLI1 silencing was observed for the E2F3 promoter, and mutation of a single ETS binding site lead to a comparable reduction of luciferase activity as knockdown of EWS-FLI1. These results validate E2F3 as a directly EWS-FLI1 regulated gene in Ewing's sarcoma. For the other EWS-FLI1 target candidates mutation analysis lead to more complex results. In the RAD51 promoter simultaneous mutation of two ETS binding sites was necessary to obtain a significant reduction of luciferase activity. Furthermore, mutation of a single E2F binding site abolished the effect of EWS-FLI1 knockdown. In promoters of two other candidates, GEMIN4 and ATAD2, mutation of the ETS site resulted in only a moderate reduction of reporter activity, and additional destruction of an E2F binding site was required to achieve a significant decrease in promoter activity. Our results imply that EWS-FLI1 uses several modes of interaction with E2F factors to control common target genes. First, EWS-FLI1 directly activates E2F3 and other E2F factors which, in turn, co-operate with EWS-FLi1 on target gene promoters in a feed-forward loop. Second, results for the RAD51 promoter are compatible with a mechanism where a repressive E2F factor is exchanged for an activating E2F factor via binding of EWS-FLI1. Finally, EWS-FLI1 might have a more indirect role in the regulation of ATAD2 and GEMIN4 where most of the transcriptional effect seems to be exerted by E2F factors. Acknowledgement: This study was funded by the 6th framework program of the European Commission, (“E.E.T. Pipeline” contract LSHC-CT-2006-037260) and by the Austrian genome research program “GEN-AU Ch.I.L.D.“ (GZ 200.136/1 - VI/1/2005). R. Schwentner is a recipient of a DOC-fFORTE-fellowship of the Austrian Academy of Sciences. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4968.

Exogenous Nef Is an Inhibitor of Mycobacterium tuberculosis-induced Tumor Necrosis Factor-α Production and Macrophage Apoptosis

Human immunodeficiency virus-1 (HIV-1) impairs tumor necrosis factor-alpha (TNF-alpha)-mediated macrophage apoptosis induced by Mycobacterium tuberculosis (Mtb). HIV Nef protein plays an important role in the pathogenesis of AIDS. We have tested the hypothesis that exogenous Nef is a factor that inhibits TNF-alpha production/apoptosis in macrophages infected with Mtb. We demonstrate that Mtb and Nef individually trigger TNF-alpha production in macrophages. However, TNF-alpha production is dampened when the two are present simultaneously, probably through cross-regulation of the individual signaling pathways leading to activation of the TNF-alpha promoter. Mtb-induced TNF-alpha production is abrogated upon mutation of the Ets, Egr, Sp1, CRE, or AP1 binding sites on the TNF-alpha promoter, whereas Nef-mediated promoter activation depends only on the CRE and AP1 binding sites, pointing to differences in the mechanisms of activation of the promoter. Mtb-dependent promoter activation depends on the mitogen-activated kinase (MAPK) kinase kinase ASK1 and on MEK/ERK signaling. Nef inhibits ASK1/p38 MAPK-dependent Mtb-induced TNF-alpha production probably by inhibiting binding of ATF2 to the TNF-alpha promoter. It also inhibits MEK/ERK-dependent Mtb-induced binding of FosB to the promoter. Nef-driven TNF-alpha production occurs in an ASK1-independent, Rac1/PAK1/p38 MAPK-dependent, and MEK/ERK-independent manner. The signaling pathways used by Mtb and Nef to trigger TNF-alpha production are therefore distinctly different. In addition to attenuating Mtb-dependent TNF-alpha promoter activation, Nef also reduces Mtb-dependent TNF-alpha mRNA stability probably through its ability to inhibit ASK1/p38 MAPK signaling. These results provide new insight into how HIV Nef probably exacerbates tuberculosis infection by virtue of its ability to dampen Mtb-induced TNF-alpha production.

Diverse modes of Drosophila tracheal fusion cell transcriptional regulation

Drosophila tracheal fusion cells play multiple important roles in guiding and facilitating tracheal branch fusion. Mechanistic understanding of how fusion cells function during development requires deciphering their transcriptional circuitry. In this paper, three genes with distinct patterns of fusion cell expression were dissected by transgenic analysis to identify the cis-regulatory modules that mediate their transcription. Bioinformatic analysis involving phylogenetic comparisons coupled with mutational experiments were employed. The dysfusion bHLH-PAS gene was shown to have two fusion cell cis-regulatory modules; one driving initial expression and another autoregulatory module to enhance later transcription. Mutational dissection of the early module identified at least four distinct inputs, and included putative binding sites for ETS and POU-homeodomain proteins. The ETS transcription factor Pointed mediates the transcriptional output of the branchless/ breathless signaling pathway, suggesting that this pathway directly controls dysfusion expression. Fusion cell cis -regulatory modules of CG13196 and CG15252 require two Dysfusion:Tango binding sites, but additional sequences modulate the breadth of activation in different fusion cell classes. These results begin to decode the regulatory circuitry that guides transcriptional activation of genes required for fusion cell morphogenesis.

Integrative genomic analyses of CXCR4: Transcriptional regulation of CXCR4 based on TGFβ, Nodal, Activin signaling and POU5F1, FOXA2, FOXC2, FOXH1, SOX17, and GFI1 transcription factors

CXCR4, CD133, CD44 and ABCG2 are representative transmembrane proteins expressed on the surfaces of normal and/or cancer stem cells. CXCR4 is co-expressed with POU5F1 in endodermal precursors and adult-tissue stem cells. CXCR4 is expressed in a variety of human tumors, such as breast cancer, prostate cancer, pancreatic cancer, and gastric cancer. CXCR4 is a G protein-coupled receptor (GPCR) for CXCL12 (SDF1) chemokine, and the CXCL12-CXCR4 signaling axis is involved in proliferation, survival, migration, and homing of cancer cells. Integrative genomic analyses of CXCR4 gene were carried out to elucidate the mechanisms of CXCR4 expression in stem cells, because CXCR4 is a key molecule occupying the crossroads of oncology, immunology, gerontology and regenerative medicine. Human CXCR4 promoter region with binding sites for HIF1alpha, ETS1, NF-kappaB and GLI was not conserved in mouse and rat Cxcr4 orthologs. Proximal enhancer region with palindromic Smad-binding sites, FOX-binding site, POU-binding site, triple SOX17-binding sites, bHLH-binding site, TCF/LEF-binding site, and double GFI1-binding sites was almost completely conserved among human, chimpanzee, mouse, and rat CXCR4 orthologs. TGFbeta, Nodal, and Activin signals induce CXCR4 upregulation based on Smad2/3 and FOX family members, such as FOXA2, FOXC2, and FOXH1. CXCR4 is expressed in endodermal precursors due to the existence of triple SOX17-binding sites around the POU-binding site instead of the POU5F1-SOX2 joint motif. Because CXCR4 is downregulated by p53-GFI1 signaling axis, p53 mutation in cancer stem cells leads to CXCR4 upregulation. CXCR4 is also upregulated by TGFbeta and Hedgehog signals in tumor cells at the invasion front. Small molecule compound or human antibody targeted to CXCR4 will be applied for cancer therapeutics focusing on cancer stem cells at the primary lesion as well as metastasis or recurrence niches, such as bone marrow and peritoneal cavity.

The Ets Transcription Factor, GABP, Is Required by Myeloid Progenitors for Normal Myeloid Differentiation.

Abstract 256GABP transcription factor regulates genes that are required for innate immunity. GABP is an obligate tetrameric protein complex that contains two molecules of GABPa, which binds to DNA through its ets domain, and two molecules of GABPb, which contains a transcription activation domain. GABP is an essential component of a multiprotein enhanceosome that is required for retinoic acid-dependent myeloid gene transcription. Disruption in mouse embryo fibroblasts of Gabpa, the mouse gene that encodes mouse Gabpa, causes profound cell cycle arrest at the G1-S boundary, due to reduced expression of DNA Polymerase a and Thymidylate Synthase, which are required for DNA synthesis, and of Skp2, a ubiquitin ligase that controls degradation of the cyclin-dependent kinase inhibitors (CDKIs), p21 and p27. Thus, GABP is a key regulator of the cell cycle. In order to define the role of GABP in myeloid differentiation, we generated mice in which exons that encode the Gabpa ets domain are flanked by loxP recombination sites, and bred these floxed mice to mice that bear the Mx1-Cre transgene. Their progeny were treated with pI-C and Gabpa was efficiently deleted in hematopoietic cells of these Gabpa−/− mice. As controls for all experiments, mice that bear Mx1-Cre but which lack the floxed Gabpa allele were also injected with pI-C. Within days, the peripheral blood white blood cell count fell in the Gabpa−/− mice compared to the controls; half of the Gabpa−/− mice died within two weeks. Gabpa−/− mice exhibited a striking loss of Gr1+, CD11b+ cells in the peripheral blood, spleen, and bone marrow. Myeloid cells of Gabpa−/− mice were immature, morphologically dysplastic, and demonstrated aberrant patterns of myeloid gene expression. Bone marrow from Gabpa−/− mice formed reduced numbers of in vitro myeloid colonies in the presence of G-CSF, M-CSF, or GM-CSF; cells isolated from in vitro colonies from Gabpa−/− mice exhibited a strong bias toward macrophage-like morphology. Multicolor flow cytometry revealed a loss of granulocyte-monocyte committed progenitor cells (GMPs) in the bone marrow of Gabpa−/− mice, and these progenitors expressed aberrant patterns of key transcription factors. Especially notable in Gabpa−/− GMPs was reduced expression of Gfi-1, a transcriptional repressor that is mutated in some congenital neutropenic syndromes, and whose genetic disruption causes abnormalities in granulocyte development. We used chromatin immunoprecipitation (ChIP) to identify ets sites in the Gfi-1 promoter that are bound by GABP in vivo. We conclude that GABP is required for proliferation or survival of committed myeloid progenitor cells and for normal maturation of granulocytes. We hypothesize that defects in myeloid cell proliferation and differentiation associated with Gabpa disruption are caused, at least in part, by its regulation of the Gfi-1 transcriptional repressor. Furthermore, we propose that the regulation of Gfi-1 by GABP constitutes a key regulatory pathway in myeloid cell development. Disclosures:No relevant conflicts of interest to declare.

Transcriptional Regulation of the Nuclear Envelope Lamin B Receptor: New Roles for GA Binding Protein (GABP) in Neutrophil Development.

Abstract 1464Poster Board I-487The essential roles that neutrophils play in innate immune responses require that these phagocytes rapidly migrate to sites of infection, adhere to and escape capillary epithelium, and then engulf and destroy invading pathogens. The capacity of neutrophils to perform these complicated functions is largely dependent on the actions of multiple hematopoietic transcription factors that coordinate the expression of critical functional genes during neutrophil development. These transcription factors include CCAAT/enhancer binding proteins C/EBPα and C/EBPε, the ETS family protein PU.1, and the GA binding protein, GABP, which is also an ETS transcription factor that acts as an obligate heterotetramer comprised of GABPα and GABPβ. In addition to the activation of functional genes, neutrophil progenitors in the bone marrow undergo profound morphologic changes that include the formation of lobulated nuclei. Although it is still unclear as to the precise purpose of nuclear lobulation, we and others have demonstrated that the nuclear envelope protein called the lamin B receptor (LBR) is essential to neutrophil nuclear lobulation and that loss of Lbr expression in mouse neutrophils leads to decreased functional responses, including a reduced respiratory burst and abnormal chemotaxis. We also have shown that Lbr gene expression is upregulated during neutrophil development, indicating that the transcriptional control of Lbr expression may play a critical role in both neutrophil morphologic maturation and function. To identify the transcriptional regulators that control Lbr activation during neutrophil development, we have isolated the promoter of the mouse Lbr gene and have assessed the roles that different regulators of neutrophil development play on Lbr activation. Previous studies demonstrated that C/EBPε directly activates the Lbr promoter, but we found that myeloid C/EBPε-/- cells exhibit normal Lbr expression. We therefore focused on identifying alternative regulators that may control Lbr gene activation. We first identified two putative ets binding sites in the Lbr gene promoter that are located near two previously identified C/EBP binding sites. We then focused on analyzing transcriptional activities of two ETS family members known to play roles in myeloid gene activation, PU.1 and GABP, on Lbr promoter sequences that contain these ets binding sites. Using promoter expression constructs that contain different sized regions of the Lbr gene promoter and Cos cell transfections, we confirm that C/EBPε alone does indeed drive Lbr expression, but that either C/EBPα or PU.1 alone fails to activate the Lbr promoter. In contrast, expression of GABP, via co-expression of GABPα and GABPβ, transcriptionally activates the Lbr promoter to levels observed with C/EBPε alone. Interestingly, GABP activates a short Lbr promoter that contains only one ets binding site but that lacks C/EBPε binding sites, indicating that a single ets site is sufficient for GABP to drive Lbr expression in the absence of C/EBPε. Furthermore, co-expression of GABP plus PU.1 significantly increased activation of the Lbr promoter to levels above that observed for either GABP alone or C/EBPε alone. This result indicates that GABP and PU.1 synergistically activate the Lbr promoter. Ongoing analyses of mutant forms of these promoter constructs will address the importance of ets binding sites to Lbr gene activation, and binding assays will be used to identify in vivo interactions between GABP and/or PU.1 with the Lbr promoter. We are also generating an EML cell line that contains flox binding sites that flank GABP gene exons, which will be used to generate myeloid progenitors that lack GABP expression. Together our studies are identifying novel mechanisms that regulate the expression of a nuclear envelope protein that is essential for neutrophil development, and may reveal important insight into how morphologic maturation of neutrophils is closely linked to neutrophil functions. DisclosuresNo relevant conflicts of interest to declare.

Flexible Stereospecific Interactions and Composition within Nucleoprotein Complexes Assembled on the TCRα Gene Enhancer

During thymocyte maturation, enhancers of genes encoding for TCRdelta (Tcrd) and TCRalpha (Tcra), Edelta(8), and Ealpha, work as a developmental switch controlling transition from Tcrd to Tcra activity at the Tcrad locus. Previous experiments revealed that an Ealpha fragment, Talpha1-Talpha2, which constitutes a well-characterized compact nucleoprotein structure led to premature activation of V(D)J recombination compared with that observed for the entire Ealpha or Talpha1-Talpha4. These experiments indicated that Talpha3-Talpha4 collaborates with factors bound to Talpha1-Talpha2 for the strict developmental regulation of Tcra rearrangement. The compact enhanceosome created on Talpha1-Talpha2 explained the molecular basis for requirement of intact Talpha2 TCF/LEF and ets sites for enhancer function. We have created a mutant version of Ealpha, EalphaMC, in which Edelta myb and runx sites have been substituted for Talpha2 runx and ets sites, that argues against the notion of a requirement for strict Ealpha enhanceosome structure for function. EalphaMC resulted in a very potent enhancer indicating that stereospecific interactions among proteins that form an Ealpha enhanceosome are rather flexible. Activation of V(D)J recombination by EalphaMC during thymocyte development resulted, however, to be premature and indistinguishable from that of Talpha1-Talpha2. These results indicate that Talpha3-Talpha4 itself is not sufficient to impart a developmental delay to a chimeric "early" enhancer, and indicate the need for functional collaboration between Talpha2 runx/ets sites binding proteins and proteins bound to Talpha3-Talpha4 for proper developmental activation. The possibility of assembly of distinct sets of proteins on Ealpha might represent a more flexible form of information processing during thymocyte development.

Involvement of GATA transcription factors in the regulation of endogenous bovine interferon‐Tau gene transcription

Expression of interferon-tau (IFNT), necessary for pregnancy establishment in ruminant ungulates, is regulated in a temporal and spatial manner. However, molecular mechanisms by which IFNT gene transcription is regulated in this manner have not been firmly established. In this study, DNA microarray/RT-PCR analysis between bovine trophoblast CT-1 and Mardin-Darby bovine kidney (MDBK) cells was initially performed, finding that transcription factors GATA2, GATA3, and GATA6 mRNAs were specific to CT-1 cells. These mRNAs were also found in Days 17, 20, and 22 (Day 0 = day of estrus) bovine conceptuses. In examining other bovine cell lines, ovary cumulus granulosa (oCG) and ear fibroblast (EF) cells, GATA2 and GATA3, but not GATA6, were found specific to the bovine trophoblast cells. In transient transfection analyses using the upstream region (-631 to +59 bp) of bovine IFNT gene (bIFNT, IFN-tau-c1), over-expression of GATA2/GATA3 did not affect the transcription of bIFNT-reporter construct in human choriocarcinoma JEG3 cells. Transfection of GATA2, GATA3, ETS2, and/or CDX2, however, was effective in the up-regulation of the bIFNT construct transfected into bovine oCG and EF cells. One Point mutation studies revealed that among six potential GATA binding sites located on the upstream region of the bIFNT gene, the one next to ETS2 site exhibited reduced luciferase activity. In CT-1 cells, endogenous bIFNT gene transcription was up-regulated by over-expression of GATA2 or GATA3, but down-regulated by siRNA specific to GATA2 mRNA. These data suggest that GATA2/3 is involved in trophoblast-specific regulation of bIFNT gene transcription.

Transcriptional Regulation of the Human Growth Hormone Receptor (hGHR) Gene V2 Promoter by Transcriptional Activators and Repressor

The V2 transcript is the major ubiquitously expressed human GH receptor (hGHR) mRNA in all tissues examined to date. In a previous investigation, we defined the V2 promoter as TATA-less and exhibiting many characteristics of a housekeeping gene promoter. We also demonstrated that its basal activity is determined by several different cis-regulatory regions within both the promoter and the V2 exon. In the present study, we used luciferase-reporter, site-directed mutagenesis, gel shift, chromatin immunoprecipitation, and quantitative RT-PCR assays to investigate the ability of certain transcription factors to regulate hGHR V2 transcription through these regions in mammalian cells, including human adipocytes. Ets1 was found to transactivate the V2 proximal promoter through specific Ets sites. Two CCAAT/enhancer-binding protein (C/EBP) family members [C/EBP-homologous protein (CHOP) and C/EBPbeta] enhanced V2 transcription via different pathways: indirectly, by association with a V2 exon region (CHOP), and directly, using a V2 proximal promoter noncanonical binding site (C/EBPbeta). The Notch signaling mediator, Hes1, potently suppressed V2 promoter activity through interaction with two Hes sites within the V2 exon. We propose that these transcriptional factors regulate hGHR V2 expression by acting as downstream nuclear effectors, linking specific signaling cascades (e.g. MAPK and Notch) triggered by different growth factor-, development-, and nutrition- as well as stress-related stimuli. Our data also suggest that these factors are likely to be important in the differentiation-induced increase in V2 mRNA expression in adipocytes, with Ets1 and CHOP functioning at the preadipocyte stage to prepare the cells for differentiation and increasing C/EBPs and decreasing Hes1 levels contributing during adipocyte maturation.

Increased Transcription of the Neuropeptide Neuromedin U by Ets-2 and C-Myb Not B-Myb in Human Myeloid Leukemia Cells Promotes Their Proliferation.

Neuromedin U (NmU) is a 25-amino acid neuropeptide that is expressed at high levels in the brain, gut, and bone marrow. In the brain, NmU modulates the central control of feeding and bone mass. In the intestine, NmU regulates the contraction of smooth muscle, local blood flow, and ion transport. In the bone marrow, the physiological function of NmU remains ill-defined. To determine the physiological function of NmU in hematopoietic cells, we utilized K562-MERT cells, which express a tamoxifen-inducible dominant negative Myb (MERT), and cell culture assays. We chose to test the function of NmU in a cell line that expresses high levels of the proto-oncogene c-myb because c-Myb encodes a transcription factor that regulates cell proliferation, survival, and differentiation through the transactivation of its target genes. By inhibiting endogenous Myb activity in K562-MERT cells by tamoxifen in methylcellulose cultures, we observed a 6-fold decrease in cell number compared to untreated K562-MERT cells. Supplementing the tamoxifen treated methylcellulose cultures of K562-MERT cells with NmU resulted in a 3.6-fold increase in K562-MERT cell number compared to K562-MERT cells treated with only tamoxifen. In liquid cultures using primary cells from patients with acute myeloid leukemia, we observed more cells in the cultures with NmU than when NmU was absent from the cultures. Finally, silencing NmU gene expression in K562 cells via RNA interference decreased the proliferation of these cells. Collectively, these data demonstrate that NmU functions to promote the proliferation of hematopoietic cells. The ability of NmU to rescue cell growth in tamoxifen treated K562-MERT cells and Myb's role in regulating hematopoietic cell proliferation led us to hypothesize that Myb mediates hematopoietic cell proliferation in part by directly regulating NmU gene expression. To test this hypothesis, we examined the DNA sequence upstream of NmU's predicted transcription start site (as noted in Genbank accession #NM_006681) for potential Myb response elements (MREs). We identified eleven potential MREs within the first 2kb upstream of NmU's transcription start site. Of these MREs, five were identified as canonical (PyAAC(G/T)G). Our search also identified potential Ets-2 binding motifs within the human NmU promoter region, which were of interest because Myb has been reported to cooperate with Ets-2 in the regulation of c-kit and CD34 gene expression. To determine if any of the potential MREs within the NmU promoter were functional, we first completed in vitro assays using luciferase reporter constructs followed by in vivo assays using chromatin immunoprecipitation (ChIP) assays. The luciferase reporter constructs were generated such that the first 2kb upstream of NmU's transcription start site was inserted upstream of the luciferase gene in pGL3-basic to yield pGL3-NmU. When pGL3-NmU was co-transfected into 293T cells with a c-Myb expression construct, we observed an average of 15-fold induction of luciferase activity compared to empty vector. There was no change in luciferase activity when 293T cells were co-transfected with the c-Myb isoform B-Myb compared to empty vector, suggesting that c-Myb and not B-Myb interact with the MREs within the NmU promoter to induce its expression. Mutation of either MRE9 or 10, which are distal to NmU's transcription start site, in pGL3-NmU prevented c-Myb from inducing luciferase gene expression, demonstrating that MRE9 and 10 are functional. When Ets-2 was co-transfected with pGL3-NmU into 293T cells, a 10-fold increase in luciferase activity was observed suggesting that Ets-2 and c-Myb may cooperate to increase the transcription of NmU. To determine the physiologic relevance of our in vitro luciferase studies, we performed ChIP assays. Following immunoprecipitation of the cross-linked chromatin with either anti-c-Myb or anti-Ets-2, a PCR product of the recovered DNA was observed using primers that flanked MRE9, MRE10, and one Ets-2 site, demonstrating that in vivo c-Myb and Ets-2 interact directly with NmU's promoter. Studies are underway to determine whether c-Myb and Ets-2 cooperate to induce NmU gene expression. Further characterization of the regulation of NmU gene expression in normal and malignant hematopoietic cells may yield new clues to Myb's role in leukemogenesis and could suggest new therapeutic targets in human myeloid leukemia cells.

Phorbol-12-myristate 13-Acetate Acting through Protein Kinase Cε Induces Translocator Protein (18-kDa) Tspo Gene Expression

Translocator protein (TSPO) is an 18-kDa cholesterol-binding protein that is expressed at high levels in steroid synthesizing and several cancer cells where it is involved in steroidogenesis and cell proliferation, respectively. The factors regulating Tspo expression are unknown. We analyzed Tspo transcriptional responses to the tumor promoter, phorbol-12-myristate 13-acetate (PMA), in cells with varying TSPO levels. PMA induced Tspo promoter activity and Tspo mRNA levels in TSPO-poor nonsteroidogenic cells (NIH-3T3 fibroblasts and COS-7 kidney) but not in TSPO-rich steroidogenic cells (MA-10 Leydig) with high basal Tspo transcriptional activity. The stimulatory effect of PMA was mediated by an 805-515-bp region upstream of the transcription start site. Electrophoretic mobility shift assay (EMSA) revealed that PMA induced binding of c-jun and GA-binding protein transcription factor (GABP-alpha) to their respective activator protein 1 (AP1) and v-ets erythroblastosis virus E26 oncogene homologue (Ets) sites in this region. Protein kinase C (PKC)-specific inhibitors blocked PMA induction of Tspo promoter activity with an inhibition profile suggestive of involvement of PKCepsilon. PKCepsilon expression correlated with TSPO content in the three cell lines. In NIH-3T3 cells, PKCepsilon overexpression induced Tspo promoter activity and mRNA levels and enhanced PMA-induced up regulation of c-jun and TSPO. In MA-10 cells, a PKCepsilon-specific translocation inhibitor peptide reduced basal Tspo promoter activity. PKCepsilon siRNA pool reduced PKCepsilon and TSPO levels in MA-10 cells indicating a role for PKCepsilon in regulating TSPO expression. Taken together, these data suggest that elevated TSPO expression in steroidogenic cells may be due to high constitutive expression of PKCepsilon that renders them unresponsive to further induction while PMA activation of PKCepsilon drives inducible TSPO expression in nonsteroidogenic cells, likely through AP1 and Ets.

DNA methylation inhibition increases T cell KIR expression through effects on both promoter methylation and transcription factors

Killer-cell immunoglobulin-like receptor (KIR) genes are a polymorphic family expressed on NK cells, and “senescent” CD28− T cells implicated in cardiovascular disease. KIR promoters are highly homologous, and NK expression is regulated by DNA methylation. T cell KIR regulation is poorly understood. We asked if epigenetic mechanisms and/or transcription factor alterations determine T cell KIR expression. DNA methylation inhibition activated multiple KIR genes in normal T cells. KIR2DL2 and KIR2DL4 were selected for further study. Expression of both was associated with promoter demethylation, and methylation of the promoters in reporter constructs suppressed expression. KIR reporter construct expression also increased in demethylated T cells and required Ets1, Sp1 and AML sites, implying effects on transcription factors. This was confirmed for Sp1. These results indicate that KIR genes are suppressed by DNA methylation in most T cells, and DNA demethylation promotes their expression through effects on both chromatin structure and transcription factors.

Retinoic acid-dependent activation of the polycystic kidney disease-1 (PKD1) promoter

The retinoic acids all-trans retinoic acid (AT-RA) and 9-cis retinoic acid (9C-RA) and the retinoic acid receptors RAR and RXR significantly induce transcriptional activity from a 200-bp PKD1 proximal promoter in transfected mammalian cells. This PKD1 promoter region contains Ets, p53, and GC box motifs, but lacks a canonical RAR/RXR motif. Mutagenesis of the Ets sites did not affect RA induction. In contrast, GC box mutations completely blocked stimulation by AT-RA and by RXRbeta or RARbeta. Mithramycin A, which prevents Sp1 binding, significantly reduced basal promoter activity and suppressed upregulation by AT-RA and RXR. The 200-bp proximal promoter could not be induced by AT-RA in Drosophila SL2 cells, which lack Sp1, but could be activated in these cells transfected with exogenous Sp1. Small interfering RNA knockdown of Sp1 in mammalian cells completely blocked RXRbeta upregulation of the promoter. These data indicate that induction of the PKD1 promoter by retinoic acid is mediated through Sp1 elements. RT-PCR showed that AT-RA treatment of HEK293T cells increased the levels of endogenous PKD1 RNA, and chromatin immunoprecipitation showed the presence of both RXR and Sp1 at the PKD1 proximal promoter. These results suggest that retinoids and their receptors may play a role in PKD1 gene regulation.

Characterization of the transcriptional regulation of the human MT1-MMP gene and association of risk reduction for focal-segmental glomerulosclerosis with two functional promoter SNPs

The matrix metalloproteinase MT1-MMP (MMP-14) is an important player in wound healing, bone development, angiogenesis, inflammation and tumour invasion. MT1-MMP also plays an important role in the development and resolution of experimental kidney diseases. The role of MT1-MMP was investigated for distinction between minimal-change glomerulonephritis (MCGN) and focal-segmental glomerulosclerosis (FSGS) that can sometimes be difficult due to sampling error in renal biopsy. We defined the transcriptional regulation of the human MT1-MMP and the influence of single nucleotide polymorphisms (SNPs) within its promoter region in renal mesangial cells with reporter gene constructs and gel sift analysis. Genomic DNA from healthy blood donors (n = 500) and from kidney biopsies with defined renal diseases (MCGN: n = 189, FSGS: n = 311) was screened for MT1-MMP promoter SNPs. Transcription of MT1-MMP is regulated by two enhancers, an Sp1 binding site and a regulatory region 1 (RR1). RR1 contains an Ets site binding the transcription factors Elf-1 and E1AF but not NFAT. The MT1-MMP promoter contains two SNPs (-378 T/C and -364 G/T) in close vicinity to the RR1. Occurrence of the SNP variant -378 C leads to strong inhibition of nuclear protein binding to the RR1 reducing its enhancer function. Appearance of either variant -378 C or variant -364 T in at least one copy of the MT1-MMP promoter was associated with a significant risk reduction for the development of FSGS (P < 0.048). Genetic testing for MT1-MMP promoter SNPs could put renal biopsy results into new perspective. An independent study will be required to verify these findings and their possible diagnostic value for differentiation between certain renal diseases.

Ets2 Maintains hTERT Gene Expression and Breast Cancer Cell Proliferation by Interacting with c-Myc

Human telomerase reverse transcriptase (hTERT) underlies cancer cell immortalization, and the expression of hTERT is regulated strictly at the gene transcription. Here, we report that transcription factor Ets2 is required for hTERT gene expression and breast cancer cell proliferation. Silencing Ets2 induces a decrease of hTERT gene expression and increase in human breast cancer cell death. Reconstitution with recombinant hTERT rescues the apoptosis induced by Ets2 depression. In vitro and in vivo analyses show that Ets2 binds to the EtsA and EtsB DNA motifs on the hTERT gene promoter. Mutation of either Ets2 binding site reduces the hTERT promoter transcriptional activity. Moreover, Ets2 forms a complex with c-Myc as demonstrated by co-immunoprecipitation and glutathione S-transferase pulldown assays. Immunological depletion of Ets2, or mutation of the EtsA DNA motif, disables c-Myc binding to the E-box, whereas removal of c-Myc or mutation of the E-box also compromises Ets2 binding to EtsA. Thus, hTERT gene expression is maintained by a mechanism involving Ets2 interactions with the c-Myc transcription factor and the hTERT gene promoter, a protein-DNA complex critical for hTERT gene expression and breast cancer cell proliferation.

The Role of Homeobox Protein Distal-Less 3 and Its Interaction with ETS2 in Regulating Bovine Interferon-Tau Gene Expression-Synergistic Transcriptional Activation with ETS21

Distal-less 3 (DLX3), a homeodomain transcription factor required for placental development in the mouse, modestly transactivates hCG-alpha subunit gene (hCGA) expression in human choriocarcinoma cells. Because hCG and interferon-tau (IFNT) are expressed in trophectoderm of primates and ruminants, respectively, we have tested the hypothesis that DLX3 regulates the genes for IFNT (IFNT). A bovine IFNT1 promoter (-457 to +66), linked to a luciferase (luc) reporter, was transactivated approximately 20-fold by overexpressing DLX3 in human JAr cells. Elimination of a potential DLX3-binding site (-54 GATAATGAG -46) by either truncation or mutagenesis abolished this effect. A sequence (-59 to -44) encompassing this site bound DLX3 specifically. Coexpression of DLX3 and ETS2, which is known to be a key regulator of IFNT expression, increased reporter activity by more than 250-fold, whereas deletion of the established ETS2 site (-79 to -70) eliminated the ability of DLX3 to transactivate the gene. Conversely, mutation of the DLX3 site significantly reduced the transactivational effects of ETS2. Both DLX3 and ETS2 are coexpressed in JAr cells and in an IFNT-producing, bovine trophoblast cell line, CT-1. The two can be immunoprecipitated together as a complex from CT-1 cells, and RNAi-mediated, partial knockdown of DLX3 expression reduced the production of IFNT by approximately 50+. Together, these results suggest that DLX3 has a central role in controlling IFNT gene expression by associating with ETS2 on the IFNT promoter.

1,25-Dihydroxyvitamin D3 Induces CCR10 Expression in Terminally Differentiating Human B Cells

In the B cell lineage, CCR10 is known to be selectively expressed by plasma cells, especially those secreting IgA. In this study, we examined the regulation of CCR10 expression in terminally differentiating human B cells. As reported previously, IL-21 efficiently induced the differentiation of activated human CD19+ B cells into IgD-CD38+ plasma cells in vitro. A minor proportion of the resulting CD19+IgD-CD38+ cells expressed CCR10 at low levels. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the active metabolite of vitamine D3, dramatically increased the proportion of CD19+IgD-CD38+ cells expressing high levels of CCR10. The 1,25-(OH)2D3 also increased the number of CCR10+ cells expressing surface IgA, although the majority of CCR10+ cells remained negative for surface IgA. Thus, 1,25-(OH)2D3 alone may not be sufficient for the induction of IgA expression in terminally differentiating human B cells. To further determine whether 1,25-(OH)2D3 directly induces CCR10 expression in terminally differentiating B cells, we next performed the analysis on the human CCR10 promoter. We identified a proximal Ets-1 site and an upstream potential vitamin D response element to be critical for the inducible expression of CCR10 by 1,25-(OH)2D3. We confirmed the specific binding of Ets-1 and 1,25-(OH)2D3-activated vitamin D receptor to the respective sites. In conclusion, 1,25-(OH)2D3 efficiently induces CCR10 expression in terminally differentiating human B cells in vitro. Furthermore, the human CCR10 promoter is cooperatively activated by Ets-1 and vitamin D receptor in the presence of 1,25-(OH)2D3.

ESE‐1 is a potent repressor of type II collagen gene (COL2A1) transcription in human chondrocytes

The epithelium-specific ETS (ESE)-1 transcription factor is induced in chondrocytes by interleukin-1beta (IL-1beta). We reported previously that early activation of EGR-1 by IL-1beta results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE-1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF-kappaB-dependent inhibition by IL-1beta. Of the ETS factors tested, this response was specific to ESE-1, since ESE-3, which was also induced by IL-1beta, suppressed COL2A1 promoter activity only weakly. In contrast, overexpression of ETS-1 increased COL2A1 promoter activity and blocked the inhibition by IL-1beta. These responses to ESE-1 and ETS-1 were confirmed using siRNA-ESE1 and siRNA-ETS1. In transient cotransfections, the inhibitory responses to ESE-1 and IL-1beta colocalized in the -577/-132 bp promoter region, ESE-1 bound specifically to tandem ETS sites at -403/-381 bp, and IL-1-induced binding of ESE-1 to the COL2A1 promoter was confirmed in vivo by ChIP. Our results indicate that ESE-1 serves a potent repressor function by interacting with at least two sites in the COL2A1 promoter. However, the endogenous response may depend upon the balance of other ETS factors such as ETS-1, and other IL-1-induced factors, including EGR-1 at any given time. Intracellular ESE-1 staining in chondrocytes in cartilage from patients with osteoarthritis (OA), but not in normal cartilage, further suggests a fundamental role for ESE-1 in cartilage degeneration and suppression of repair.

Combinatorial Roles of Protein Kinase A, Ets2, and 3′,5′-Cyclic-Adenosine Monophosphate Response Element-Binding Protein-Binding Protein/p300 in the Transcriptional Control of Interferon-τ Expression in a Trophoblast Cell Line

In ruminants, conceptus interferon-tau (IFNT) production is necessary for maintenance of pregnancy. We examined the role of protein kinase A (PKA) in regulating IFNT expression through the activation of Ets2 in JAr choriocarcinoma cells. Although overexpression of the catalytic subunit of PKA or the addition of 8-bromo-cAMP had little ability to up-regulate boIFNT1 reporter constructs on their own, coexpression with Ets2 led to a large increase in gene expression. Progressive truncation of reporter constructs indicated that the site of PKA/Ets2 responsiveness lay in a region of the promoter between -126 and -67, which lacks a cAMP response element but contains the functional Ets2-binding site and an activator protein 1 (AP1) site. Specific mutation of the former reduced the PKA/Ets2 effects by more than 98%, whereas mutation of an AP1-binding site adjacent to the Ets2 site or pharmacological inhibition of MAPK kinase 2 led to a doubling of the combined Ets2/PKA effects, suggesting there is antagonism between the Ras/MAPK pathway and the PKA signal transduction pathway. Although Ets2 is not a substrate for PKA, lowering the effective concentrations of the coactivators, cAMP response element-binding protein-binding protein (CBP)/p300, known PKA targets, reduced the ability of PKA to synergize with Ets2, suggesting that PKA effects on IFNT regulation might be mediated through CBP/p300 coactivation, particularly as CBP and Ets2 occupy the proximal promoter region of IFNT in bovine trophoblast CT-1 cells. The up-regulation of IFNT in the elongating bovine conceptus is likely due to the combinatorial effects of PKA, Ets2, and CBP/p300 and triggered via growth factors released from maternal endometrium.

A combinatorial code of maternal GATA, Ets and β-catenin-TCF transcription factors specifies and patterns the early ascidian ectoderm

Our understanding of the maternal factors that initiate early chordate development, and of their direct zygotic targets, is still fragmentary. A molecular cascade is emerging for the mesendoderm, but less is known about the ectoderm, giving rise to epidermis and nervous tissue. Our cis-regulatory analysis surprisingly places the maternal transcription factor Ci-GATAa (GATA4/5/6) at the top of the ectodermal regulatory network in ascidians. Initially distributed throughout the embryo, Ci-GATAa activity is progressively repressed in vegetal territories by accumulating maternal beta-catenin. Once restricted to the animal hemisphere, Ci-GATAa directly activates two types of zygotic ectodermal genes. First, Ci-fog is activated from the 8-cell stage throughout the ectoderm, then Ci-otx is turned on from the 32-cell stage in neural precursors only. Whereas the enhancers of both genes contain critical and interchangeable GATA sites, their distinct patterns of activation stem from the additional presence of two Ets sites in the Ci-otx enhancer. Initially characterized as activating elements in the neural lineages, these Ets sites additionally act as repressors in non-neural lineages, and restrict GATA-mediated activation of Ci-otx. We thus identify a precise combinatorial code of maternal factors responsible for zygotic onset of a chordate ectodermal genetic program.