Proximal Regulatory Element Research Articles

The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression.

Human prostate cancer can result from chromosomal rearrangements that lead to aberrant ETS gene expression. The mechanisms that lead to fusion-independent ETS factor upregulation and prostate oncogenesis remain relatively unknown. Here, we show that two neighboring transcription factors, Capicua (CIC) and ETS2 repressor factor (ERF), which are co-deleted in human prostate tumors can drive prostate oncogenesis. Concurrent CIC and ERF loss commonly occur through focal genomic deletions at chromosome 19q13.2. Mechanistically, CIC and ERF co-bind the proximal regulatory element and mutually repress the ETS transcription factor, ETV1. Targeting ETV1 in CIC and ERF-deficient prostate cancer limits tumor growth. Thus, we have uncovered a fusion-independent mode of ETS transcriptional activation defined by concurrent loss of CIC and ERF.

Active endogenous retroviral elements in human pluripotent stem cells play a role in regulating host gene expression

Human endogenous retroviruses, also called LTR elements, can be bound by transcription factors and marked by different histone modifications in different biological contexts. Recently, individual LTR or certain subclasses of LTRs such as LTR7/HERVH and LTR5_Hs/HERVK families have been identified as cis-regulatory elements. However, there are still many LTR elements with unknown functions. Here, we dissected the landscape of histone modifications and regulatory map of LTRs by integrating 98 ChIP-seq data in human embryonic stem cells (ESCs), and annotated the active LTRs enriching enhancer/promoter-related histone marks. Notably, we found that MER57E3 functionally acted as proximal regulatory element to activate respective ZNF gene. Additionally, HERVK transcript could mainly function in nucleus to activate the adjacent genes. Since LTR5_Hs/LTR5 was bound by many early embryo-specific transcription factors, we further investigated the expression dynamics in different pluripotent states. LTR5_Hs/LTR5/HERVK exhibited higher expression level in naïve ESCs and extended pluripotent stem cells (EPSCs). Functionally, the LTR5_Hs/LTR5 with high activity could serve as a distal enhancer to regulate the host genes. Ultimately, our study not only provides a comprehensive regulatory map of LTRs in human ESCs, but also explores the regulatory models of MER57E3 and LTR5_Hs/LTR5 in host genome.

Chromosome level reference of Atlantic halibut Hippoglossus hippoglossus provides insight into the evolution of sexual determination systems.

Changes in the genetic mechanisms that control sexual determination have occurred independently across the tree of life, and with exceptional frequency in teleost fishes. To investigate the genomic changes underlying the evolution of sexual determination, we sequenced a chromosome-level genome, multitissue transcriptomes, and reduced representation population data for the Atlantic halibut (Hippoglossus hippoglossus), which has an XY/XX sex determination mechanism and has recently diverged (0.9-3.8Ma) from the Pacific halibut (Hippoglossus stenolepis), which has a ZZ/ZW system. We used frequency and coverage-based population approaches to identify a putative sex-determining factor, GSDF. We characterized regions with elevated heterozygosity and linkage disequilibrium indicating suppression of recombination across a nascent sex chromosome. We detected testis-specific expression of GSDF, the sequence of which is highly conserved across flatfishes. Based on evidence from genome-wide association, coverage, linkage disequilibrium, testis and brain transcriptomes, and sequence conservation with other flatfishes, we propose a mechanism for the recent evolution of an XY sex-determination mechanism in Atlantic halibut. Changes to the ancestral sex-determining gene DMRT1 in regulating the downstream gene GSDF probably coincided with GSDF, or a proximal regulatory element of it, becoming the primary sex-determining factor. Our results suggest changes to a small number of elements can have drastic repercussions for the genomic substrate available to sex-specific evolutionary forces, providing insight into how certain elements repeatedly evolve to control sex across taxa. Our chromosome-level assembly, multitissue transcriptomes, and population genomic data provide a valuable resource and understanding of the evolution of sexual systems in fishes.

Capicua Suppresses  <i>YAP1</i> To Limit Tumorigenesis and Maintain Drug Sensitivity in Human Cancer

Inactivation of Capicua (CIC) or upregulation of yes-associated protein 1, YAP1, leads to broad RAS-RAF-MEK-ERK inhibitor resistance and tumor progression in multiple human cancers. Despite these shared malignant phenotypes, it remains unknown if CIC and YAP1 are mechanistically linked. Here, we show that the ERK-regulated transcription factor CIC can directly repress YAP1 transcription through non-consensus GGAAGGAA DNA-binding motifs in a proximal YAP1 regulatory element. Through binding at GGAA repeats, CIC regulates YAP1 transcriptional output in both normal and human cancer cells. Silencing YAP1 in CIC -deficient cells restores MAPK inhibitor sensitivity and suppresses tumor growth. Thus, we have uncovered a molecular link between the MAPK-ERK effector CIC and YAP1 in human cells and establish YAP1 inhibition as a strategy to target CIC-deficient cancers.

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The expression pattern of MHC class I genes in trophoblast cells at the feto–maternal interface is thought to be the basis of the maintenance of pregnancy by protecting the fetus from maternal immune rejection. Transcription of classical HLA class I genes is low or undetectable in most trophoblast cells as well as in JEG-3 and BeWo trophoblast-derived choriocarcinoma cells. The aim of this study was to characterize the regulatory mechanisms that repress HLA-A transcription in these cell types. We show that the −335 to ATG region of the HLA-A11 gene promoter is inactive in JEG-3 and BeWo cells while able to control efficient reporter gene transcription in other cell types, indicating that this region is the target for downregulation of HLA-A expression in choriocarcinoma cell lines. The regulatory sequence involved in HLA-A gene repression was further mapped to a proximal regulatory element within the −107 to +2 ATG region of the promoter. We show that the HLA-A promoter activity cannot be induced by interferon-γ (IFN-γ) and that exogenous MHC class II transactivator CIITA is able to induce HLA class I promoter activity in these cells. Stringent transcriptional regulatory mechanisms, implicating the lack of basal and IFN-γ-inducible class I promoter activity, are thus involved in the downregulation of HLA-A expression in JEG-3 and BeWo trophoblast-derived choriocarcinoma cells.

Lrrc10 is a novel cardiac-specific target gene of Nkx2-5 and GATA4

Cardiac gene expression is precisely regulated and its perturbation causes developmental defects and heart disease. Leucine-rich repeat containing 10 (Lrrc10) is a cardiac-specific factor that is crucial for proper cardiac development and deletion of Lrrc10 in mice results in dilated cardiomyopathy. However, the mechanisms regulating Lrrc10 expression in cardiomyocytes remain unknown. Therefore, we set out to determine trans-acting factors and cis-elements critical for mediating Lrrc10 expression. We identify Lrrc10 as a transcriptional target of Nkx2-5 and GATA4. The Lrrc10 promoter region contains two highly conserved cardiac regulatory elements, which are functional in cardiomyocytes but not in fibroblasts. In vivo, Nkx2-5 and GATA4 endogenously occupy the proximal and distal cardiac regulatory elements of Lrrc10 in the heart. Moreover, embryonic hearts of Nkx2-5 knockout mice have dramatically reduced expression of Lrrc10. These data demonstrate the importance of Nkx2-5 and GATA4 in regulation of Lrrc10 expression in vivo. The proximal cardiac regulatory element located at around −200bp is synergistically activated by Nkx2-5 and GATA4 while the distal cardiac regulatory element present around −3kb requires SRF in addition to Nkx2-5 and GATA4 for synergistic activation. Mutational analyses identify a pair of adjacent Nkx2-5 and GATA binding sites within the proximal cardiac regulatory element that are necessary to induce expression of Lrrc10. In contrast, only the GATA site is functional in the distal regulatory element. Taken together, our data demonstrate that the transcription factors Nkx2-5 and GATA4 cooperatively regulate cardiac-specific expression of Lrrc10.

PuF, an antimetastatic and developmental signaling protein, interacts with the Alzheimer's amyloid-β precursor protein via a tissue-specific proximal regulatory element (PRE).

BackgroundAlzheimer’s disease (AD) is intimately tied to amyloid-β (Aβ) peptide. Extraneuronal brain plaques consisting primarily of Aβ aggregates are a hallmark of AD. Intraneuronal Aβ subunits are strongly implicated in disease progression. Protein sequence mutations of the Aβ precursor protein (APP) account for a small proportion of AD cases, suggesting that regulation of the associated gene (APP) may play a more important role in AD etiology. The APP promoter possesses a novel 30 nucleotide sequence, or “proximal regulatory element” (PRE), at −76/−47, from the +1 transcription start site that confers cell type specificity. This PRE contains sequences that make it vulnerable to epigenetic modification and may present a viable target for drug studies. We examined PRE-nuclear protein interaction by gel electrophoretic mobility shift assay (EMSA) and PRE mutant EMSA. This was followed by functional studies of PRE mutant/reporter gene fusion clones.ResultsEMSA probed with the PRE showed DNA-protein interaction in multiple nuclear extracts and in human brain tissue nuclear extract in a tissue-type specific manner. We identified transcription factors that are likely to bind the PRE, using competition gel shift and gel supershift: Activator protein 2 (AP2), nm23 nucleoside diphosphate kinase/metastatic inhibitory protein (PuF), and specificity protein 1 (SP1). These sites crossed a known single nucleotide polymorphism (SNP). EMSA with PRE mutants and promoter/reporter clone transfection analysis further implicated PuF in cells and extracts. Functional assays of mutant/reporter clone transfections were evaluated by ELISA of reporter protein levels. EMSA and ELISA results correlated by meta-analysis.ConclusionsWe propose that PuF may regulate the APP gene promoter and that AD risk may be increased by interference with PuF regulation at the PRE. PuF is targeted by calcium/calmodulin-dependent protein kinase II inhibitor 1, which also interacts with the integrins. These proteins are connected to vital cellular and neurological functions. In addition, the transcription factor PuF is a known inhibitor of metastasis and regulates cell growth during development. Given that APP is a known cell adhesion protein and ferroxidase, this suggests biochemical links among cell signaling, the cell cycle, iron metabolism in cancer, and AD in the context of overall aging.

LEF-1 Negatively Controls Interleukin-4 Expression through a Proximal Promoter Regulatory Element

Lymphoid enhancer-binding factor 1 (LEF-1) and T cell factor (TCF-1) are downstream effectors of the Wnt signaling pathway and are involved in the regulation of T cell development in the thymus. LEF-1 and TCF-1 are also expressed in mature peripheral primary T cells, but their expression is down-regulated following T cell activation. Although the decisive roles of LEF-1 and TCF-1 in the early stages of T cell development are well documented, the functions of these factors in mature peripheral T cells are largely unknown. Recently, LEF-1 was shown to suppress Th2 cytokines interleukin-4 (IL-4), -5, and -13 expression from the developing Th2 cells that overexpress LEF-1 through retrovirus gene transduction. In this study, we further investigated the expression and functions of LEF-1 and TCF-1 in peripheral CD4+ T cells and revealed that LEF-1 is dominantly expressed in Th1 but not in Th2 cells. We identified a high affinity LEF-1-binding site in the negative regulatory element of the IL-4 promoter. Knockdown LEF-1 expression by LEF-1-specific small interfering RNA resulted in an increase in the IL-4 mRNA expression. This study further confirms a negative regulatory role of LEF-1 in mature peripheral T cells. Furthermore, we found that IL-4 stimulation possesses a negative effect on the expressions of LEF-1 and TCF-1 in primary T cells, suggesting a positive feedback effect of IL-4 on IL4 gene expression.

Characterization of the sequence and architectural constraints of the regulatory and core regions of the human interleukin-2 promoter

The cytokine interleukin-2 (IL-2) is produced by T cells when they recognize a foreign antigen. Transcription of the IL-2 gene is tightly controlled by the combined actions of multiple transcriptional activators. However, the contribution of sequences in the IL-2 core promoter and the architecture of the IL-2 regulatory region to setting levels of IL-2 transcription are not understood. We have probed these properties of the human IL-2 promoter to understand how the regulatory and core promoter regions cooperate in response to T cell stimulation, thereby setting high levels of inducible transcription. We found that the IL-2 core promoter contains a TATA box that is critical for inducible expression. Moreover, the spacing and orientation between the IL-2 regulatory and core promoter regions is important for setting the level of transcription. The regulatory region of the IL-2 promoter is capable of mediating high levels of expression even when the helical phasing between transcription factor binding sites is perturbed. Although long considered an enhancer, our studies indicate that the regulatory region in the IL-2 promoter is better considered as a proximal regulatory element, since it lacks multiple properties associated with enhancer elements.

Hepatocyte nuclear factor 4a isoform originated from the P1 promoter regulates CAR expression in adult human liver.

CAR (Constitutive Androstane Receptor, NR1I3) and PXR (Pregnane X Receptor, NR1I2) are nuclear receptors that control both endogenous and exogenous toxic compounds metabolism and elimination. In contrasts to PXR, CAR is expressed only in adult liver and not in fetal liver. Factors regulating human CAR (hCAR) gene expression remain largely unexplored. We focused our attention on the effect of Hepatocyte Nuclear Factor (HNF)-4a isoforms onto hCAR gene promoter. HNF-4a gene possesses two promoters, proximal P1 and distal P2, whose use results in HNF-4a1 and HNF4a7 transcripts, respectively. While HNF-4a7 is mainly expressed in the embryonic liver, HNF-4a1 is almost exclusively in the adult liver. Transient transfections showed that HNF-4a1, but not HNF-4a7, enhances hCAR promoter activity. 5′-Deletion and mutation analysis of hCAR promoter identified a proximal DR1 regulatory element, termed CAR HNF-4a-RE (−114ccAGGCCTtTGCCCTga). HNF-4a1 increased the activity of a heterologous promoter driven by two copies of the CAR HNF-4a-RE. We demonstrate that in vitro synthetized HNF-4a binds to this element in gel shift assays, while endogenous HNF4-a interacts with hCAR promoter in chromatin immunoprecipitation studies performed with human hepatocytes. In addition, we observed that glucocorticoid receptor and HNF-4a1 (but not HNF4-a7) cooperate to maximally transactivate CAR gene expression, while HNF4-a7 repressed this cooperation. Finally, we observed a strong correlation between CAR and HNF-4a1 mRNA expression level in human liver tissu samples, and an inverse correlation between CAR and HNF-4a7 in human hepatoma suggesting that, while HNF-4a1 positively regulates CAR expression in adult, the fetal HNF-4a7 isoform represses CAR gene expression.

Molecular Basis of Estrogen-Induced Cyclooxygenase Type 1 Upregulation in Endothelial Cells

Estrogen upregulates cyclooxygenase-1 (COX-1) expression in endothelial cells. To determine the basis of this process, studies were performed in ovine endothelial cells transfected with the human COX-1 promoter fused to luciferase. Estradiol (E2) caused activation of the COX-1 promoter with maximal stimulation at 10(-8) mol/L E2, and the response was mediated by either ERalpha or ERbeta. Mutagenesis revealed a primary role for a putative Sp1 binding motif at -89 (relative to the ATG codon) and lesser involvement of a consensus Sp1 site at -111. Electrophoretic mobility shift assays yielded a single complex with the site at -89, and supershift analyses implicated AP-2alpha and ERalpha, and not Sp1, in protein-DNA complex formation. In endothelial cells with minimal endogenous ER, the transfection of ERalpha mutants lacking the DNA binding domain or primary nuclear localization signals caused 4-fold greater stimulation of promoter activity with E2 than wild-type ERalpha. In contrast, mutant ERalpha lacking the A-B domains was inactive. Thus, estrogen-mediated upregulation of COX-1 in endothelium is uniquely independent of direct ERalpha-DNA binding and instead entails protein-DNA interaction involving AP-2alpha and ERalpha at a proximal regulatory element. In addition, the process may be initiated by cytoplasmic ERalpha, and critical receptor elements reside within the amino terminus.

TEF-1 transcription factors regulate activity of the mouse mammary tumor virus LTR

The mouse mammary tumor virus long terminal repeat (LTR) is a potent transcriptional enhancer. We identified several putative binding sites for the TEF-1 family of transcription factors (TEF-1, RTEF-1, DTEF-1, and ETF) in the proximal negative regulatory element of the LTR. Gel mobility shift assays revealed strong TEF-1 factor binding to one site using nuclear extracts from CV-1 cells and from the human breast cancer cell line MCF-7. Mutation of this site increased basal activity of the LTR. In transient transfection assays, TEF-1 squelched the basal LTR activity and completely abrogated the response to the glucocorticoid dexamethasone. RTEF-1 and DTEF-1 had little effect on the basal activity, whereas ETF activated the LTR. These TEF-1 factors also interfered with the response to dexamethasone. Taken together, our results reveal an important new role for TEF-1 factors in regulating MMTV LTR activity and suggest that TEF-1 factors might participate in mammary tumorigenesis.

Function of GATA transcription factors in hydroxyurea-induced HEL cells.

HEL cells, a human erythroleukemia cell line, mainly express the fetal (gamma) globin gene and trace amount of the embryonic (epsilon) globin gene, but not adult (beta) globin gene. Here we show that hydroxyurea (HU) can induce HEL cells to express adult (beta) globin gene and lead these cells to terminal differentiation. Results showed in Gel mobility shift assays that GATA factors could specifically bind to the regulatory elements of human beta-globin gene, including the proximal regulatory element (the beta-promoter) and the distal regulatory elements (the DNase I hypersensitive sites in the LCR, HS2-HS4 core sequences). However, the DNA binding patterns of GATA factors were quite different between HU-induced and uninduced HEL cells. Western-blot analysis of nuclear extracts from both the uninduced and HU-induced HEL cells revealed that the level of GATA-2 transcription factor decreased, whereas the level of GATA-1 transcription factor increased following the time of hydroxyurea induction. Furthermore, using RT-PCR analysis the expression of human beta-globin gene in HU-induced HEL cells could be blocked again when HEL cells were incubated in the presence of antisense oligonucleotides for hGATA-1, suggesting that the upregulation of hGATA-1 transcription factor might be critical for the expression of human beta-globin gene in HU-induced HEL cells.

Induction of Secreted Type IIA Phospholipase A2 Gene Transcription by Interleukin-1β: ROLE OF C/EBP FACTORS

Secreted type IIA phospholipase A(2), which is involved in arachidonic acid release, is abundantly produced by chondrocytes and secreted in the synovial fluids of patients affected by rheumatoid arthritis. Transfection experiments showed that interleukin-1beta stimulates the phospholipase A(2) [-1614; +20] promoter activity by 6-7-fold and that the [-210; -176] fragment is critical for this stimulation. CAAT enhancer-binding protein (C/EBP) beta and C/EBPdelta transcription factors bind to this element as shown by bandshift experiments. Interleukin-1beta increased the levels of C/EBPdelta mRNA as soon as 2 h and up to 24 h without affecting those of C/EBPbeta. Higher amounts of C/EBPdelta proteins correlate with the stimulation of C/EBPdelta mRNA. Mutations or 5' deletions in the upstream [-247; -210] region reduced by 2-fold the basal and interleukin-1beta-stimulated transcription activities. Two types of factors bind to overlapping sequences on this fragment: NF1-like proteins and the glucocorticoid receptor. The glucocorticoid receptor is responsible for a moderate stimulation of the promoter activity by dexamethasone and may interact with C/EBP factors to achieve a full transcription activity in basal conditions and in the presence of interleukin-1beta. A [-114; -85] proximal regulatory element forms three complexes in bandshift experiments, the slowest mobility one involving the Sp1 zinc finger factor. Mutation of this sequence reduced to 2-fold the stimulation of the promoter activity by interleukin-1beta or the C/EBP factors. Induction of the transcription of secreted type IIA phospholipase A(2) gene by interleukin-1beta in chondrocytes absolutely requires C/EBPbeta and C/EBPdelta factors but does not involve NF-kappaB.

Downregulation of HLA Class I Gene Transcription in Choriocarcinoma Cells is Controlled by the Proximal Promoter Element and Can be Reversed by CIITA

The expression pattern of MHC class I genes in trophoblast cells at the feto–maternal interface is thought to be the basis of the maintenance of pregnancy by protecting the fetus from maternal immune rejection. Transcription of classical HLA class I genes is low or undetectable in most trophoblast cells as well as in JEG-3 and BeWo trophoblast-derived choriocarcinoma cells. The aim of this study was to characterize the regulatory mechanisms that repress HLA-A transcription in these cell types. We show that the −335 to ATG region of the HLA-A11 gene promoter is inactive in JEG-3 and BeWo cells while able to control efficient reporter gene transcription in other cell types, indicating that this region is the target for downregulation of HLA-A expression in choriocarcinoma cell lines. The regulatory sequence involved in HLA-A gene repression was further mapped to a proximal regulatory element within the −107 to +2 ATG region of the promoter. We show that the HLA-A promoter activity cannot be induced by interferon-γ (IFN-γ) and that exogenous MHC class II transactivator CIITA is able to induce HLA class I promoter activity in these cells. Stringent transcriptional regulatory mechanisms, implicating the lack of basal and IFN-γ-inducible class I promoter activity, are thus involved in the downregulation of HLA-A expression in JEG-3 and BeWo trophoblast-derived choriocarcinoma cells.

Purification and characterization of TEF1, a transcription factor that controls the human transforming growth factor-α promoter

Transforming growth factor-α (TGF-α) is a member of the epidermal growth factor family. It activates signal transduction pathways leading to cell proliferation through the interaction with cell surface epidermal growth factor receptor. The overexpression of TGF-α has been found in many types of cancers and is thought to be involved in the genesis and maintenance of these tumors. Recent results also implicate this growth factor in the development of certain diabetic complications, such as atherosclerosis. The function of TGF-α can be tightly controlled at the level of transcription of its gene. We have previously characterized the proximal TGF-α promoter and identified two neighboring regulatory elements that appeared to cooperate with each other in the regulation of TGF-α transcription. The transcription factor that functions through the distal element was identified as AP-2, a protein that was found to be induced by the oncoprotein, Ras. However, what factor binds and controls the proximal regulatory element (PRE) is still unclear. Here, we report the purification and preliminary characterization of the PRE-binding transcription factor TEF1 by sequence-specific DNA-affinity chromatography from rat kidney nuclear extracts. The purified TEF1 migrates on the SDS–PAGE at a molecular mass of about 36 kDa. It specifically interacts with the PRE and was able to strongly activate transcription from the TGF-α promoter in HeLa cell nuclear extracts in an in vitro transcription assay. The UV cross-linking experiment confirmed that this 36 kDa protein is indeed the protein that specifically binds the PRE. We also show that the spacing between the AP-2 and the TEF1 sites in the TGF-α promoter has little effect on the transcription from the TGF-α promoter. The purification of TEF1 furthers our understanding of how TGF-α expression is regulated and may help us understand the upstream signaling events that lead to the elevated expression of this growth factor.

IL-4 and prostaglandin E 2 inhibit hypomethylation of the 5′ regulatory region of IFN-γ gene during differentiation of naive CD4 + T cells

We have previously shown that prostaglandin E 2 (PGE2) and IL-4 inhibit the priming of IFN-γ-production during the differentiation of naive CD4 + T cells from human cord blood by different signal-transducing mechanisms. To compare and analyse the molecular mechanisms by which PGE2 and IL-4 inhibit the priming of IFN-γ production, we investigated the effects of PGE2 and IL-4 on the methylation of the IFN-γ gene during the in vitro differentiation of naive CD4 + T cells. In human naive CD4 + T cells, which produce primarily IL-2 and a little amount of IFN-γ, the IFN-γ gene was methylated. After stimulation via TCR, CD4 + T cells produced IFN-γ and the CpG dinucleotide contained within the TATA proximal regulatory element of the IFN-γ gene was partially hypomethylated. Both IL-4 and PGE2 inhibited the hypomethylation of this site and the acquisition of IFN-γ-producing ability. In contrast to the SnaBI site in the TATA proximal regulatory element, the HpaII site in the first intron of the IFN-γ gene of the CD4 + T cells from cord blood was completely methylated even after stimulation via TCR. 5-azacytidine restored the IFN-γ-producing ability of these cells treated with IL-4 and PGE2. These findings suggest that, although the signal transduction that inhibits the priming of IFN-γ-production is different for each reagent, the protection from hypomethylation of the regulatory region of the IFN-γ gene is involved in the molecular mechanisms by which these reagents inhibit the priming of IFN-γ-production during the differentiation of human naive CD4 + T cells.

IL-4 and prostaglandin E2 inhibit hypomethylation of the 5' regulatory region of IFN-gamma gene during differentiation of naive CD4+ T cells.

We have previously shown that prostaglandin E2 (PGE2) and IL-4 inhibit the priming of IFN-gamma-production during the differentiation of naive CD4+ T cells from human cord blood by different signal-transducing mechanisms. To compare and analyse the molecular mechanisms by which PGE2 and IL-4 inhibit the priming of IFN-gamma production, we investigated the effects of PGE2 and IL-4 on the methylation of the IFN-gamma gene during the in vitro differentiation of naive CD4+ T cells. In human naive CD4+ T cells, which produce primarily IL-2 and a little amount of IFN-gamma, the IFN-gamma gene was methylated. After stimulation via TCR, CD4+ T cells produced IFN-gamma and the CpG dinucleotide contained within the TATA proximal regulatory element of the IFN-gamma gene was partially hypomethylated. Both IL-4 and PGE2 inhibited the hypomethylation of this site and the acquisition of IFN-gamma-producing ability. In contrast to the SnaBI site in the TATA proximal regulatory element, the HpalI site in the first intron of the IFN-gamma gene of the CD4+ T cells from cord blood was completely methylated even after stimulation via TCR. 5-azacytidine restored the IFN-gamma-producing ability of these cells treated with IL-4 and PGE2. These findings suggest that, although the signal transduction that inhibits the priming of IFN-gamma-production is different for each reagent, the protection from hypomethylation of the regulatory region of the IFN-gamma gene is involved in the molecular mechanisms by which these reagents inhibit the priming of IFN-gamma-production during the differentiation of human naive CD4+ T cells.

Transcription Factor AP-2 Controls Transcription of the Human Transforming Growth Factor-α Gene

The epidermal growth factor receptor is vital for normal development and plays a role in oncogenesis. The level of activation of this receptor by transforming growth factor-alpha (TGF-alpha) is controlled, in part, by the rate of transcription of the TGF-alpha gene. In the characterization of the proximal TGF-alpha promoter by DNase I footprinting, a 43-base pair element (-88 to -130 relative to the transcription start site), designated TalphaRE I, was found that was specifically protected by nuclear proteins from human mammary carcinoma MDA468 cells. TalphaRE I was essential for the maximal expression of the TGF-alpha gene as indicated by deletion and mutagenesis analyses. TalphaRE I consists of two cis-acting elements, a proximal regulatory element (PRE, -89 to -103) and a distal regulatory element (DRE, -121 to -128). Both elements were able to form specific complexes with protein from MDA468 cell nuclear extracts and are necessary for the full activity of the entire 1.1-kilobase pair TGF-alpha promoter. Competition and antibody studies determined that the DRE contains a binding site for the transcription factor AP-2, while the protein that binds to the PRE has yet to be identified. When linked upstream to the heterologous herpes simplex thymidine kinase promoter, the TalphaRE I enhanced transcription up to 11-fold in MDA468 cells. Cotransfection of an AP-2 expression vector was able to activate transcription from the TalphaREI-TK construct in a DRE-dependent manner. These results further our understanding of how TGF-alpha transcription is regulated.

The Proximal Regulatory Element of the Interferon-γ Promoter Mediates Selective Expression in T Cells

Interferon-gamma (IFN-gamma) is produced by natural killer cells and certain subsets of T cells, but the basis for its selective expression is unknown. Within the region between -108 and -40 base pairs of the IFN-gamma promoter are two conserved and essential regulatory elements, which confer activation-specific expression in T cells. This report describes studies indicating that the most proximal of these two regulatory elements is an important determinant of its restricted expression. The proximal element is a composite site that binds members of the CREB/ATF, AP-1, and octamer families of transcription factors. Jun is essential for activation-induced transcription and binds preferably as a heterodimer with ATF-2. In contrast, CREB appears to dampen transcription from this element. The CpG dinucleotide in this element is selectively methylated in Th2 T cells and other cells that do not express IFN-gamma, and methylation markedly reduces transcription factor binding. As a target for DNA methylation and for binding of transcription factors that mediate or impede transcription, this element appears to play a central role in controlling IFN-gamma expression.