E2A and HEB activate the pre-TCR alpha promoter during immature T cell development.
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The pre-TCRalpha (pTalpha) is exclusively expressed in immature thymocytes and constitutes the pre-TCR complex with TCRbeta, which regulates early T cell differentiation. Despite the recent identification of the pTalpha enhancer, the contribution of the promoter region, the direct DNA-protein interaction, and the regulation of such interaction along with T cell development have not been investigated. We analyzed the pTalpha promoter region and identified the critical elements for transcription of the pTalpha gene. The pTalpha promoter was found to contain two consecutive E-box elements that are critical for pTalpha transcription. The E-box elements in the promoter region formed the specific DNA-protein complex that was exclusively observed in immature thymocytes, not in mature thymocytes and T cells. The E proteins in this complex were identified as E2A and HeLa E-box binding protein (HEB), and overexpression of E2A and HEB resulted in activation of the pTalpha promoter. The binding complex in the consecutive E-boxes in the pTalpha promoter changed along with T cell development, as a distinct DNA-binding complex was observed in mature T cells. Comparing the E-box regions in the enhancer and the promoter, those in the promoter appear to make a greater contribution to pTalpha gene transcription. (+info)
Regulation of E-box DNA binding during in vivo and in vitro activation of rat and human hepatic stellate cells.
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BACKGROUND: Activation of hepatic stellate cells (HSCs) to a myofibroblastic phenotype is a key event in liver fibrosis. Identification of transcription factors with activities that are modulated during HSC activation will improve our understanding of the molecular events controlling HSC activation. AIMS: To determine if changes in E-box DNA binding activity occur during in vitro and in vivo activation of rat and human HSCs and to investigate mechanisms underlying any observed changes. METHODS: Nuclear extracts were prepared from rat HSCs isolated and cultured from normal and carbon tetrachloride injured rat livers and from HSCs isolated from human liver. EMSA analysis of E-box DNA binding activity was performed on nuclear extracts to determine changes during HSC activation. Western and northern blot analysis of MyoD and Id1 basic helix-loop-helix (bHLH) proteins was performed to confirm expression in HSC. RESULTS: HSC activation was associated with inducible expression of two low mobility E-box binding complexes that were immunoreactive with an anti-MyoD antibody. MyoD mRNA expression was found at similar levels in freshly isolated and activated HSCs; in contrast, MyoD protein expression was elevated in activated HSCs. Activation of rat HSCs was accompanied by reduced expression of the inhibitory bHLH protein Id1. CONCLUSIONS: In vitro and in vivo activation of rat and human HSCs is accompanied by induction of MyoD binding to E-box DNA sequences which appears to be mechanistically associated with elevated MyoD protein expression and reduced expression of the inhibitory Id1 protein. Clarification of the role of MyoD and Id1 proteins in HSC activation and liver fibrogenesis is now required. (+info)
Transcriptional regulation of cyclooxygenase-2 gene in ovine large luteal cells.
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There is positive feedback pathway in the ovine large luteal cell, such that prostaglandin (PG) F(2 alpha) stimulation induces intraluteal PGF(2 alpha) production as the result of induction of one of the rate-limiting enzymes in PG production, cyclooxygenase-2 (Cox-2). The objective of the present study was to evaluate the intracellular effector systems and important DNA transcriptional element(s) involved in regulating the Cox-2 gene in ovine large luteal cells. In transient transfection assays, Cox-2 promoter was rapidly induced (4 h) by phorbol didecanoate (a protein kinase [PK] C activator), ionomycin, and cloprostenol (PGF(2 alpha) analogue), with a peak induction at 12 h. Cloprostenol-mediated promoter activation was not blocked by inhibition of various second messenger systems, including PKA, calcium calmodulin kinase II, or mitogen-activated protein kinases. However, myristoylated PKC pseudosubstrate peptide inhibited cloprostenol stimulation of Cox-2 promoter, indicating the critical role of PKC in this stimulation. The Cox-2 promoter could be reduced to 282 base pairs (bp) of the 5' flanking sequence with retention of full inducibility by cloprostenol. Mutation of three critical cis-responsive elements within this 282-bp region (C/EBP, cAMP responsive element [CRE], and E-box) indicated that E-box was critical in both basal and cloprostenol-induced promoter activity. However, there was also significant but less dramatic inhibition of cloprostenol stimulation by mutation of C/EBP and CRE in the Cox-2 promoter, and mutation of all three elements eliminated cloprostenol induction of this promoter. Electrophoretic mobility shift assays of nuclear extracts from large luteal cells revealed that upstream stimulatory factor (USF)-1 and USF-2 bound to the E-box in Cox-2. Thus, PKC directly regulates transcription of the Cox-2 gene in large luteal cells by acting through DNA elements close to the putative transcriptional start point, particularly an E-box region at -50 bp. (+info)
Isolation and characterization of the activated B-cell factor 1 homolog in Caenorhabditis elegans.
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Members of the basic helix-loop-helix (bHLH) family of transcription factors regulate a wide array of developmental processes in many cell types, including cell fate specification, differentiation and morphogenesis. Our studies describe the cloning of a gene from the nematode Caenorhabditis elegans that is closely related to the vertebrate-activated B-cell factor (ABF) gene. The nematode gene product CeABF-1 was detected by northern blot analysis from RNA isolated from pooled nematodes representing different developmental stages. The developmental expression profile of CeABF-1 was shown by RT-PCR analysis to be predominantly expressed in the larval stages L3 and L4, with lower levels observed in the L2 larval stage and adult. We also show that CeABF-1 is capable of forming heterodimers with E2A proteins and binding E-box target sites. Mammalian cells transfected with CeABF-1 expression plasmids were capable of blocking E2A-mediated gene transcription, but full repression activity required the presence of two conserved amino acid residues found within the first helix of the CeABF-1 bHLH domain. These results suggest a conserved mechanism of gene repression between certain class II bHLH and class I bHLH proteins found in vertebrates and invertebrates. (+info)
Transcriptional repression of the human p53 gene by cobalt chloride mimicking hypoxia.
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The switch to an angiogenic phenotype is known to be a fundamental determinant of neoplastic growth and tumor progression. We herein report that the transcription of the human p53 gene was repressed by treatment with a hypoxia-mimicking concentration of cobalt chloride and alone by hypoxia-inducible factor 1alpha. Analyses of serial deletions, site-directed mutageneses and heterologous promoter systems showed that the site responsible for the repression by both factors was the E-box element in the promoter of the p53 gene. These results alongside previous data suggest that the loss of p53 including the transcriptional repression may play an important role in the angiogenic switch during tumorigenesis. (+info)
TNFalpha decreases alphaMHC expression by a NO mediated pathway: role of E-box transcription factors for cardiomyocyte specific gene regulation.
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OBJECTIVE: Tumor necrosis factor alpha(TNFalpha) is thought to play a key role in the pathogenesis of cardiac failure. In the myocardium, TNFalpha enhances the expression of inducible nitric oxide synthase (iNOS). Nitric oxide (NO) has been shown to affect beta-agonist-dependent cardiac contractility and relaxation. It is not clear, however, whether TNFalpha mediated NO release has sustained cardiac effects, by altering expression of cardiomyocyte specific genes such as alpha-myosin heavy chain (alphaMHC). METHODS: Neonatal rat ventricular cardiomyocytes (CM) were stimulated with TNFalpha and/or the NOS inhibitor nitro-L-arginine (L-NNA). Protein binding to the E-box enhancer element in the alphaMHC promoter was evaluated by electrophoretic mobility shift assay (EMSA) and transcriptional activity of the E-box consensus motif was determined by luciferase assay. mRNA levels of the endogenous alphaMHC gene were assessed by RT-PCR. In vivo studies were performed in transgenic mice with cardiac specific over-expression of TNFalpha. RESULTS: CM treated with TNFalpha exhibited decreased levels of alphaMHC transcripts (69 +/- 8% of control), the effect of TNFalpha was reversed by L-NNA (94 +/- 14% of control). As shown by EMSA, TNFalpha reduced protein binding to the alphaMHC E-box enhancer motif via NO dependent pathways. Addition of the NO-donor sodium nitroprusside (SNP) to CM nuclear extracts dose dependently disrupted protein binding to the alphaMHC E-box. Furthermore, exposure of CM to TNFalpha or SNP decreased transcription from an E-box luciferase-reporter construct (TNFalpha: 74 +/- 12%; SNP 250 microM: 72 +/- 10%; SNP 500 microM: 66 +/- 11% of control). In myocardial tissue of TNFalpha transgenic mice, increased nitrotyrosine staining, decreased protein binding to the alphaMHC E-box motif and reduced expression of alphaMHC (62 +/- 26%) were observed. CONCLUSIONS: The present study shows that TNFalpha reduces alphaMHC transcript levels in cardiomyocytes. Our data obtained in cultured CM and in TNFalpha transgenic mice support the notion that TNFalpha exerts these effects by NO and E-box dependent mechanisms in vitro and possibly in vivo. (+info)
The E-box motif in the proximal ABCA1 promoter mediates transcriptional repression of the ABCA1 gene.
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To identify regulatory elements in the proximal human ATP-binding cassette transporter A1 (hABCA1) gene promoter we transfected RAW cells with plasmids containing mutations in the E-box, AP1, and liver X receptor (LXR) elements as well as the two Sp1 motifs. Point mutations in either Sp1 site or in the AP1 site had only a minor effect whereas mutation of the LXR element decreased promoter activity. In contrast, mutation or deletion of the E-box motif caused a 3-fold increase in transcriptional activity under basal conditions. Gel shift and DNaseI footprint analysis showed binding of a protein or protein complex to this region. Preincubation of nuclear extracts with antibodies established that USF1, USF2, and fos related antigen (Fra) 2 bind to DNA sequences in the human ABCA1 promoter that contains the intact E-box but not the mutant or deleted E-box. Co-transfection of USF1 and USF2 enhanced, but Fra2 repressed, ABCA1 promoter activity. Thus, a complex consisting of USF1, USF2, and Fra2 binds the E-box motif 147 bp upstream of the transcriptional start site and facilitates repression of the human ABCA1 promoter. These combined studies identify a novel site in the human ABCA1 promoter involved in the regulation of ABCA1 gene expression. (+info)
The SLUG zinc-finger protein represses E-cadherin in breast cancer.
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Loss of expression of the E-cadherin cell-cell adhesion molecule is important in carcinoma development and progression. Because previous data suggest that loss of E-cadherin expression in breast carcinoma may result from a dominant transcriptional repression pathway acting on the E-cadherin proximal promoter, we pursued studies of cis sequences and transcription factors regulating E-cadherin expression in breast cancer cells. E-box elements in the E-cadherin promoter were found to play a critical negative regulatory role in E-cadherin gene transcription in breast cancer cell lines lacking E-cadherin transcription. The E-box elements had a minimal role in E-cadherin transcription in breast cancer cell lines expressing E-cadherin. Two zinc-finger transcription factors known to bind E-box elements, SLUG and SNAIL, repressed E-cadherin-driven reporter gene constructs containing wild-type promoter sequences but not those with mutations in the E-box elements. Additionally, both SLUG and SNAIL repressed endogenous E-cadherin expression. These findings suggest SLUG and SNAIL are potential repressors of E-cadherin transcription in carcinomas lacking E-cadherin expression. Analysis of the expression patterns of SLUG, SNAIL, and E-cadherin in breast cancer cell lines demonstrated that expression of SLUG was strongly correlated with loss of E-cadherin transcripts. Taken together, the data indicate the E-box elements in the proximal E-cadherin promoter are critical in transcriptional repression of the E-cadherin gene, and SLUG is a likely in vivo repressor of E-cadherin in breast cancer. (+info)