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At least 1,747 records · Page 97Linked to original sources
PubMed1997

Neurotensin agonist induces differential regulation of neurotensin receptor mRNA. Identification of distinct transcriptional and post-transcriptional mechanisms.

The binding of neurotensin (NT) to specific receptors triggers the multiple functions that NT exerts in both periphery and brain. By studying the effect of the concentration and time of NT agonist exposure, two separate regulatory mechanisms were detected for the neurotensin receptor (NTR) gene in human colonic adenocarcinoma cells (HT-29). The incubation of cells for 6 h with the NT agonist, JMV 449, resulted in an increase of 270% in NTR mRNA levels. These changes were the direct result of new NTR gene transcription, as indicated by run-on and half-life experiments. In addition, the transcriptional activation of the NTR gene was dependent on NT-receptor complex internalization and de novo protein synthesis. A second response was detected with prolonged exposure to JMV 449. In this case, a decrease of 70% was detected in NTR mRNA levels. Unlike the initial phase, this change was mediated by a post-transcriptional event as the half-life of NTR mRNA from treated cells decreased by 50% as compared with control cells. NT agonist appears to regulate the synthesis of NTR mRNA. In HT-29 cells, this feedback is exerted by a biphasic response. These phases are apparently independent and mediated by two separate mechanisms.

Analgesics↗
PubMed1998

rut Sites in the nascent transcript mediate Rho-dependent transcription termination in vivo.

The in vitro function of the coliphage lambda tR1 Rho-dependent terminator is governed primarily by a tripartite upstream sequence element designated rut. To determine the contribution of the different components of the rut site to terminator function in the normal context of coupled translation of the nascent cro message, tR1 variants lacking different rut site sequences were tested for terminator function in vivo. Intact rutA and rutB sequences were both necessary for efficient termination. However, deletion of the upstream rutA was far more detrimental than deletion of rutB. The intervening boxB, which encodes a short RNA stem and loop, could be deleted without reducing termination or detectably altering Rho's interaction with the corresponding cro transcript. The relative importance of these sequence elements was also the same in a minimal in vitro termination assay system. Rut sequences are therefore essential for terminator function in vivo and rutA contributes substantially more to tR1 function than does rutB. The relative contribution of these elements can be ascribed to differences in Rho's binding affinity for the encoded transcripts. If other cellular factors also bind the rut element RNA, they do not alter the relative contribution of its two regions to Rho-dependent transcription termination in vivo.

Adenosine Triphosphatases↗
PubMed1999

The transcription factor CCAAT/enhancer-binding protein beta regulates gluconeogenesis and phosphoenolpyruvate carboxykinase (GTP) gene transcription during diabetes.

CCAAT/enhancer-binding protein (C/EBP) beta and C/EBPalpha are members of the c/ebp gene family and are highly expressed in mammalian liver and adipose tissue. C/EBPalpha is essential for adipogenesis and neonatal gluconeogenesis, as shown by the C/EBPalpha knockout mouse. C/EBPbeta binds to several sequences of the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter with high affinity, and C/EBPbeta protein is increased 200% in the livers of streptozotocin-diabetic mice, concurrent with increased PEPCK mRNA. To elucidate the role of C/EBPbeta in the control of gluconeogenesis during diabetes, we studied the levels of plasma metabolites and hormones related to energy metabolism during diabetes in adult mice heterozygous and homozygous for a null mutation of the gene for C/EBPbeta. We also examined the expression of PEPCK and glucose 6-phosphatase mRNAs and regulation of blood glucose, including the contribution of gluconeogenesis to blood glucose in c/ebpbeta-/- mice. C/EBPbeta was not essential to basal PEPCK mRNA levels. However, C/EBPbeta deletion affected streptozotocin-diabetic response by: (a) delaying hyperglycemia, (b) preventing the increase of plasma free fatty acids, (c) limiting the full induction of PEPCK and glucose 6-phosphatase genes, and (d) preventing the increase in gluconeogenesis rate. Gel supershifts of transcription factor C/EBPalpha, bound to CRE, P3I, and AF-2 sites of the PEPCK promoter, was not increased in diabetic c/ebpbeta-/- mouse liver nuclei, suggesting that C/EBPalpha does not substitute for C/EBPbeta in the diabetic response of liver gene transcription. These results link C/EBPbeta to the metabolic and gene regulatory responses to diabetes and implicate C/EBPbeta as an essential factor underlying glucocorticoid-dependent activation of PEPCK gene transcription in the intact animal.

Animals↗
PubMed1999

A binding site for the transcription factor Grainyhead/Nuclear transcription factor-1 contributes to regulation of the Drosophila proliferating cell nuclear antigen gene promoter.

The Drosophila proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including upstream regulatory element (URE), DNA replication-related element, E2F recognition sites, and three common regulatory factor for DNA replication and DNA replication-related element-binding factor genes recognition sites. In nuclear extracts of Drosophila embryos, we detected a protein factor, the URE-binding factor (UREF), that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE. Analyses in Drosophila Kc cells and transgenic flies revealed that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies. A yeast one-hybrid screen using URE as a bait allowed isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (GRH/NTF-1). The nucleotide sequence required for binding to GRH was indistinguishable from that for UREF detected in embryo nuclear extracts. Furthermore, a specific antibody to GRH reacted with UREF in embryo nuclear extracts. From these results we conclude that GRH is identical to UREF. Although GRH has been thought to be involved in regulation of differentiation-related genes, this study demonstrates, for the first time, involvement of a GRH-binding site in regulation of the DNA replication-related proliferating cell nuclear antigen gene.

Animals↗
PubMed2000

The hematopoietic transcription factor PU.1 represses gelatinase A transcription in glomerular mesangial cells.

The matrix metalloproteinase gelatinase A plays a key role in the evolution of glomerular injury and is a major contributing factor to the development of glomerulosclerosis. Prior studies have focused on a potent cis-acting enhancer element located in the near 5'-flanking region of the rat and human gelatinase A genes (Harendza, S., Pollock, A. S., Mertens, P. R., and Lovett, D. H. (1995) J. Biol. Chem. 270, 18286-18796; Mertens, P. R., Alfonso-Jaume, M. A., Steinmann, K., and Lovett, D. H. (1999) J. Am. Soc. Nephrol. 10, 2480-2487). Given the combinatorial nature of transcriptional regulation, we examined additional regions of the 5'-flanking region of the rat gelatinase A gene to identify further regulatory elements. In this study the identification of a silencing element located between -1903 and -1847 base pairs of the 5'-flanking region of the rat gelatinase A gene is reported. Sequence analysis, electrophoretic mobility studies, and transfection experiments demonstrate that a specific binding sequence for the hematopoietic transcription factor PU.1 is present within the silencing sequence. PU.1 activity is absolutely required for the expression of silencing activity within the context of transfected glomerular mesangial cells. Western blots identify the PU.1 protein within nuclear extracts of mesangial cells, and cotransfection with a PU.1 expression vector directly augments silencing activity. These studies underscore the complex patterns of gelatinase A transcriptional regulation and also strongly suggest that glomerular mesangial cells are ultimately derived from bone marrow cells.

Animals↗
PubMed2000

Differential association of products of alternative transcripts of the candidate tumor suppressor ING1 with the mSin3/HDAC1 transcriptional corepressor complex.

The candidate tumor suppressor ING1 was identified in a genetic screen aimed at isolation of human genes whose expression is suppressed in cancer cells. It may function as a negative growth regulator in the p53 signal transduction pathway. However, its molecular mechanism is not clear. The ING1 locus encodes alternative transcripts of p47(ING1a), p33(ING1b), and p24(ING1c). Here we report differential association of protein products of ING1 with the mSin3 transcriptional corepressor complex. p33(ING1b) associates with Sin3, SAP30, HDAC1, RbAp48, and other proteins, to form large protein complexes, whereas p24(ING1c) does not. The ING1 immune complexes are active in deacetylating core histones in vitro, and p33(ING1b) is functionally associated with HDAC1-mediated transcriptional repression in transfected cells. Our data provide basis for a p33(ING1b)-specific molecular mechanism for the function of the ING1 locus.

3T3 Cells↗
PubMed2001

Sumo-1 modification regulates the DNA binding activity of heat shock transcription factor 2, a promyelocytic leukemia nuclear body associated transcription factor.

Heat shock transcription factor 2 (HSF2) is a transcription factor that regulates heat shock protein gene expression, but the mechanisms regulating the function of this factor are unclear. Here we report that HSF2 is a substrate for modification by the ubiquitin-related protein SUMO-1 and that HSF2 colocalizes in cells with SUMO-1 in nuclear granules. Staining with anti-promyelocytic leukemia antibodies indicates that these HSF2-containing nuclear granules are PML bodies. Our results identify lysine 82 as the major site of SUMO-1 modification in HSF2, which is located in a "wing" within the DNA-binding domain of this protein. Interestingly, SUMO-1 modification of HSF2 results in conversion of this factor to the active DNA binding form. This is the first demonstration that SUMO-1 modification can directly alter the DNA binding ability of a transcription factor and reveals a new mechanism by which SUMO-1 modification can regulate protein function.

Biological Transport↗
PubMed2001

Analysis of gene induction and arrest site transcription in yeast with mutations in the transcription elongation machinery.

In vitro, transcript elongation by RNA polymerase II is impeded by DNA sequences, DNA-bound proteins, and small ligands. Transcription elongation factor SII (TFIIS) assists RNA polymerase II to transcribe through these obstacles. There is however, little direct evidence that SII-responsive arrest sites function in living cells nor that SII facilitates readthrough in vivo. Saccharomyces cerevisiae strains lacking elongation factor SII and/or containing a point mutation in the second largest subunit of RNA polymerase II, which slows the enzyme's RNA elongation rate, grow slowly and have defects in mRNA metabolism, particularly in the presence of nucleotide-depleting drugs. Here we have examined transcriptional induction in strains lacking SII or containing the slow polymerase mutation. Both mutants and a combined double mutant were defective in induction of GAL1 and ENA1. This was not due to an increase in mRNA degradation and was independent of any drug treatment, although treatment with the nucleotide-depleting drug 6-azauracil exacerbated the effect preferentially in the mutants. These data are consistent with mutants in the Elongator complex, which show slow inductive responses. When a potent in vitro arrest site was transcribed in these strains, there was no perceptible effect upon mRNA accumulation. These data suggest that an alternative elongation surveillance mechanism exists in vivo to overcome arrest.

Adenosine Triphosphatases↗
PubMed2002

Differential cytostatic and apoptotic effects of ecteinascidin-743 in cancer cells. Transcription-dependent cell cycle arrest and transcription-independent JNK and mitochondrial mediated apoptosis.

We have found that ecteinascidin-743 (ET-743) inhibited cell proliferation at 1-10 ng/ml, leading to S and G(2)/M arrest and subsequent apoptosis, and induced early apoptosis without previous cell cycle arrest at 10-100 ng/ml in cancer cells. ET-743-mediated apoptosis, did not involve Fas/CD95. ET-743 induced c-Jun NH(2)-terminal kinase (JNK) and caspase-3 activation, and JNK and caspase inhibition prevented ET-743-induced apoptosis. ET-743 failed to promote apoptosis in caspase-3-deficient MCF-7 cells, further implicating caspase-3 in its proapoptotic action. Overexpression of bcl-2 by gene transfer abrogated ET-743-induced apoptosis, but cells underwent cell cycle arrest. ET-743 triggered cytochrome c release from mitochondria that was inhibited by Bcl-2 overexpression. Inhibition of transcription or protein synthesis did not prevent ET-743-induced apoptosis, but abrogated ET-743-induced cell cycle arrest. Microarray analyses revealed changes in the expression of a small number of cell cycle-related genes (p21, GADD45A, cyclin G2, MCM5, and histones) that suggested their putative involvement in ET-743-induced cell cycle arrest. These data indicate that ET-743 is a very potent anticancer drug showing dose-dependent cytostatic and proapoptotic effects through activation of two different signaling pathways, namely a transcription-dependent pathway leading to cell cycle arrest and a transcription-independent route leading to rapid apoptosis that involves mitochondria, JNK, and caspase-3.

Antineoplastic Agents↗
PubMed2002

Cyclooxygenase-2 expression in cultured cortical thick ascending limb of Henle increases in response to decreased extracellular ionic content by both transcriptional and post-transcriptional mechanisms. Role of p38-mediated pathways.

We showed previously that decreased extracellular salt or chloride up-regulates the cortical thick ascending limb of Henle (cTALH) COX-2 expression via a p38-dependent pathway. The present studies determined that low salt medium increased COX-2 mRNA expression 3.9-fold control by 6 h in cultured cTALH, which was blocked by actinomycin D pretreatment, suggesting transcriptional regulation. Luciferase activity (normalized to beta-galactosidase activity) of the full-length (-3400) COX-2 promoter in cTALH increased from 1.8 +/- 0.3 in control media to 5.8 +/- 0.7 in low salt (n = 9; p < 0.01). Low chloride medium had similar effects as low salt has on COX-2 promoter activity. Deletion constructs -815, -512, and -410 were similarly stimulated, but -385 could not be stimulated significantly by low salt (1.8 +/- 0.3 versus 2.4 +/- 0.5, n = 10). This suggested involvement of an NF-kappaB cis-element located in this region, which was confirmed by utilizing a construct with a point mutation of this NF-kappaB-binding site that was not stimulated by low salt medium. Co-incubation of the specific p38 inhibitor, SB203580 or PD169316, inhibited a low salt-induced increase in luciferase activity of the intact COX-2 promoter (5.8 +/- 0.7 versus 1.1 +/- 0.2, n = 8 and 1.4 +/- 0.4, n = 4 respectively, p < 0.01). Mobility shift assays indicated that the low salt medium stimulated NF-kappaB binding activity, and this stimulation was inhibited by p38 inhibitors. To test whether p38 also increased COX-2 expression by increasing mRNA stability, cTALH were incubated in low salt for 2 h, and actinomycin was then added with or without SB203580. p38 inhibition led to a decreased half-life of COX-2 mRNA (from 68 to 18 min, n = 4-7, p < 0.05). Therefore, these studies indicate that p38 stimulates COX-2 expression in cTALH and macula densa by transcriptional regulation predominantly via a NF-kappaB-dependent pathway and by post-transcriptional increases in mRNA stability.

Animals↗
PubMed2006

Transcription regulation of ompF and ompC by a single transcription factor, OmpR.

The ompF and ompC genes of Escherichia coli are reciprocally regulated by a single transcription factor, phosphorylated OmpR (OmpR-P), depending upon medium osmolarity. This regulation involves activation of ompF and its repression with concomitant activation of ompC. This occurs through OmpR-P binding to four (F1, F2, F3, and F4) and three (C1, C2, and C3) sites located upstream of the ompF and ompC promoters, respectively, through a novel mechanism. Here we show that there is a distinct OmpR-P binding hierarchy within F1, F2, and F3 sites as well as within C1, C2, and C3 sites. Each of these sites contains two tandem 10-bp OmpR-P-binding subsites, a-site and b-site (from 5' to 3' direction). OmpR-P has higher affinity to the downstream b-site than to the upstream a-site in each case. Six OmpR-P molecules bind to F and C sites two-by-two in a discontinuous "galloping" manner. We propose that this tight hierarchical binding of a transcription factor, OmpR, allows distinct stepwise regulation of ompF and ompC transcription, which minimizes their overlapping expression upon changes in the medium osmolarity to achieve the reciprocal expression of ompF and ompC.

Bacterial Proteins↗
PubMed2000

c-Jun inhibits transforming growth factor beta-mediated transcription by repressing Smad3 transcriptional activity.

Transforming growth factor beta (TGF-beta) is a pleiotropic cytokine that exerts its effects through a heteromeric complex of transmembrane serine/threonine kinase receptors. At least two intracellular pathways are activated by TGF-beta as follows: the SAPK/JNK, involving the MEKK1, MKK4, and JNK cascade, and the Smad pathway. Here, we report that the SAPK/JNK pathway inhibits the Smad3 pathway. Expression of dominant negative or constitutively active mutants of kinases of the SAPK/JNK pathway, respectively, activates or represses a TGF-beta-induced reporter containing Smad3-binding sites. This effect is not dependent on blocking of Smad3 nuclear translocation but involves a functional interaction between Smad3 and c-Jun, a transcription factor activated by the SAPK/JNK pathway. Overexpression of constitutively active MEKK1 or MKK4 mutants stabilizes the physical interaction between Smad3 and c-Jun, whereas dominant negative mutants inhibit this interaction. Moreover, overexpression of wild-type c-Jun inhibits Smad3-dependent transcription. However, c-Jun does not inhibit Smad3 binding to DNA in vitro. The repression obtained with a c-Jun mutant unable to activate transcription through AP-1 sites indicates that the inhibitory mechanism does not rely on the induction of a Smad3 repressor by c-Jun, suggesting that c-Jun could act as a Smad3 co-repressor. The inhibition of the Smad3 pathway by the SAPK/JNK pathway, both triggered by TGF-beta, could participate in a negative feedback loop to control TGF-beta responses.

Animals↗
PubMed2005

Functional dissection of the HNF-1alpha transcription factor: a study on nuclear localization and transcriptional activation.

Hepatocyte nuclear factor-1alpha (HNF-1alpha) is a homeodomain-containing transcription factor regulating the expression of liver and pancreas-specific genes. Mutations in the HNF-1alpha-encoding gene TCF1 cause maturity-onset diabetes of the young, type 3 (MODY3). These mutations may affect nuclear import or reduce the ability of HNF-1alpha to stimulate transcription. We performed a functional dissection of HNF-1alpha, attempting both to define its nuclear localization signals (NLSs) and to identify important elements of the Cterminal transactivation domain. Three HNF-1alpha regions, A (amino acids 158-171), B (197-205), and C (271-282), highly similar to consensus NLSs, were studied by immunolocalization in HeLa cells. Region B could be identified as the most critical for correct nuclear localization. Deletion of two subregions (amino acids 398-470 and 544-631, respectively) in the HNF-1alpha C-terminal transactivation domain, resulted in the greatest reduction in stimulation of transcription compared to wild-type protein. However, this domain probably consists of many elements that work in concert to give the full transactivation potential of the protein.

Amino Acid Sequence↗
PubMed1993

Physical and transcriptional mapping of DXS56-PGK1 1 Mb region: identification of three new transcripts.

Several new techniques for isolation expressed sequences have been recently described considerably speeding up the identification of unknown genes. Here we present a transcriptional map of the 1 Mb DXS56-PGK1 region in Xq13.3. Rare cutter restriction site mapping, direct cDNA selection on membrane discs and probing of Northern blots with total YAC DNA, were the methods explored in order to achieve this goal. In addition to three known genes from this region which have been recloned, two new cDNA clones corresponding to two new genes were isolated, mapped and characterized. Moreover one more transcript, highly expressed in placenta, has been detected in the region with a total YAC as a probe. In summary there are at least six genes known to reside in the DXS56-PGK1 region. As several human disease gene loci (i.e. SCID, CMTX1, WWS, MRX, XDP, ASB) were tightly linked to the markers from the region (PGK, CA repeats), the three new transcripts may be considered as their potential candidate genes.

Base Sequence↗
PubMed1993

Transcriptional and post-transcriptional mechanisms are involved in the absence of CD4 surface expression in two HIV-1 chronically infected T cell lines.

In vivo infection of human T cell lymphocytes by HIV-1 is mediated by the specific binding of the HIV-1 envelope glycoprotein gp120 to the T cell CD4 receptor. One of the post-infection events observed in vivo is the progressive loss of CD4+ T cells. One possible mechanism is the production of infected T cells which are lacking in surface expression of the CD4 receptor protein. We have analysed this possibility utilizing the two HIV-1 chronically-infected CD4- cell lines, 8E5 and ACH-2, both of which are derived from a CD4+ parental strain (A3.01) after HIV-1 infection. In each cell (8E5 and ACH-2) the loss of CD4 surface expression was found to occur by different mechanisms. In ACH-2 cells, neither CD4 protein nor the 3 kb CD4 RNA transcript could be detected. However, treatment of ACH-2 cells with cycloheximide elicited production of the 3 kb transcript suggesting the possibility for a repressor protein(s) to act at the level of transcription and/or stability of the 3 kb mRNA. In contrast, in 8E5 cells the level of the 3 kb CD4 RNA was comparable with that found in the CD4+ A3.01 parental strain. Analysis of the 8E5 strain revealed the presence of a CD4- gp160 bimolecular protein complex sequestered internally in the rough endoplasmic reticulum (RER). Finally, the protein tyrosine kinase p56lck, normally associated with the cellular membrane, appeared to be linked to the RER and bound to the CD4- gp160 proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Far upstream sequences are required for efficient transcription from the adenovirus-2 E1A transcription unit.

We have investigated the requirement for sequences located upstream from the TATA box for efficient transcription from the Adenovirus-2 (Ad2) E1A promoter. A series of deletions located within the E1A promoter upstream sequences were introduced into recombinants which contain or do not contain the E1A structural sequences. The amount of E1A-specific RNA produced after transfection into HeLa cells was determined by quantitative S1 nuclease analysis. We demonstrate that sequences located more than 231 bp upstream from the E1A capsite are required for efficient transcription from the E1A promoter. However, the requirement for these stimulatory sequences is less pronounced in recombinants which contain the E1A structural sequences than in those in which these sequences have been deleted. We demonstrate also that these Ad2 stimulatory sequences activate transcription in cis when inserted upstream from the heterologous -34 to +33 Ad2 major late promoter (Ad2MLP) element which is otherwise inactive when transfected into HeLa cells. These results suggest that the 270 bp Ad2 left-terminal segment contains an enhancer-like element.

Adenoviruses, Human↗
PubMed1985

5-Methylcytidylic modification of in vitro transcript from the rat identifier sequence; evidence that the transcript forms a tRNA-like structure.

Previous studies showed that the rat identifier sequence, the rodent type 2 Alu family, the rabbit C family and the bovine or goat 73 bp repeat show remarkable resemblance with a few specific tRNA molecules (Sakamoto and Okada, J. Mol. Evol. in press). This paper reports 5-methylcytidylic modification of the in vitro transcript from the ID sequence, and provides further evidence that the transcript forms a tRNA-like structure in vitro. Fingerprint analysis and oligonucleotide mapping suggested that the sequence of the oligonucleotide containing 5-methylcytidine is CpCpm5CpUpGp, which corresponds to the extra and T psi stem regions in the secondary structure of phenylalanine tRNA. Since the sequence of the corresponding oligonucleotide in the phenylalanine tRNA is Cpm5CpCpUpGp, these results suggests that a tRNA (cytosine-5) methyl transferase recognizes the secondary structure of the transcript from the ID sequence rather than the primary sequence. The significance of this modification in relation to the functional role of the ID sequence is discussed.

Animals↗
PubMed1990

A simple and sensitive method for determining transcription initiation site: identification of two transcription initiation sites in rat group II phospholipase A2 gene.

We developed a simple and sensitive method for assigning transcriptional initiation sites, and applied it to characterize the transcriptional unit of rat group II phospholipase A2 (PLA2) gene. Our method involves the primer extension reaction followed by detection of its products by hybridization. Using this method, we were able to map two transcriptional initiation sites on the nucleotide sequence of the core promoter region of PLA2 gene with one-base resolution without any difficulties.

Animals↗