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PubMed1990

Interleukin 2-induced expression of proliferating cell nuclear antigen is regulated by transcriptional and post-transcriptional mechanisms.

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein required for leading strand DNA synthesis by polymerase delta. Steady-state levels of PCNA RNA increase during interleukin 2 stimulated G1 activation of cloned T cells (L2 cells) and correlate with expression of the PCNA protein. We demonstrate that the initial (G1) rapid rise in PCNA RNA levels is predominantly related to increased transcription. The slower rate of increase in PCNA RNA at the initial G1-S transition is related to transcription as well as increased stability of the PCNA message and partially requires protein synthesis. These data provide a foundation for further investigation of the mechanisms of increased stability and transcription of PCNA.

Animals↗
PubMed1991

Rho-dependent transcription termination. Characterization of the requirement for cytidine in the nascent transcript.

By substituting template segments encoding AU-rich, GU-rich, and CA-rich transcripts for natural sequences upstream of the phage lambda rho-dependent tR1 termination site, we demonstrate that cytidines are required in the upstream RNA for rho-dependent termination to occur. These results are extended through in vitro mutagenesis of a template encoding an inactive AU-rich upstream sequence: certain mutant templates encoding new cytidines are able to activate rho-dependent termination. Cytidines must be dispersed over a region of the transcript in order for rho to be activated, although no specific pattern of cytidines appears to be required. The results show that no local clustering or regular spacing of cytidines is necessary and that cytidines are not used as a "ruler" to determine the location of termination sites. Rho is somewhat sensitive to the relative positions of cytidines since slightly different nascent transcripts activate rho termination activity to various degrees. A model is presented in which hexameric rho binds 78 nucleotides of contiguous RNA in a primary site, such that each monomer interacts with at least one cytidine somewhere in the 13 nucleotides allotted to the monomeric primary site.

Bacteriophage lambda↗
PubMed1990

Transcriptional activator LEU3 of yeast. Mapping of the transcriptional activation function and significance of activation domain tryptophans.

The LEU3 protein of yeast activates a number of genes in the branched chained amino acid pathways. Native LEU3 is modulated by alpha-isopropylmalate, an intermediate in leucine biosynthesis. alpha-Isopropylmalate is needed for transcriptional activation, but not for DNA binding. We show here that the transcriptional activation function of LEU3 resides within the C-terminal 32 amino acids. An adjacent stretch of 81 residues is dispensable and apparently forms a connecting link between the activation domain and a large central region previously identified as important for modulation. The newly defined activation domain contains a cluster of three tryptophan residues, each of which was changed to alanine by site-directed mutagenesis. Surprisingly, all three Trp----Ala mutations affect modulation. One of them, Trp-864----Ala, creates a LEU3 molecule that is largely unmodulated and also is a better transcriptional activator than is wild type LEU3 ("hyperactivator"). The other two mutations (Trp-861----Ala and Trp-870----Ala) change the modulation ratio but have no effect on the maximal activation efficiency of the activator. We propose that the activation domain of LEU3 is kept silent by association with the central region of the protein and that an alpha-isopropylmalate-induced conformational change in the central region releases and thus activates the activation domain.

Base Sequence↗
PubMed1985

Transcriptional and post-transcriptional regulation of L-type pyruvate kinase in diabetic rat liver by insulin and dietary fructose.

Regulation of the expression of the hepatic L-type pyruvate kinase gene by insulin and dietary fructose was studied in diabetic rats. Insulin increased the levels of putative nuclear RNA precursor species of this enzyme in parallel with that of total cellular pyruvate kinase L mRNA. These changes occurred more slowly than those induced by dietary fructose. Insulin caused a 3-fold increase in transcription of the pyruvate kinase L gene after 6 h and a 6-fold increase after 16 h. The increase caused by insulin was inhibited by glucagon, but not by adrenalectomy. Cycloheximide inhibited the induction caused by insulin, suggesting that insulin may stimulate transcription of the pyruvate kinase L gene by stimulating synthesis of some unknown protein. On the other hand, feeding fructose had no effect on transcription of the pyruvate kinase L gene. We previously showed that increases in the levels of putative nuclear RNA precursor species of the pyruvate kinase L after fructose feeding preceded changes in the levels of cytosolic pyruvate kinase L mRNA (Inoue, H., Noguchi, T., and Tanaka, T. (1984) J. Biochem. (Tokyo) 96, 1457-1462). Thus, dietary fructose may increase the levels of pyruvate kinase L mRNA by stabilizing nuclear RNA species. Glucagon inhibited the increase in pyruvate kinase L mRNA caused by dietary fructose. However, plasma levels of glucagon and thyroid hormones were not decreased in diabetic rats after fructose feeding. In addition, treatment with triiodo-L-thyronine caused no change in the pyruvate kinase L mRNA level. Furthermore, adrenalectomy did not impair enzyme induction by fructose in diabetic rats. Thus, the effect of fructose on pyruvate kinase L seems to be directly on the liver.

Adrenalectomy↗
PubMed1988

[Determination of the direction of the transcription of bacteriophage T4 genes by heat induction of the transcription from recombinant plasmid Pl-promoter: 2 directions in the region of genes 25-29].

The bacterial strains with recombinant plasmids constructed on the basis of vector plasmid pSCC31 and containing BglII fragment of bacteriophage T4 DNA with genes 25-29 have been used in this study. Restriction analysis and subcloning demonstrated that in the case of the recombinant plasmid pRL705, the phage DNA fragment had right orientation for phage late genes transcription, while the opposite one in the plasmid pRL707. Heat induction of plasmids transcription under control of pL promoter led to the substantial change in phage burst size and the production of recombinants, as shown in complementation experiments, the changes in the burst size being dependent both on the host strain and the amber mutant used. On the basis of these data, the conclusion has been drawn that the genes 26 and 25, in contrast to genes 51, 27, 28 and 29, are being transcribed in the counter-clockwise direction on the genomic map, i.e. in the direction of transcription of T4 early genes.

Bacterial Proteins↗
PubMed1986

Transcription and post-transcriptional regulation of avian HSP70 gene expression.

Transient incubation of chicken lymphoblastoid (MSB) cells at elevated temperatures induces the synthesis of three heat shock proteins of 89,000 Da (HSP89), 70,000 Da (HSP70), and 23,000 Da (HSP23). We have examined the effects of heat shock on the transcription and post-transcriptional regulation of the chicken HSP70 and beta-actin genes. The rate of HSP70 transcription is rapidly induced by heat shock, reaches maximal levels by 60 min, and thereafter decreases. The level of HSP70 mRNA increases 20-fold by 60 min and remains constant through 6 h of heat shock. Upon return of heat-shocked cells to normal growth temperatures, the level of HSP70 mRNA rapidly decreases to pre-heat shock levels. These results suggest that HSP70 mRNA is stably maintained and translated during heat shock, but rapidly degraded during recovery from heat shock. The effect of heat shock on beta-actin mRNA is opposite to the apparent stabilizing effects of elevated temperatures on HSP70 mRNA.

Actins↗
PubMed1983

Dinucleotide primers facilitate convenient identification of the mouse ribosomal DNA transcription initiation site. A general method for analysis of transcription by RNA polymerases I and III.

The in vitro initiation site for RNA polymerase I on the mouse rRNA gene was identified using a new method that is generally applicable to the study of other eukaryotic transcripts. First, the 5' end of mouse rRNA was located to an ApC . . . by high resolution S1 nuclease mapping. Dinucleotide primers were then used in transcription reactions to demonstrate that this position is the actual de novo initiation site, and not a rapid RNA processing site. For this analysis, initiation was inhibited by reduced rXTP concentration, and, upon supplementation with various dinucleotides, only ApC stimulated correct synthesis. Independently confirming its role as the initiating nucleotide, ATP was shown to be required at a much higher concentration than the other rXTPs for RNA initiation, but not for elongation. These results also demonstrate a marked sequence conservation of rRNA initiation sites between the mouse and frog, two species that violate the general rule of species specificity in RNA polymerase I initiation. Extending these studies to RNA polymerase III, the initiation site for 5 S RNA can be similarly located by dinucleotide analysis and confirmed from the concentration requirements of each rXTP. In addition to allowing initiation at suboptimal rXTP concentration, dinucleotide primers can also circumvent the need for a factor normally required for initiation, suggesting their potential value in dissecting the mechanism of eukaryotic transcription.

Animals↗
PubMed1994

Dual mechanism for the control of inducible-type NO synthase gene expression in macrophages during activation by interferon-gamma and bacterial lipopolysaccharide. Transcriptional and post-transcriptional regulation.

Production of nitric oxide (NO) by macrophages is enhanced upon activation by bacterial endotoxins and cytokines mainly via an increase of the intracellular content of the inducible isoform of nitric oxide synthase (i-NOS). We have studied in detail the effect of several modulators of macrophage activity on steady state levels of i-NOS mRNA in the mouse macrophage-like cell line RAW 264.7. Bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) were found to be effective inducers of i-NOS mRNA, in accordance with their known ability to stimulate both i-NOS activity and NO production in macrophages from different sources, while TNF-alpha, IL-1, or IL-6 was ineffective in this regard. Accumulation of i-NOS mRNA in response to either LPS or IFN-gamma stimulation was accompanied by increased i-NOS gene transcription, as detected both by using a nuclear "run-on" transcription assay and by transient transfection of the cloned gene promoter in RAW 264.7 cells. Co-stimulation of the cells with both inducers resulted in higher steady state levels of i-NOS mRNA in the absence, however, of a corresponding potentiation of the rate of gene transcription. This was due primarily to a considerable effect of LPS on i-NOS mRNA stability, with prolongation of its half-life from 1-1.5 h, in the presence of IFN-gamma alone, to 4-6 h in the presence of both LPS and IFN-gamma.

Amino Acid Oxidoreductases↗
PubMed1994

Serum-inducible factors binding to an activating transcription factor motif regulate transcription of the Id2A promoter during myogenic differentiation.

Expression of Id, a dominant negative regulator of helix-loop-helix transcription factors, is tightly regulated during cellular differentiation. In this study, we have defined the sequences responsible for the transcriptional regulation of the human Id2A gene. 5' deletion and site-specific mutation analyses of the Id2A promoter showed that both an Sp-1 site and an activating transcription factor (ATF)-like site (referred to as IdATF sites) are required for expression in C2 cells, whereas these sites are not essential for the expression in nonmuscle cells, including HeLa and 10T1/2 cells. Gel shift assays revealed nuclear factors with specific binding to IdATF sites in both C2 and HeLa cells, which are efficiently competed by either legitimate ATF- or AP-1 binding sites. DNA binding activity to IdATF sites was clearly decreased during C2 cell differentiation and rapidly reactivated by treatment with either phorbol 12-myristate 13-acetate or serum, which correlated with the induction of endogenous Id2A mRNA expression. In addition, we show that overexpression of the catalytic domain of protein kinase C leads to the activation of the Id2A promoter through IdATF sites in C2 cells. These results suggest a model in which down-modulation of IdATF site binding activity by mitogen depletion contributes to the down-regulation of Id2A gene expression that is essential for myogenic differentiation.

Base Sequence↗
PubMed1993

Expression of endogenous NMDAR1 transcripts without receptor protein suggests post-transcriptional control in PC12 cells.

Expression of RNA for the NMDAR1 subunit of the N-methyl-D-aspartate receptor was detected by Northern hybridization in both nerve growth factor-differentiated and undifferentiated rat pheochromocytoma (PC12) cells. The NMDA receptor type 1 (NMDAR1) message in PC12 cells was similar in size to that expressed in hippocampal neurons. PC12 cell cDNAs that were amplified by polymerase chain reaction with primers flanking the coding region of NMDAR1 corresponded to the NMDAR1 splice variant NMDA receptor type 1 isoform C (NMDAR1C). Using calcium imaging or patch-clamp recording, no functional NMDA-gated ion channels were found in PC12 cells. A monoclonal antibody against NMDAR1 was developed in order to investigate whether or not NMDAR1 protein was present in PC12 cells. Only trace amounts of NMDAR1 protein were found in native PC12 cells. However, expression of NMDAR1 protein was detected in PC12 cells that were transfected with an expression vector containing an NMDAR1C clone under control of a cytomegalovirus promoter. These findings suggest that the expression of NMDAR1 protein in PC12 cells may be controlled by post-transcriptional mechanisms. The PC12 cell line may serve as a model system for the study of the transcriptional, post-transcriptional, and translational regulation of NMDAR1. Furthermore, the presence of NMDAR1 RNA in a particular cell type may not necessarily indicate expression of NMDAR1 protein.

Alternative Splicing↗
PubMed1993

Transcriptional regulation in the testis: a role for transcription factor AP-1 complexes at various stages of spermatogenesis.

The products of two proto-oncogenes, c-fos and c-jun, have been implicated in signal transduction pathways as regulators of gene expression. Both proto-oncogenes are members of gene families encoding closely related proteins that together make up transcription factor AP-1. The expression of members of this transcription factor has been associated with cellular pathways that result in both mitosis and differentiation. We have been studying the process of spermatogenesis, which is a complex, continual cycle of cell renewal, proliferation and differentiation. Using a seasonal breeder, the European red fox (Vulpes vulpes), as our model, we have examined the expression of five AP-1 family members (c-fos, fra-1, fra-2, c-jun and junB) with a view to elucidating their role in the regulation of spermatogenesis. Unique patterns of expression, falling into three broad categories, were observed for the five genes: (i) continuous expression throughout the spermatogenic cycle (c-fos); (ii) expression only at times corresponding to the onset and shutdown of spermatogenesis (fra-1, fra-2 and c-jun); and (iii) expression only at the onset of the cycle (junB). Furthermore, the proteins were expressed in both premeiotic and post-meiotic cell types, suggesting a role in haploid, as well as diploid, gene expression in this tissue. The data suggest distinct, although not necessarily unrelated, roles for the different components of transcription factor AP-1 in the regulation of spermatogenesis.

Animals↗
PubMed1997

Ethanol inhibits inducible nitric oxide synthase transcription and post-transcriptional processes in vivo.

The effects of ethanol (ETOH) on post-transcriptional regulation of inducible nitric oxide synthase (iNOS) in vivo has not been demonstrated. We examined the effect of ETOH on iNOS mRNA, protein, and the production of the nitrate and nitrite anion (RNI) in rat lung alveolar macrophages (AM) in vivo when stimulated by lipopolysaccharide (LPS) and dibutyryl cyclic AMP (DB-cAMP). Sprague-Dawley rats (225-250 g) (n = 5-7/gp) were given intratracheal LPS (0.6 mg/kg) or DB-cAMP (0.1 and 1 mg/kg) 30 min after ETOH (4 mg/kg, intraperitoneally (ip)) or phosphate-buffered sterile saline (PBS) (5 ml/kg, ip) or pyrrolidine dithiocarbamate (PDTC 10 mg/kg, intratracheally) or 15 min after diethyl dithiocarbamate (DETC) (5 mg/kg, intratracheally). At selected times after administration of LPS or DB-cAMP, the animals were anesthetized, the lungs with the heart attached were removed and the lungs subjected to bronchoalveolar lavage (BAL). The BAL fluid was assayed for tumor necrosis factor alpha (TNF alpha) and RNI. The BAL fluid AM were isolated and analyzed for iNOS mRNA and protein with competitor-equalized polymerase chain reaction (PCR) and Western blots, respectively. The ex vivo incubates of AM were assayed for RNI and TNF alpha. LPS and DB-cAMP each increased iNOS mRNA and protein in AM and RNI in the BAL fluid and ex vivo incubates of AM. However, the peak of the increase of iNOS mRNA occurred at 2 hr for DB-cAMP and 4 to 6 hr for LPS. Only LPS increased the concentrations of TNF alpha in BAL fluid and ex vivo incubates of AM. ETOH attenuated LPS-mediated up-regulation of iNOS mRNA and TNF alpha and iNOS protein and RNI produced by the AM. In contrast, pretreatment of rats with ETOH did not affect DB-cAMP-mediated increases of iNOS mRNA in AM at any time but suppressed the amount of iNOS protein and RNI produced in DB-cAMP-stimulated AM. DETC, but not PDTC, attenuated LPS-mediated up-regulation of iNOS mRNA without effects on that produced by DB-cAMP. Since ETOH and DETC, but not PDTC, suppressed LPS-mediated but not DB-cAMP-mediated transcription of iNOS, we conclude that two distinct pathways exist for induction of iNOS mRNA by these agonists. ETOH and DETC may inhibit the LPS-mediated activation pathway by acting as antioxidants. Also, since ETOH inhibited DB-cAMP-mediated increases in iNOS protein without affecting iNOS mRNA ETOH also acts at a post-transcriptional or translational site to inhibit iNOS protein in rat AM in vivo.

Animals↗
PubMed2000

The thyroid transcription factor 2 (TTF-2) is a promoter-specific DNA-binding independent transcriptional repressor.

The thyroid transcription factor TTF-2 is a forkhead-containing protein involved in thyroid-specific gene expression and necessary for thyroid morphogenesis. In this paper, we demonstrate that TTF-2 is able to inhibit the activity of the thyroid-specific transcription factors TTF-1 and Pax-8 only on certain promoters. We identified the minimal protein domain responsible for repressor activity, which behaves as an independent functional domain, and we show that repression by TTF-2 is DNA-binding independent. We suggest that TTF-2 is able to interfere with a specific cofactor required for TTF-1 and Pax-8 activity.

DNA↗
PubMed1996

The multifunctional transcription factors Abf1p, Rap1p and Reb1p are required for full transcriptional activation of the chromosomal PGK gene in Saccharomyces cerevisiae.

We have identified two new transcription factor binding sites upstream of the previously defined UAS within the phosphoglycerate kinase (PGK) gene promoter in Saccharomyces cerevisiae. These sites are bound in vitro by the multifunctional factors Cpf1p and Reb1p. We have generated targeted deletions of Rap1p, Abf1p and Reb1p binding sites in the promoter of the chromosomal copy of the PGK gene. Northern blot analysis confirmed that most PGK promoter activity is mediated through the Rap1p binding site. However, significant effects are also mediated through both the Reb1p and Abf1p sites. In contrast, when the promoter is present on a high-copy-number plasmid, both the Abf1p and Reb1p sites play no role in transcriptional activation. The role of Cpf1p was examined using a cpf1 null strain. Cpf1p was found to have little if any, effect on activation of either the chromosomal or plasmid-borne PGK gene.

Base Sequence↗
PubMed2004

Overexpression of octamer transcription factors 1 or 2 alone has no effect on HIV-1 transcription in primary human CD4 T cells.

We explored the binding of octamer (Oct) transcription factors to the HIV-1 long terminal repeat (LTR) by gel shift assays and showed none of the previously identified four potential Oct binding sites bound Oct-1 or Oct-2. Overexpression of Oct-1 or Oct-2 had no effect on HIV-1 LTR activity in transiently transfected primary human CD4 T cells. Next, primary human CD4 T cells were co-transfected with a green fluorescent protein (GFP)-expression vector and an Oct-1 or Oct-2 expression plasmid. The transfected cells were stimulated for 2 days and then infected with the NL4-3 strain of HIV-1. After 3 days of infection, there were no differences in HIV-1 p24 supernatant levels. Apoptosis of infected or bystander cells overexpressing Oct-1 or Oct-2 compared to control was also unaffected. Our studies demonstrate that Oct-1 and Oct-2 fail to bind to the HIV-1 LTR and have no effect on HIV-1 transcription in primary human CD4 T cells.

Apoptosis↗
PubMed1998

An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription.

The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

Binding Sites↗
PubMed1996

Calmodulin-dependent protein kinase II potentiates transcriptional activation through activating transcription factor 1 but not cAMP response element-binding protein.

Activating transcription factor 1 (ATF1) and the cAMP response element-binding protein (CREB) are members of the CREB/ATF family implicated in cAMP- and calcium-induced transcriptional activation. Although ATF1 and CREB share extensive homology, the function of ATF1 is poorly understood. Its phosphorylation state and activation by Ca2+- and calmodulin-dependent protein kinase (CaMK) II were therefore examined. Phosphopeptide mapping analysis and Western blotting studies demonstrated that in vitro, CaMK II phosphorylates only Ser63 (corresponding to Ser133 of CREB), which is essential for the activation, and not Ser72 (corresponding to Ser142 of CREB), which is a negative regulation site. Both ATF1 and CREB bound CBP in a phosphorylation-dependent manner. As expected from these in vitro studies, transient transfection studies revealed that ATF1 is activated by CaMK II. Our findings suggest that CaMK II mediates transactivation of cAMP responsive genes via ATF1.

Activating Transcription Factor 1↗
PubMed1999

Context-dependent transcriptional cooperation mediated by cardiac transcription factors Csx/Nkx-2.5 and GATA-4.

Although the cardiac homeobox gene Csx/Nkx-2.5 is essential for normal heart development, little is known about its regulatory mechanisms. In a search for the downstream target genes of Csx/Nkx-2. 5, we found that the atrial natriuretic peptide (ANP) gene promoter was strongly transactivated by Csx/Nkx-2.5. Deletion and mutational analyses of the ANP promoter revealed that the Csx/Nkx-2.5-binding element (NKE2) located at -240 was required for high level transactivation by Csx/Nkx-2.5. We also found that Csx/Nkx-2.5 and GATA-4 displayed synergistic transcriptional activation of the ANP promoter, and in contrast to previous reports (Durocher, D., Charron, F., Warren, R., Schwartz, R. J., and Nemer, M. (1997) EMBO J. 16, 5687-5696; Lee, Y., Shioi, T., Kasahara, H., Jobe, S. M., Wiese, R. J., Markham, B., and Izumo, S (1998) Mol. Cell. Biol. 18, 3120-3129), this synergism was dependent on binding of Csx/Nkx-2.5 to NKE2, but not on GATA-4-DNA interactions. Although GATA-4 also potentiated the Csx/Nkx-2.5-induced transactivation of the artificial promoter that contains multimerized Csx/Nkx-2.5-binding sites, Csx/Nkx-2.5 reduced the GATA-4-induced transactivation of the GATA-4-dependent promoters. These findings indicate that the cooperative transcriptional regulation mediated by Csx/Nkx-2.5 and GATA-4 is promoter context-dependent and suggest that the complex cis-trans interactions may fine-tune gene expression in cardiac myocytes.

Animals↗

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