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At least 451 records · Page 25Linked to original sources
PubMed2000

Identification of acute myeloid leukemia patients with diminished expression of CD13 myeloid transcripts by competitive reverse transcription polymerase chain reaction (RT-PCR).

Normal myeloid cells of monocytic and granulocytic origin express the metallopeptidase cluster of differentiation 13 (CD13) on the surface just as leukemic blasts in most acute myeloid leukemias (AML). A minor percentage of AML patients, however, lack the surface expression of CD13 antigen. To study this difference in CD13 surface expression, specific CD13 mRNA from 44 individuals were quantified by competitive reverse transcription polymerase chain reaction (RT-PCR). Absolute values for CD13 transcripts were normalised against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript levels to control for variations in sample preparation and mRNA degradation. By correlating normalised CD13 transcript levels and CD13 surface expression, a subgroup of AML patients was identified, having simultaneous diminished levels of myeloid CD13 transcripts and surface expression of the corresponding antigen. For this subgroup we suggest CD13/aminopeptidase N (APN) gene expression to be restricted primarily by limited amounts of transcripts. For the majority of AML patients determinants in addition to transcript levels must be involved in regulating CD13/APN gene expression.

Adult↗
PubMed1999

Biallelic transcription of Igf2 and H19 in individual cells suggests a post-transcriptional contribution to genomic imprinting.

The H19 and insulin-like growth factor 2 (Igf2) genes in the mouse are models for genomic imprinting during development. The genes are located only 90 kb apart in the same transcriptional orientation [1], but are reciprocally imprinted: Igf2 is paternally expressed while H19 is maternally expressed. It has been suggested that expression of H19 and repression of Igf2 (or the converse) on a given chromosome are mechanistically linked and that the parental imprint operates at the level of transcription [2]. Although expression of Igf2 and H19 is thought to be monoallelic, the data have so far been obtained exclusively by looking at steady-state RNA levels using techniques that reflect the average activity of the genes in a cell population [3] [4]. Here, we have adapted a fluorescent in situ hybridisation (FISH) method to detect nascent RNA molecules of Igf2 and H19 at the initial transcription sites in the nuclei of wild-type mouse embryonic liver cells. Nine different transcription patterns were observed, reflecting a high heterogeneity of transcription at the single-cell level. Our observations suggest that regulation of Igf2 and H19 by parental imprinting is much more complex than previously proposed and acts at both transcriptional and post-transcriptional levels.

Alleles↗
PubMed1996

Down-regulation of cytokine-induced cyclo-oxygenase-2 transcript isoforms by dexamethasone: evidence for post-transcriptional regulation.

The immediate-early cyclo-oxygenase-2 (Cox-2) gene encodes an inducible prostaglandin synthase enzyme that has been implicated in inflammatory and proliferative diseases. We have shown that the inflammatory cytokine interleukin-1 (IL-1) induces the Cox-2 gene in a sustained manner and that post-transcriptional mRNA stabilization is an important even [Ristimäki, Garfinkel, Wessendorf, Maciag and Hla (1994) J. Biol. Chem. 269, 11769-11775]. The anti-inflammatory glucocorticoid dexamethasone potently down-regulates IL-1-induced Cox-2 mRNA expression. Kinetic studies suggest that antagonism of IL-1-induced mRNA stabilization is, at least in part, responsible for the suppression of Cox-2 mRNA. The Cox-2 gene produces two major transcript isoforms, namely Cox-2(4.6) (4.6 kb) and Cox-2(2.8) (2.8 kb), which are derived by alternative polyadenylation in the 3'-untranslated region (UTR). In response to dexamethasone, the short Cox-2(2.8) transcript isoform, which lacks a highly conserved AU-rich region, decays with a longer half-life than the Cox-2(4.6) isoform. Furthermore, heterologous expression of the hybrid Cox-1 open reading frame and the Cox-2 3'-UTR results in the accumulation of high levels of the short isoform and lower levels of the long isoform. These data suggest that multiple elements in the 3'-UTR of the Cox-2 gene are involved in the determination of the differential mRNA stabilities of Cox-2 transcript isoforms. Because dexamethasone destabilizes the Cox-2 transcript, and because the decay of Cox-2 transcript isoforms induced by dexamethasone occurs with different half-lives, post-transcriptional mRNA destabilization may be an important mechanism in the action of anti-inflammatory glucocorticoids.

Base Sequence↗
PubMed2000

Regulation of the CYB2 gene expression: transcriptional co-ordination by the Hap1p, Hap2/3/4/5p and Adr1p transcription factors.

Expression of the Saccharomyces cerevisiae nuclear gene CYB2 encoding the mitochondrial enzyme L-(+)-lactate-cytochrome c oxidoreductase (EC 1.2.2.3) is subject to several strict metabolic controls at the transcriptional level: repression due to glucose fermentation, derepression by ethanol, induction by lactate and inhibition under anaerobic conditions or in response to deficiency of haem biosynthesis. In this respect, the data obtained from the transcriptional analysis of the CYB2 gene contribute to a better understanding of the control of mitochondrial biogenesis. In this study, we show that Hap1p is the main transcriptional activator involved in the control of CYB2 transcription. We found that Hap1p activity, known to be oxygen dependent, is effected by DNA-protein interaction with two binding sites present in the CYB2 promoter. Control is moreover dependent on carbon sources. This regulation by the carbon substrates is subordinate to the activity of the complex Hap2/3/4/5p, which counteracts the negative effect of the URS1 element. Finally, our results suggest that the Adr1p transcriptional activator is also required in CYB2 transcription control. This work provides new data which allows a better understanding of the molecular mechanisms implicated in the co-regulation at the transcriptional level of the genes encoding proteins involved in various aspects of oxidative metabolism.

Animals↗
PubMed2003

Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription.

Ribonucleotide reductase is essential for the synthesis of all four dNTPs required for DNA replication. The enzyme is composed of two proteins, R1 and R2, which are both needed for activity. Expression of the R1 and R2 mRNAs is restricted to the S-phase of the cell cycle, but the R1 and R2 promoters show no obvious sequence homologies that could indicate coordination of transcription. Here we study initiation of transcription at the natural mouse R2 promoter, which contains an atypical TATA-box with the sequence TTTAAA, using a combination of in vivo reporter gene assays and in vitro transcription. Our results indicate that in constructs where sequences from the R2 5'-UTR are present, the mouse R2 TATA-box is dispensable both for unregulated, basal transcription from the R2 promoter and for S-phase specific activity. Instead, initiation of R2 transcription is directed by sequences downstream from the transcription start. We report that this region contains a conserved palindrome sequence that interacts with TAFIIs. This interaction down-regulates basal transcription from the R2 promoter, both in the absence and in the presence of the TATA-box.

5' Untranslated Regions↗
PubMed1998

Chromatin and transcription--how transcription factors battle with a repressive chromatin environment.

The last year has seen much progress in our understanding of chromatin and transcription. Transcriptionally active chromatin has long been correlated with a higher level of histone acetlyation. The discovery of a nuclear histone acetyltransferase activity encoded by factors with a role in transcription raises the possibility that the cell is able to dynamically modulate the (local) level of histone acteylation, switching chromatin templates from inactive to transcriptionally active states. Furthermore, histone acetylation states have shown to play a role in determining the efficacy of transcriptionally silenced chromatin in both yeast and Drosophila. The advances in our knowledge regarding the role of the origin-recognition complex in the establishment of silencing, and the requirement for a locally concentrated zone of the silence information regulator proteins in the nucleus has provided insights into the complex architecture of silenced chromatin. The goal of understanding the mechanisms by which the cell is able to 'open' repressive chromatin structures has prompted the discovery of multiple chromatin remodelling activities. These large protein complexes identified from organisms as diverse as yeast, mouse, fly and man demonstrate the ubiquity and fundamental importance of the ability to perturb the structure of chromatin allowing transcription of the desired genes. These data provide the latest and potentially most significant demonstration of the importance of the nucleosome in the regulation of transcription.

Acetyltransferases↗
PubMed1998

A role for the architectural transcription factors HMGI(Y) in cytokine gene transcription in T cells.

The ability of CD4- T helper (Th) cells to differentiate into two phenotypes distinguished by their cytokine profile is a major determinant of the type of immune response elicited by bacterial, viral or parasitic infections. The development of Th1 cells is associated with delayed-type hypersensitivity and cell-mediated immune responses while Th2 responses are associated with humoral immunity and allergic inflammation. While these phenotypes exist at the extremes of the immune response and are associated with pathological conditions, there is an enormous plasticity that allows reversibility and the development of a wide array of cytokine profiles. There has been considerable interest in determining the signals and transcription factors that govern the differential production of the Th1 and Th2 cytokines. There are now several candidate transcription factors that may play a role in skewing the cytokine profile in a distinct direction. Because of the plasticity of the system, these transcription factors must be able to respond to environmental signals in a very subtle manner and not simply be on/off switches for expression of the cytokine genes. The architectural transcription factor high mobility group (HMG) I(Y) is a modulator of the function of many of the transcription factors that control cytokine gene transcription. HMGI(Y) appears to play either a positive or negative role depending on the cytokine promoter and its ratio to other transcription factors. It is proposed that HMGI(Y) may have a role in regulating the production of cytokines in favour of a given immune response.

Cell Differentiation↗
PubMed1982

Transcription termination at the tryptophan operon attenuator is decreased in vitro by an oligomer complementary to a segment of the leader transcript.

A DNA oligomer 15 nucleotides long was used to probe the involvement of RNA secondary structure in the control of transcription termination at the attenuator of the tryptophan (trp) operon of Escherichia coli. This 15-mer is perfectly complementary to a segment of trp RNA that is thought to play a role in regulation of attenuation. When added to an in vitro transcription reaction mixture containing wild-type E. coli or Salmonella typhimurium trp operon templates, the complementary 15-mer caused a 4-fold increase in read-through transcription. By contrast, the 15-mer did not affect attenuation when a mutant E. coli template was used that does not allow formation of a crucial RNA secondary structure. Control experiments established that oligomers that were not complementary to E. coli trp leader RNA did not affect attenuation and that the 15-mer did not reduce termination when the transcript lacked a complementary region. Other experiments established that the 15-mer did not increase read-through transcription by allowing RNA polymerase molecules that might have already stopped at the attenuator to resume transcription. These findings provide direct support for the view that alternate base-paired structures control transcription termination at the trp attenuator.

DNA, Bacterial↗
PubMed1991

Footprinting analysis of mammalian RNA polymerase II along its transcript: an alternative view of transcription elongation.

Ternary complexes of RNA polymerase II, bearing the nascent RNA transcript, are intermediates in the synthesis of all eukaryotic mRNAs and are implicated as regulatory targets of factors that control RNA chain elongation and termination. Information as to the structure of such complexes is essential in understanding the catalytic and regulatory properties of the RNA polymerase. We have prepared complexes of purified RNA polymerase II halted at defined positions along a DNA template and used RNase footprinting to map interactions of the polymerase with the nascent RNA. Unexpectedly, the transcript is sensitive to cleavage by RNases A and T1 at positions as close as 3 nucleotides from the 3'-terminal growing point. Ternary complexes in which the transcript has been cleaved to give a short fragment can retain that fragment and remain active and able to continue elongation. Since DNA.RNA hybrid structures are completely resistant to cleavage under our reaction conditions, the results suggest that any DNA.RNA hybrid intermediate can extend for no more than 3 base pairs, in dramatic contrast to recent models for transcription elongation. At lower RNase concentrations, the transcript is protected from cleavage out to about 24 nucleotides from the 3' terminus. We interpret this partial protection as due to the presence of an RNA binding site on the polymerase that binds the nascent transcript during elongation, a model proposed earlier by several workers in preference to the hybrid model. The properties of this RNA binding site are likely to play a central role in the process of transcription elongation and termination and in their regulation.

Animals↗
PubMed1995

Effects of the antiterminator BoxA on transcription elongation kinetics and ppGpp inhibition of transcription elongation in Escherichia coli.

It has been shown previously that two different mRNA chains (lacZ and infB) are elongated at a rate of approximately 40 nucleotides (nt)/s during steady state growth on minimal medium and that the rate of mRNA chain elongation is inhibited by ppGpp in vivo. On the other hand, it was found that a truncated ribosomal RNA chain was elongated at a rate of approximately 80 nt/s, independent of growth condition (Vogel, U., and Jensen, K. F. (1994) J. Biol. Chem. 269, 16236-16241). We reasoned that the different transcriptional behavior of mRNA genes and rRNA operons might be caused by the antiterminator sequences present in the rRNA operons. To test this possibility, we have (a) inserted the minimal antiterminator boxA sequence between the promoter and the lacZ and infB genes and (b) deleted the antiterminator sequences from the rRNA transcription unit and measured transcription elongation rates in vivo on the resulting hybrid genes. We found that insertion of boxA in front of the coding region of lacZ increased the transcription elongation rate from 42 nt/s to 69 nt/s during steady state growth and that it eliminated the ppGpp-dependent decrease in the transcription elongation rate during the stringent response. On the other hand, deletion of the antiterminator sequences from the rRNA operon resulted in a reduced transcription elongation rate, but the elongation rate was still insensitive to changes in the ppGpp pool. These results are consistent with the hypothesis that the antiterminator boxA is a primary determinant of the rate of transcription elongation rate.

Base Sequence↗
PubMed1998

A cyclic AMP response element in the angiotensin-converting enzyme gene and the transcription factor CREM are required for transcription of the mRNA for the testicular isozyme.

The angiotensin-converting enzyme (ACE) gene produces two mRNA species from tissue-specific promoters. The transcription start site of the mRNA for the smaller testicular isozyme (ACET) is located within an intron of the larger transcription unit that encodes the pulmonary isozyme (ACEP).We have previously demonstrated that a 298-base pair DNA fragment, 5' to the rabbit ACET mRNA transcription initiation site, can activate the testicular expression of a transgenic reporter gene. In the current study, using the same transgenic reporter system, we identified a putative cyclic AMP response element present within this DNA fragment to be absolutely essential for transcriptional activation. Moreover, we observed that ACET mRNA was not expressed in the testes of mice homozygous for a null mutation in the transcription factor CREM. However, in the same mice, ACEP mRNA was abundantly expressed in the lung. Our observations indicate that ACET mRNA expression in the testes is regulated by the putative cyclic AMP response element present 5' to the transcription start site and the corresponding transcription factor CREM.

Animals↗
PubMed1998

Lack of transcription-coupled repair of acetylaminofluorene DNA adducts in human fibroblasts contrasts their efficient inhibition of transcription.

The N-(deoxyguanosine-8-yl)-2-acetylaminofluorene (dG-C8-AAF) lesion is among the most helix distorting DNA lesions. In normal fibroblasts dG-C8-AAF is repaired rapidly in transcriptionally active genes, but without strand specificity, indicating that repair of dG-C8-AAF by global genome repair (GGR) overrules transcription-coupled repair (TCR). Yet, dG-C8-AAF is a very potent inhibitor of transcription. The target size of inhibition (45 kilobases) suggests that transcription inhibition by dG-C8-AAF is caused by blockage of initiation rather than elongation. Cockayne's syndrome (CS) cells appear to be extremely sensitive to the cytotoxic effects of dG-C8-AAF and are unable to recover inhibited RNA synthesis. However, CS cells exhibit no detectable defect in repair of dG-C8-AAF in active genes, indicating that impaired TCR is not the cause of the enhanced sensitivity of CS cells. These and data reported previously suggest that the degree of DNA helix distortion determines the rate of GGR as well as the extent of inhibition of transcription initiation. An interchange of the transcription/repair factor TFIIH from promoter sites to sites of damage might underlie inhibition of transcription initiation. This process is likely to occur more rapidly and efficiently in the case of strongly DNA helix distorting lesions, resulting in a very efficient GGR, a poor contribution of TCR to repair of lesions in active genes, and an efficient inhibition of transcription.

2-Acetylaminofluorene↗
PubMed1999

The Tup1-Cyc8 protein complex can shift from a transcriptional co-repressor to a transcriptional co-activator.

Cyc8(Ssn6)-Tup1, a general co-repressor complex, is recruited to promoter DNA via interactions with DNA-binding regulatory proteins and inhibits the transcription of many different yeast genes. Previous studies have established that repression function of the complex is performed by one subunit of the complex, the Tup1 protein, and requires specific components of the RNA polymerase II holoenzyme such as Sin4 and Rgr1. In this study we test the transcriptional activity of the Cyc8 subunit using a lexA operator-containing reporter. We show that a LexA-Cyc8 hybrid stimulates transcription when expressed in a tup1Delta, a sin4Delta, or a rgr1Delta strain, suggesting that transcriptional activation is an intrinsic property of the Cyc8-Tup1 co-repressor. In support of this notion we demonstrate that Cyc8-Tup1 has a dual function on CIT2, a gene encoding a citrate synthase that is expressed upon mitochondrial dysfunction. First, we show that Cyc8-Tup1 is tethered to CIT2 promoter by interacting with the activation domain of Rtg3, a bHLH/L-Zip DNA-binding transactivator of CIT2. Next we demonstrate that Cyc8-Tup1 activates CIT2 transcription in response to mitochondrial dysfunction, and this stimulatory effect is mediated by Cyc8. In contrast, basal (noninduced) expression of this gene is inhibited by Tup1. These findings establish a positive role for the Cyc8-Tup1 complex in transcription and support a model by which specific metabolic signals may convert the Cyc8-Tup1 transcriptional co-repressor to a co-activator of certain promoters.

DNA-Binding Proteins↗
PubMed2003

Functional dissection of transcription factor ZBRK1 reveals zinc fingers with dual roles in DNA-binding and BRCA1-dependent transcriptional repression.

The breast- and ovarian-specific tumor suppressor BRCA1 has been implicated in both activation and repression of gene transcription by virtue of its direct interaction with sequence-specific DNA-binding transcription factors. However, the mechanistic basis by which BRCA1 mediates the transcriptional activity of these regulatory proteins remains largely unknown. To clarify this issue, we have examined the functional interaction between BRCA1 and ZBRK1, a BRCA1-dependent KRAB eight zinc finger transcriptional repressor. We report here the identification and molecular characterization of a portable BRCA1-dependent transcriptional repression domain within ZBRK1 composed of zinc fingers 5-8 along with sequences in the unique ZBRK1 C terminus. This C-terminal repression domain functions in a BRCA1-, histone deacetylase-, and promoter-specific manner and is thus functionally distinguishable from the N-terminal KRAB repression domain in ZBRK1, which exhibits no BRCA1 dependence and broad promoter specificity. Significantly, we also find that the BRCA1-dependent transcriptional repression domain on ZBRK1 includes elements that modulate its sequence-specific DNA binding activity. These findings thus reveal the presence within ZBRK1 of functionally bipartite zinc fingers with dual roles in sequence-specific DNA-binding and BRCA1-dependent transcriptional repression. We discuss the implications of these findings for the role of BRCA1 as ZBRK1 co-repressor.

BRCA1 Protein↗
PubMed2004

Post-transcriptional regulation of endothelial nitric-oxide synthase by an overlapping antisense mRNA transcript.

Endothelial nitric-oxide synthase (eNOS) mRNA levels are abnormal in diseases of the cardiovascular system, but changes in gene expression cannot be accounted for by transcription alone. We found evidence for the existence of an antisense mRNA (sONE) that is derived from a transcription unit (NOS3AS) on the opposite DNA strand from which the human eNOS (NOS3) mRNA is transcribed at human chromosome 7q36. The genes are oriented in a tail-to-tail configuration, and the mRNAs encoding sONE and eNOS are complementary for 662 nucleotides. The mRNA for sONE could be detected in a variety of cell types, both in vivo and in vitro, but not vascular endothelial cells. In contrast, expression of eNOS is highly restricted to vascular endothelium. Most surprisingly, interrogation of transcriptional events across NOS3/NOS3AS genomic regions, using single- and double-stranded probes for nuclear run-off analyses and chromatin immunoprecipitation-based assessments of RNA polymerase II distribution, indicated that NOS3 and NOS3AS gene transcription did not correlate with steady-state mRNA levels. We found strong evidence supporting a role for NOS3AS in the post-transcriptional regulation of NOS3 expression. RNA interference-mediated inhibition of sONE expression in vascular smooth muscle cells increased eNOS expression. Overexpression of sONE in endothelial cells blunted eNOS expression. Finally, the histone deacetylase inhibitor trichostatin A is known to regulate the expression of eNOS via a post-transcriptional mechanism. We found that trichostatin A treatment of vascular endothelial cells increased expression of sONE mRNA levels prior to the observed decrease in eNOS mRNA expression. Taken together, these results indicate that an antisense mRNA (sONE) participates in the post-transcriptional regulation of eNOS and provide a newer model for endothelial cell-specific gene expression.

Base Sequence↗
PubMed1989

Relative V beta transcript levels in thymus and peripheral lymphoid tissues from various mouse strains. Inverse correlation of I-E and Mls expression with relative abundance of several V beta transcripts in peripheral lymphoid tissues.

We have measured the relative levels of transcripts for 15 of the 22 known V beta gene segments. The level of transcripts for the highest and lowest expressed V beta gene segment differed by greater than 20-fold in the thymus and an even larger difference was observed in the periphery. The levels of expressions were unrelated to the order of the V beta genes on the chromosome. For most of the V beta gene segments, the relative transcript levels were the same in the thymus and periphery, suggesting that thymic selection in general does not act solely upon the V beta gene segment. One V beta gene segment in the BALB and B10 mice strains was an exception to this rule. V beta 5.2 expression in the periphery of BALB and B10 mice inversely correlated with the expression of the MHC class II molecule I-E. Five V beta gene segments had reduced transcript levels in the periphery of Mls-1a mice compared with their thymic levels or to the levels found in Mls-1b mice. The peripheral level of V beta 3 transcripts vary with MHC and Mls-2 haplotypes. The observation that certain V beta transcript levels are reduced in the periphery when compared with the thymus favors the hypothesis that self tolerance at the T cell level results in the elimination of self-reactive T cells, rather than paralysis by a block at some post-transcriptional step. Finally, the wide variability of V beta gene segment expression in the thymus suggests mechanisms exist to import an early bias to the repertoire. Whether this bias results from differential V beta segment rearrangement rates, differential V beta expression rates, or events occurring after TCR-alpha/beta expression on immature/nonmature thymocyte cell surfaces is yet to be determined.

Animals↗
PubMed1995

Transcription of the 5'-terminal cap nucleotide by RNA-dependent DNA polymerase: possible involvement in retroviral reverse transcription.

The possible transcription of the 5'-terminal cap nucleotide of mRNA by RNA-dependent DNA polymerase was examined by a single-step assay, based on the generation of a hairpin structure during reverse transcription of globin mRNA. Using this approach, we demonstrated that the 5'-terminal cap nucleotide of mRNA is indeed transcribed by RNA-dependent DNA polymerase into a 3'-terminal residue of cDNA and were able to measure the extent of such transcription. The observed transcription of the cap nucleotide raises a number of questions that may be addressed using the relatively simple single-step assay employed in the present study. Cap nucleotide transcription by RNA-dependent DNA polymerase may have important implications for our understanding of the mechanism of action of reverse transcriptases. It may represent a selection mechanism for only partial transcription of the 5' repeat element of viral RNA genome, thus generating a RNA fragment that may play a role in priming of second (plus) DNA strand during retroviral reverse transcription. Moreover, the demonstrated ability of complementary nucleotides to form hydrogen bonds, even when in a parallel orientation, may have interesting and important consequences.

Animals↗
PubMed1997

Histone acetylation: influence on transcription, nucleosome mobility and positioning, and linker histone-dependent transcriptional repression.

We demonstrate using a dinucleosome template that acetylation of the core histones enhances transcription by RNA polymerase III. This effect is not dependent on an increased mobility of the core histone octamer with respect to DNA sequence. When linker histone is subsequently bound, we find both a reduction in nucleosome mobility and a repression of transcription. These effects of linker histone binding are independent of core histone acetylation, indicating that core histone acetylation does not prevent linker histone binding and the concomitant transcriptional repression. These studies are complemented by the use of a Xenopus egg extract competent both for chromatin assembly on replicating DNA and for RNA polymerase III transcription. Incorporation of acetylated histones and lack of linker histones together facilitate transcription by >10-fold in this system; however, they have little independent effect on transcription. Thus core histone acetylation significantly facilitates transcription, but this effect is inhibited by the assembly of linker histones into chromatin.

Acetylation↗