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PubMed1999

Cell cycle-regulated transcription in fission yeast: Cdc10-Res protein interactions during the cell cycle and domains required for regulated transcription.

In Schizosaccharomyces pombe the MBF (DSC1) complex mediates transcriptional activation at Start and is composed of a common subunit called Cdc10 in combination with two alternative DNA-binding partners, Res1 and Res2. It has been suggested that a high-activity MBF complex (at G1/S) is switched to a low-activity complex (in G2) by the incorporation of the negative regulatory subunit Res2. We have analyzed MBF protein-protein interactions and find that both Res proteins are associated with Cdc10 throughout the cell cycle, arguing against this model. Furthermore we demonstrate that Res2 is capable of interacting with a mutant form of Cdc10 that has high transcriptional activity. It has been shown previously that both Res proteins are required for periodic cell cycle-regulated transcription. Therefore a series of Res1-Res2 hybrid molecules was used to determine the domains that are specifically required to regulate periodic transcription. In Res2 the nature of the C-terminal region is critical, and in both Res1 and Res2, a domain overlapping the N-terminal ankyrin repeat and a recently identified activation domain is important for mediating cell cycle-regulated transcription.

Cell Cycle↗

PubMed2000

A zinc-finger protein, Rst2p, regulates transcription of the fission yeast ste11(+) gene, which encodes a pivotal transcription factor for sexual development.

Schizosaccharomyces pombe ste11 encodes a high-mobility group family transcriptional activator that is pivotal in sexual development. Transcription of ste11 is induced by starvation of nutrients via a decrease of the cAMP-dependent protein kinase (PKA) activity. Here we report the identification of a novel transcription factor, Rst2p, that directly regulates ste11 expression. Cells in which the rst2 gene was disrupted expressed ste11 poorly and were sterile, and this sterility could be suppressed by artificial expression of ste11. Disruption of rst2 suppressed hypermating and hypersporulation in the PKA-null mutant, whereas overexpression of rst2 induced sexual development in the PKA-activated mutant. Cloning analysis indicated that Rst2p was a Cys(2)His(2) zinc-finger protein carrying 567 amino acid residues. Rst2p could bind specifically to a stress response element-like cis element located in the ste11 promoter region, which was important for ste11 expression. Meanwhile, transcription of ste11 was reduced significantly by a defective mutation in itself. An artificial supply of functional Ste11p circumvented this reduction. A complete Ste11p-binding motif (TR box) found in the promoter region was necessary for the full expression of ste11, suggesting that Ste11p is involved in the activation of ste11. We conclude that transcription of ste11 is under autoregulation in addition to control through the PKA-Rst2p pathway.

Amino Acid Sequence↗

PubMed1998

Transcription elongation factor P-TEFb mediates Tat activation of HIV-1 transcription at multiple stages.

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcription elongation through recognition of the transactivation response (TAR) RNA stem-loop structure at the 5' end of nascent viral transcripts. Recently, a human transcription elongation factor P-TEFb, consisting of CDK9 kinase, cyclin T and other associated factors, has been shown to interact with Tat to restore Tat activation in HeLa nuclear extract depleted of P-TEFb. Here, we report the purification of a P-TEFb complex fraction containing epitope-tagged wild-type CDK9 or kinase-inactive CDK9 and five tightly associated polypeptides. Only wild-type P-TEFb complex with an active CDK9 kinase was able to hyperphosphorylate the C-terminal domain of RNA polymerase II and mediate Tat transactivation in P-TEFb-depleted HeLa nuclear extract. Tat also stimulated transcription elongation by recruitment of the P-TEFb complex to the HIV-1 promoter through a Tat-TAR interaction. A possible mechanism for P-TEFb to become associated with polymerase elongation complexes and function as a general elongation factor was demonstrated by an interaction of P-TEFb with double-stranded RNA molecules through an 87 kDa subunit. Finally, P-TEFb was found to interact with and phosphorylate Tat-SF1, a Tat cofactor required for Tat transactivation. Our data indicate that the various subunits of the human P-TEFb complex may play distinct roles at multiple stages to mediate Tat activation of HIV-1 transcription elongation.

Base Sequence↗

PubMed1998

Isolation of cDNAs encoding novel transcription coactivators p52 and p75 reveals an alternate regulatory mechanism of transcriptional activation.

Transcriptional activation in human cell-free systems containing RNA polymerase II and general initiation factors requires the action of one or more additional coactivators. Here, we report the isolation of cDNAs encoding two novel human transcriptional coactivators (p52 and p75) that are derived from alternatively spliced products of a single gene and share a region of 325 residues, but show distinct coactivator properties. p52 and p75 both show strong interactions with the VP16 activation domain and several components of the general transcriptional machinery. p52, like the previously described PC4, is a potent broad-specificity coactivator, whereas p75 is less active for most activation domains. These results suggest that p52 is a general transcriptional coactivator that mediates functional interactions between upstream sequence-specific activators and the general transcription apparatus, possibly through a novel mechanism.

Adaptor Proteins, Signal Transducing↗

PubMed1985

Transcription of a cloned rainbow trout protamine gene is accurately initiated following transfection into HeLa cells but the majority of the transcripts fail to polyadenylate at the correct site.

The expression of a cloned trout protamine gene transfected into mammalian cells in culture has been studied. This small intronless gene has a consensus TATA-box, a classical AATAAA sequence and the cap and polyadenylation sites are separated by only 228 base pairs (Gregory et al., ref 10). When 1kb of cloned trout genomic DNA containing this sequence was introduced into HeLa cells, S1-mapping showed that transcripts of the protamine gene were accurately initiated at the in vivo cap site but were not polyadenylated at the authentic 3'-site. Replacement of the 3'-end of the protamine transcription unit with a fragment of SV40 containing the small-t intron and early polyadenylation site resulted in only a modest increase in transcript levels over the wild-type gene in HeLa cells. However, transcripts of a fusion gene in which the 5'-end of the protamine gene was replaced by the SV40 early promoter were present at extremely low levels in transfected COS cells. The data are discussed in the context of the involvement of RNA processing events in the stabilisation of eukaryotic gene transcripts.

Animals↗

PubMed1994

Novel clustering of Sp1 transcription factor binding sites at the transcription initiation site of the human muscle phosphofructokinase P1 promoter.

The regulatory sequence elements of the human muscle phosphofructokinase (HPFKM) p1 promoter from -655 to +78 were cloned and characterized. In the human cervical carcinoma cell line, HeLa S3, the HPFKM type C RNA initiated from a single predominant transcription initiation site and the HPFKM p1 promoter displayed transcriptional activity in transient transfection assays. The HPFKM p1 promoter region was shown to possess eight binding sites for the Sp1 transcription factor by DNase I footprinting and gel retardation analysis. The functional importance of these interactions was examined by transient transfection analysis in Drosophila SL2 and HeLa S3 cells. This analysis demonstrated that the HPFKM p1 promoter sequence between +12 and +78 retained Sp1-dependent transcriptional activity in Drosophila SL2 cells and retained promoter activity in HeLa S3 cells. These results suggest that the Sp1 binding site (site 8 between +12 and +21) immediately adjacent to the transcription initiation site represents an important regulatory element of this promoter at least in the context of the minimal HPFKM p1 promoter. However mutagenesis of the Sp1 site 8 demonstrated that, in the context of a larger HPFKM p1 promoter region containing Sp1 sites 1 to 7, it now contributed very little to the total promoter activity. Therefore it appears the Sp1 sites in the HPFKM p1 promoter display functional redundancy.

Animals↗

PubMed2001

Analysis of intergenic spacer transcripts suggests 'read-around' transcription of the extrachromosomal circular rDNA in Euglena gracilis.

We report here the sequence of the 1743 bp intergenic spacer (IGS) that separates the 3'-end of the large subunit ribosomal RNA (rRNA) gene from the 5'-end of the small subunit (SSU) rRNA gene in the circular, extrachromosomal ribosomal DNA (rDNA) of Euglena gracilis. The IGS contains a 277 nt stretch of sequence that is related to a sequence found in ITS 1, an internal transcribed spacer between the SSU and 5.8S rRNA genes. Primer extension analysis of IGS transcripts identified three abundant reverse transcriptase stops that may be analogous to the transcription initiation site (TIS) and two processing sites (A' and A0) that are found in this region in other eukaryotes. Features that could influence processing at these sites include an imperfect palindrome near site A0 and a sequence near site A' that could potentially base pair with U3 small nucleolar RNA. Our identification of the TIS (verified by mung bean nuclease analysis) is considered tentative because we also detected low-abundance transcripts upstream of this site throughout the entire IGS. This result suggests the possibility of 'read-around' transcription, i.e. transcription that proceeds multiple times around the rDNA circle without termination.

Animals↗

PubMed2005

Regulation of metabolic transcriptional co-activators and transcription factors with acute exercise.

Endurance exercise improves insulin sensitivity and increases fat oxidation, which are partly facilitated by the induction of metabolic transcription factors. Next to exercise, increased levels of FFA's also increase the gene expression of transcription factors, hence making it difficult to discern the effects from contractile signals produced during exercise, from those produced by increased circulatory FFA's. We aimed to investigate, in human skeletal muscle, whether acute exercise affects gene expression of metabolic transcriptional co-activators and transcription factors, including PGC-1alpha, PRC, PPARalpha, beta/delta, and gamma and RXR, SREBP-1c and FKHR, and to discern the effect of exercise per se from those of elevated levels of FFA. Two hours of endurance exercise was performed either in the fasted state, or following carbohydrate ingestion prior to and during exercise, thereby blunting the fasting-induced increase in FA availability and oxidation. Of the genes measured, PGC-1alpha and PRC mRNA increased immediately after, while PPARbeta/delta and FKHR mRNA increased 1-4 h after exercise, irrespective of the increases in FFA's. Our results suggest that the induction in vivo of metabolic transcription factors implicated in mitochondrial biogenesis are under the control of inherent signals, (PGC-1alpha, PRC), while those implicated in substrate selection are under the control of associated signals (PPARbeta/delta, FKHR) stimulated from the contracting skeletal muscle that are independent of circulating FFA levels.

Dietary Carbohydrates↗

PubMed2003

Gene expression in plant mitochondria: transcriptional and post-transcriptional control.

The informational content of the mitochondrial genome in plants is, although small, essential for each cell. Gene expression in these organelles involves a number of distinct transcriptional and post-transcriptional steps. The complex post-transcriptional processes of plant mitochondria such as 5' and 3' RNA processing, intron splicing, RNA editing and controlled RNA stability extensively modify individual steady-state RNA levels and influence the mRNA quantities available for translation. In this overview of the processes in mitochondrial gene expression, we focus on confirmed and potential sites of regulatory interference and discuss the evolutionary origins of the transcriptional and post-transcriptional processes.

Cell Nucleus↗

PubMed1998

Transcription and transcript processing in the sdhCDAB-sucABCD operon of Escherichia coli.

The genes encoding succinate dehydrogenase (sdhCDAB), the specific components of the 2-oxoglutarate dehydrogenase complex (ODH, E1o and E2o; sucAB) and succinyl-CoA synthetase (sucCD) form a cluster containing two promoters at 16.3 min in the chromosome of Escherichia coli: Psdh sdhCDAB-Psuc sucAB-sucCD. The gene encoding the lipoamide dehydrogenase component of both the 2-oxoglutarate and pyruvate dehydrogenase complexes (E3; lpdA) is the distal gene of another cluster containing two promoters located at 2.7 min: Ppdh pdhR-aceEF-Plpd lpdA. The responses of the suc and lpd promoters to different environmental conditions and to regulator defects were investigated with appropriate lacZ fusions, in order to understand how expression of the sucAB genes is co-regulated with other genes in the sdhCDAB-sucABCD cluster and with lpdA expression. Expression from the suc promoter was repressed by IHF and partially activated by sigma 38 but it was not regulated by ArcA, FNR, CRP, FruR or Fis, and not repressed by glucose or anaerobiosis, indicating that the well-established catabolite and anaerobic repression of ODH synthesis is imposed elsewhere. In contrast, the lpd promoter was repressed by both glucose (via a CRP-independent mechanism) and anaerobiosis (mediated by ArcA), and activated by Fis, but it was not regulated by FNR, FruR, IHF or sigma 38. These observations support the view that transcription of the sucABCD genes is primarily initiated and regulated at the upstream sdh promoter, and that the lpd promoter is independently co-regulated with Psdh (primarily by ArcA-mediated repression) rather than with Psuc. Direct evidence for co-transcription of the entire sdhCDAB-sucABCD region from Psdh was obtained by detecting a 10 kb transcript in rnc and rne mutants, but not in the parental strains. Three RNaseIII-specific processing sites, which contribute to the extreme instability of the readthrough transcript, were identified in the sdhCDAB-sucABCD intergenic region. Other sites of endonuclease processing were located by interpreting the patterns of transcript subfragments observed in Northern blotting.

Base Sequence↗

PubMed1987

Contributions of transcriptional and post-transcriptional mechanisms to the regulation of c-myc expression in mouse erythroleukemia cells.

Chemically induced differentiation of mouse erythroleukemia (MEL) cells leads to complex changes in c-myc mRNA levels. Within 1-2 hr after the addition of the inducer hexamethylene bisacetamide (HMBA), c-myc mRNA levels decrease 10- to 20-fold and remain low until 12-24 hr, at which time the mRNA reaccumulates to its original level. Thereafter as the cells undergo terminal differentiation, c-myc mRNA again declines to a low level. We have investigated the regulation of these changes by measuring c-myc gene transcription and mRNA turnover. We find that the early rapid decline in c-myc mRNA is due to an increase in the block to elongation of transcription within the c-myc first exon. Effective c-myc transcription is then restored after 2 hr of HMBA treatment to the level present in uninduced cells and is maintained throughout the remainder of the differentiation program. These results demonstrate that, except for the rapid decline in c-myc mRNA immediately following inducer treatment, all subsequent regulation of message levels occurs through post-transcriptional mechanisms. Studies of c-myc mRNA turnover suggest that some post-transcriptional regulation is nuclear.

Acetamides↗

PubMed2001

Specific association of transcripts of tbzF and tbz17, tobacco genes encoding basic region leucine zipper-type transcriptional activators, with guard cells of senescing leaves and/or flowers.

Induction by low temperature is a common feature of the lip19 subfamily members of the basic region leucine zipper gene family in plants. Here, we characterize two tobacco (Nicotiana tabacum) genes, tbzF and tbz17, belonging to the lip19 subfamily, whose gene products, TBZF and TBZ17, show 73% identity and are located in nuclei. They preferentially bind to DNA fragments spanning A-box/G-box and C-box/G-box hybrid motifs and show transactivation activity in cobombarded tobacco BY-2 cells, indicating they function as transcriptional activators. Transcripts of tbzF were detected at a high level in senescing leaves and flowers. In contrast, tbz17 transcripts could be shown to accumulate in aged leaves but not in flowers. In situ hybridization analysis revealed transcripts of tbzF and tbz17 to be predominantly located in guard cells and vascular tissues of senescing leaves. These results suggest that TBZF and TBZ17 are both involved in controlling gene transcription related to functions of guard cells in senescing leaves and that TBZF bifunctionally acts in floral development.

Amino Acid Sequence↗

PubMed1995

Transcriptional and post-transcriptional regulation of granulocyte-macrophage colony-stimulating factor production in human growth factor dependent M-07e cells.

To elucidate the regulatory mechanisms of granulocyte-macrophage colony-stimulating factor (GM-CSF) production in human myeloid leukaemic cells we studied GM-CSF gene transcription, mRNA expression and GM-CSF secretion in human growth factor dependent M-07e cells. GM-CSF transcript was detected in cells cultured in the presence of interleukin-3 (IL-3). GM-CSF or mast cell growth factor (MGF), whereas it was undetectable in growth factor deprived cells. Growth factor re-addition induced, within 2 h, the appearance of GM-CSF mRNA. Nuclear run-on experiments demonstrated that the increase of GM-CSF mRNA levels depends on GM-CSF gene transcription. The simultaneous addition, to deprived cells, of the growth factor, and of cycloheximide (CHX) for 2 h inhibited GM-CSF mRNA expression, suggesting the requirement for newly made proteins for GM-CSF gene transcription. By means of the M-07e bioassay, which allows the detection of GM-CSF, IL-3 and MGF activities, and neutralizing antibodies to each of these factors, GM-CSF activity was detected in the cell-free extract of both IL-3- and MGF-sustained cells and of cells deprived for 24 h. This finding demonstrates that M-07e cells produce and store biologically active GM-CSF in response to both IL-3 and MGF. In contrast, analysis of the growth stimulatory activity present in the culture supernatants revealed that MGF, unlike IL-3, is able to induce the secretion of consistent amounts of GM-CSF. Taken together, our results suggest that, in M-07e cells, GM-CSF gene transcription and GM-CSF production are mediated, unlike its secretion, by mechanisms shared by IL-3 and MGF.

Blotting, Northern↗

PubMed2004

The tobacco bZIP transcription factor BZI-1 binds the GH3 promoter in vivo and modulates auxin-induced transcription.

Summary In order to establish the biological function of the tobacco basic leucine zipper (bZIP) transcription factor BZI-1 in hormone signalling, we have analysed transgenic plants which were altered with respect to the protein level or the activation potential of BZI-1. Overexpression of a dominant-negative derivative of BZI-1, lacking the N-terminal activation domain, resulted in plants displaying reduced internode size, enhanced lateral shoot formation and small, curly leaves. The response to auxin monitored with reference to root organogenesis, epinastic leaf curvature and transcription of the auxin-induced GH3 gene was reduced. In vitro, BZI-1 specifically binds to ACGT elements (ACEs) present in the GH3 promoter. In vivo, binding to the GH3 promoter was confirmed by chromatin immunoprecipitation (ChIP). Overexpression of BZI-1 in transgenic plants did not lead to a significant activation of the GH3 target gene. In contrast, plants expressing a VP16 (Herpes simplex virion protein 16)-BZI-1 fusion protein showed enhanced auxin-induced GH3 transcription. However, VP16-BZI-1 is insufficient to trigger GH3 expression independently of the auxin stimulus. Whereas auxin responsiveness has been shown to be mediated by ARF (auxin response factor) transcription factors, we discuss a function of BZI-1 assisting in fine-tuning of auxin-induced transcription.

Base Sequence↗

PubMed1996

The transcriptional apparatus required for mRNA encoding genes in the yeast Saccharomyces cerevisiae emerges from a jigsaw puzzle of transcription factors.

The number of identified yeast factors involved in transcription has dramatically increased in recent years and the understanding of the interplay between the different factors has become more and more puzzling. Transcription initiation at the core promoter of mRNA encoding genes consisting of upstream, TATA and initiator elements requires an approximately ribosome-sized complex of more than 50 polypeptides. The recent identification and isolation of an RNA polymerase holoenzyme which seems to be preassembled before interacting with a promoter allowed a better understanding of the roles, assignments and interplays of the various constituents of the basal transcription machinery. Recruitment of this complex to the promoter is achieved by numerous interactions with a variety of DNA-bound proteins. These interactions can be direct or mediated by additional adaptor proteins. Other proteins negatively affect transcription by interrupting the recruitment process through protein-protein or protein-DNA interactions. Some basic features of cis-acting elements, the transcriptional apparatus and various trans-acting factors involved in the initiation of mRNA synthesis in yeast are summarized.

Biological Evolution↗

PubMed1999

Both inducible and constitutive activator protein-1-like transcription factors are used for transcriptional activation of the galanin gene by different first and second messenger pathways.

We investigated trans-acting factors mediating galanin (GAL) gene activation by protein kinase-dependent signal transduction pathways in chromaffin cells. GAL mRNA up-regulation via the protein kinase A (PKA) pathway (25 microM forskolin) required new protein synthesis. Stimulation via protein kinase C (0.1 microM phorbol myristate acetate) did not. The involvement of activator protein-1(AP-1) and cAMP response element-binding protein (CREB) in serine/threonine protein kinase activation of GAL gene transcription was assessed. Cotransfection of a GAL reporter gene along with expression plasmids encoding c-Jun plus c-Fos, or the catalytic subunit of PKA (PKAbeta), resulted in a 4- to 8-fold enhancement of GAL reporter gene transcription. Transcriptional activation required the galanin 12-O-tetradecanoylphorbol-13-acetate (phorbol-12-myristate-13-acetate) response element (GTRE) octamer sequence (TGACGCGG) in the proximal enhancer of the GAL gene, previously shown to confer phorbol ester responsiveness in chromaffin cells. CREB coexpression did not stimulate GAL gene transcription or increase transcriptional activation by PKAbeta. The GTRE preferentially bound in vitro synthesized Jun and Fos-Jun, compared with CREB, in electrophoretic mobility shift assays. The GTRE preference for binding AP-1-immunoreactive protein compared with CREB was even more pronounced in chromaffin cell nuclear extracts, in which the majority of GTRE-bound protein in electrophoretic mobility shift assays was supershifted with anti-Fos and anti-Jun antibodies. Thus, GAL gene regulation mediated by protein kinase activation appears to involve both constitutively expressed and inducible AP-1-related proteins. Elevated potassium stimulation of GAL mRNA was completely blocked, but pituitary adenylyl cyclase-activating polypeptide and histamine stimulations were only partially blocked, by cycloheximide. Both inducible and constitutive pathways are therefore used by physiologically relevant first messengers that stimulate GAL biosynthesis in vivo.

Animals↗

PubMed2003

Transcription elongation factors repress transcription initiation from cryptic sites.

Previous studies have suggested that transcription elongation results in changes in chromatin structure. Here we present studies of Saccharomyces cerevisiae Spt6, a conserved protein implicated in both transcription elongation and chromatin structure. Our results show that, surprisingly, an spt6 mutant permits aberrant transcription initiation from within coding regions. Furthermore, transcribed chromatin in the spt6 mutant is hypersensitive to micrococcal nuclease, and this hypersensitivity is suppressed by mutational inactivation of RNA polymerase II. These results suggest that Spt6 plays a critical role in maintaining normal chromatin structure during transcription elongation, thereby repressing transcription initiation from cryptic promoters. Other elongation and chromatin factors, including Spt16 and histone H3, appear to contribute to this control.

Chromatin↗

PubMed2006

Translation control of trpG from transcripts originating from the folate operon promoter of Bacillus subtilis is influenced by translation-mediated displacement of bound TRAP, while translation control of transcripts originating from a newly identified trpG promoter is not.

Bacillus subtilis trpG encodes a glutamine amidotransferase subunit that participates in the biosynthesis of both tryptophan and folic acid. TRAP inhibits translation of trpG in response to tryptophan by binding to a site that overlaps the trpG Shine-Dalgarno sequence, thereby blocking ribosome binding. Similar mechanisms regulate trpP and ycbK translation. The equilibrium binding constants of tryptophan-activated TRAP for the trpG, ycbK, and trpP transcripts were determined to be 8, 3, and 50 nM, respectively. Despite TRAP having a higher affinity for the trpG transcript, TRAP exhibited the least control of trpG expression. The trpG Shine-Dalgarno sequence overlaps the stop codon of the upstream pabB gene, while six of nine triplet repeats within the TRAP binding site are located upstream of the pabB stop codon. Thus, ribosomes translating the upstream pabB cistron could be capable of reducing TRAP-dependent control of TrpG synthesis by displacing bound TRAP. Expression studies using pabB-trpG'-'lacZ fusions in the presence or absence of an engineered stop codon within pabB suggest that translation-mediated displacement of bound TRAP reduces TRAP-dependent inhibition of TrpG synthesis from transcripts originating from the folate operon promoter (P(pabB)). A new trpG promoter (P(trpG)) was identified in the pabB coding sequence that makes a larger contribution to trpG expression than does P(pabB). We found that TRAP-dependent regulation of trpG expression is more extensive for a transcript originating from P(trpG) and that transcripts originating from P(trpG) are not subject to translation-mediated displacement of bound TRAP.

Anthranilate Synthase↗