Showing papers on "Upstream activating sequence published in 1999"

Regulation of interleukin-8 gene expression.

Abstract: Interleukin-8 (IL-8), a member of the CXC chemokine family, is an important activator and chemoattractant for neutrophils and has been implicated in a variety of inflammatory diseases. IL-8 is secreted in a stimulusspecific manner by a wide variety of cell types and is regulated primarily at the level of gene transcription. Functional studies indicate that IL-8 transcriptional responses to proinflammatory mediators are rapid and require only 100 nucleotides of 5'-flanking DNA upstream of the TATA box. Within the IL-8 promoter sequence are DNA binding sites for the inducible transcription factors AP-1, NF-IL-6, and NF-kappa B. Transcription factors in these families bind the IL-8 promoter as dimers, and several distinct subunit combinations have been identified as important for IL-8 transcription. In addition, these factors can act in concert to synergistically activate the IL-8 promoter. AP-1 and NF-IL-6 physically interact with NF- kappa B, and functional cooperativity among the factors appears to be cri...

Compilation and analysis of sigma(54)-dependent promoter sequences.

Abstract: Promoters recognized by the RNA-polymerase with the alternative sigma factor sigma(54) (Esigma54) are unique in having conserved positions around -24 and -12 nucleotides upstream from the transcriptional start site, instead of the typical -35 and -10 boxes. Here we compile 186 -24/-12 promoter sequences reported in the literature and generate an updated and extended consensus sequence. The use of the extended consensus increases the probability of identifying genuine -24/-12 promoters. The effect of several reported mutations at the -24/-12 elements on RNA-polymerase binding and promoter strength is discussed in the light of the updated consensus.

A Transcriptional Switch in the Expression of Yeast Tricarboxylic Acid Cycle Genes in Response to a Reduction or Loss of Respiratory Function

Abstract: The Hap2,3,4,5p transcription complex is required for expression of many mitochondrial proteins that function in electron transport and the tricarboxylic acid (TCA) cycle. We show that as the cells' respiratory function is reduced or eliminated, the expression of four TCA cycle genes, CIT1, ACO1, IDH1, and IDH2, switches from HAP control to control by three genes, RTG1, RTG2, and RTG3. The expression of four additional TCA cycle genes downstream of IDH1 and IDH2 is independent of the RTG genes. We have previously shown that the RTG genes control the retrograde pathway, defined as a change in the expression of a subset of nuclear genes, e.g., the glyoxylate cycle CIT2 gene, in response to changes in the functional state of mitochondria. We show that the cis-acting sequence controlling RTG-dependent expression of CIT1 includes an R box element, GTCAC, located 70 bp upstream of the Hap2,3,4,5p binding site in the CIT1 upstream activation sequence. The R box is a binding site for Rtg1p-Rtg3p, a heterodimeric, basic helix-loop-helix/leucine zipper transcription factor complex. We propose that in cells with compromised mitochondrial function, the RTG genes take control of the expression of genes leading to the synthesis of alpha-ketoglutarate to ensure that sufficient glutamate is available for biosynthetic processes and that increased flux of the glyoxylate cycle, via elevated CIT2 expression, provides a supply of metabolites entering the TCA cycle sufficient to support anabolic pathways. Glutamate is a potent repressor of RTG-dependent expression of genes encoding both mitochondrial and nonmitochondrial proteins, suggesting that it is a specific feedback regulator of the RTG system.

The gene search system. A method for efficient detection and rapid molecular identification of genes in Drosophila melanogaster.

Abstract: We have constructed a P-element-based gene search vector for efficient detection of genes in Drosophila melanogaster. The vector contains two copies of the upstream activating sequence (UAS) enhancer adjacent to a core promoter, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the basis of phenotypic changes caused by GAL4-dependent forced expression of vector-flanking DNA, and the transcripts were identified with reverse transcriptase PCR (RT-PCR) using the vector-specific primer and followed by direct sequencing. The system had a greater sensitivity than those already in use for gain-of-function screening: 64% of the vector insertion lines (394/613) showed phenotypes with forced expression of vector-flanking DNA, such as lethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% matched the sequences of cloned genes, and 18% matched reported expressed sequence tags (ESTs). Of the insertions in cloned genes, 83% were upstream of the protein-coding region. We discovered two new genes that showed sequence similarity to human genes, Ras-related protein 2 and microsomal glutathione S-transferase. The system can be useful as a tool for the functional mapping of the Drosophila genome.

UASrpg can function as a heterochromatin boundary element in yeast

Abstract: The HM loci in Saccharomyces cerevisiae constitute region-specific but gene-nonspecific repression domains, as a number of heterologous genes transcribed by RNA polymerase II or III are silenced when placed at these loci. The promoters of the Ashbya gossypii TEF gene and the S. cerevisiae TEF1 and TEF2 genes, however, are resistant to transcriptional silencing by the HM silencers in yeast. Moreover, when interposed between the HML α genes and the E silencer, certain segments of these promoters block the repression effect of the silencer on the α genes. All of these fragments contain UASrpg (upstream activation sequence of ribosome protein genes) composed of multiple binding sites for Rap1. In fact, a 149-bp segment consisting essentially of only three tandem Rap1-binding sites from the UASrpg of yeast TEF2 exhibits silencer-blocking activity. This element also exhibits insulating activity and orientation dependence characteristic of known chromatin boundary elements. Finally, the element blocks the physical spread of heterochromatin initiated at a silencer. This segment provides the first example of chromatin domain boundary or insulator elements in yeast.

Dynamic visualization of nervous system in live Drosophila

Abstract: We have constructed transgenic Drosophila melanogaster lines that express green fluorescent protein (GFP) exclusively in the nervous system. Expression is controlled with transcriptional regulatory elements present in the 5′ flanking DNA of the Drosophila Na+,K+-ATPase β-subunit gene Nervana2 (Nrv2). This regulatory DNA is fused to the yeast transcriptional activator GAL4, which binds specifically to a sequence motif termed the UAS (upstream activating sequence). Drosophila lines carrying Nrv2-GAL4 transgenes have been genetically recombined with UAS–GFP (S65T) transgenes (Nrv2-GAL4+UAS–GFP) inserted on the same chromosomes. We observe strong nervous system-specific fluorescence in embryos, larvae, pupae, and adults. The GFP fluorescence is sufficiently bright to allow dynamic imaging of the nervous system at all of these developmental stages directly through the cuticle of live Drosophila. These lines provide an unprecedented view of the nervous system in living animals and will be valuable tools for investigating a number of developmental, physiological, and genetic neurobiological problems.

Nrg1 is a transcriptional repressor for glucose repression of STA1 gene expression in Saccharomyces cerevisiae.

Abstract: Expression of genes encoding starch-degrading enzymes is regulated by glucose repression in the yeast Saccharomyces cerevisiae. We have identified a transcriptional repressor, Nrg1, in a genetic screen designed to reveal negative factors involved in the expression of STA1, which encodes a glucoamylase. The NRG1 gene encodes a 25-kDa C2H2 zinc finger protein which specifically binds to two regions in the upstream activation sequence of the STA1 gene, as judged by gel retardation and DNase I footprinting analyses. Disruption of the NRG1 gene causes a fivefold increase in the level of the STA1 transcript in the presence of glucose. The expression of NRG1 itself is inhibited in the absence of glucose. DNA-bound LexA-Nrg1 represses transcription of a target gene 10.7-fold in a glucose-dependent manner, and this repression is abolished in both ssn6 and tup1 mutants. Two-hybrid and glutathione S-transferase pull-down experiments show an interaction of Nrg1 with Ssn6 both in vivo and in vitro. These findings indicate that Nrg1 acts as a DNA-binding repressor and mediates glucose repression of the STA1 gene expression by recruiting the Ssn6-Tup1 complex.

Functional analysis of upstream regulating regions from the Yarrowia lipolytica XPR2 promoter

Abstract: Summary: The XPR2 gene from Yarrowia lipolytica encodes an inducible alkaline extracellular protease. Its complex regulation involves pH, carbon, nitrogen and peptones. Two previously identified upstream activating sequence (UAS) regions were analysed in a reporter system, outside the XPR2 context. Fragments from the UAS regions were inserted upstream of a minimal LEU2 promoter directing the expression of a reporter gene. The activity of the hybrid promoters was assessed following integration into the Y. lipolytica genome. This study confirmed the presence of two UASs composed of several interacting elements. Within the distal UAS (UAS1), a TUF/RAP1 binding site exhibited a UAS activity, which was enhanced by the presence of two adjacent repeats, overlapping sites similar to the CAR1 upstream repressing sequence from Saccharomyces cerevisiae. Within the proximal UAS (UAS2), the UAS activity required the interaction of both an ABF1-like binding site and a decameric repeat, containing Aspergillus nidulans PacC site consensus sequences. This decameric repeat was able to mediate repression due to carbon and/or nitrogen sources as well as pH-dependent activation. A study in the context of trans-regulatory mutations in the Y. lipolytica RIM101 gene showed that the PacC-like sites, potential binding sites for YIRim101p, were implicated in the derepression of UAS2-driven expression at neutral-alkaline pH. The in vivo response of the PacC-like decamers to external pH was dependent on the status of the pH-regulated activator YIRim101p, which is homologous to the A. nidulans PacC regulator. The carbon/nitrogen regulation imposed on the decamers was shown to be independent of YIRim101p and to override its effects.

Candidate regulatory sequence elements for cell cycle-dependent transcription in Saccharomyces cerevisiae.

Abstract: The recent surge in the availability of complete genome sequences, as well as the development of technologies such as DNA microarrays, is ushering in a new era in the analysis of gene regulation. The yeast Saccharomyces cerevisiae, whose genome is the first to be fully sequenced from a eukaryote, is ideal for these studies. A number of groups have begun to merge the previously gathered wealth of biochemical information about yeast with its newly published complete genome sequence. One way to analyze gene expression is to scan the genome for the consensus-binding sequence of a known transcription factor to predict additional genes that may be regulated by that factor. This method has been used to look for Gcn4p-binding sites (Schuldiner et al. 1998) and stress-response elements (Moskvina et al. 1998; Treger et al. 1998). Another strategy focuses on the identification of upstream gene regulatory sites in groups of coregulated genes. Van Helden et al. (1998) and Brazma et al. (1998) looked at groups of coregulated genes to find over-represented oligonucleotide sequences. Both groups detected new candidate regulatory sites, as well as sites that had already been characterized. Brazma et al. (1998) also identified sequence patterns that occur more frequently in promoter regions than in other regions of the genome. The availability of the complete yeast genome sequence has also facilitated whole genome expression analyses in which the expression levels of the ∼6200 yeast genes are assayed in different conditions. Recent studies have identified the genes that are expressed during growth on rich and minimal medium (Wodicka et al. 1997), during the diauxic shift from anaerobic to aerobic metabolism (DeRisi et al. 1997), and when the transcriptional corepressor Tup1p is deleted or the transcriptional activator Yap1p is overexpressed (DeRisi et al. 1997). The role of key components of the transcriptional machinery on the population of yeast genes was assessed by Holstege et al. (1998). Chu et al. (1998) assayed genes induced during sporulation in yeast. Roth et al. (1998) measured transcript abundance in the galactose response, heat shock, and mating type regulation systems, and took the further step of using a modified Gibbs sampling strategy to identify sequences that may be involved in the gene regulation. Cho et al. (1998) identified 420 genes whose mRNA expression exhibits cell cycle periodicity. They classified the genes by whether their expression peaked in the early G1, late G1, S, G2, or M phase of the cell cycle. This analysis paves the way for a new type of experiment—a computational prediction of the sequence elements involved in cell cycle regulation. A preliminary analysis of these elements was described previously (Cho et al. 1998). In this manuscript, we present a more detailed interpretation. We have developed a statistical technique to predict short sequence elements (i.e., oligomers) that may be involved in the expression of groups of coregulated genes. Our strategy is to look for pentamers and hexamers that are over-represented among the upstream regions of genes whose expression peaks at a particular phase of the cell cycle. We have identified 9 hexamers and 12 pentamers that may play a role in cell cycle regulation. Some of these oligomers may function in an orientation-dependent manner; the function of others may be dependent on their position within the promoter. The highest scoring hexamer to come out of this study, by all criteria, is the previously characterized sequence element MCB, which is known to control the expression of genes expressed in late G1 (McIntosh 1993). Thus, this method is able to select biologically relevant sequence elements, and we predict that the other elements we identified also play roles in cell cycle regulation. A companion website to this manuscript is available from http://www.ncbi.nlm.nih.gov/CBBresearch/Landsman/Cell_cycle_data. This site includes the upstream sequence of each of the cell cycle-regulated yeast genes, electronic versions of Tables ​Tables11–4 with links from the oligomer to the names of the genes that contain the oligomer, and the 50–100 top scoring oligomers for each analysis, including some sequences that are not statistically significant. Table 1 Position-Independent Hexamers (Over-Represented Hexamers) Table 4 Position-Dependent Pentamers (Clustered Pentamers)

Cat8p, the activator of gluconeogenic genes in Saccharomyces cerevisiae, regulates carbon source-dependent expression of NADP-dependent cytosolic isocitrate dehydrogenase (Idp2p) and lactate permease (Jen1p).

Abstract: The yeast transcriptional activator Cat8p has been identified as a factor that is essential for the derepression of genes involved in gluconeogenesis (like FBP1, PCK1, ACR1, ICL1 and MLS1) when only non-fermentable carbon sources are provided. Cat8p-dependent expression is mediated by cis-acting elements in the respective promoters, which are named UAS/CSREs (upstream activating sequence/carbon source responsive element). To establish whether the function of Cat8p is restricted to the activation of gluconeogenesis or is also involved in the regulation of a greater variety of genes, we investigated the transcriptional regulation of two genes, IDP2 and JEN1, which exhibit a similar expression pattern to gluconeogenic genes, although IDP2 at least is not linked directly to the gluconeogenic pathway. We identified functional UAS/CSRE elements in the promoters of both genes. Expression studies revealed that JEN1 is regulated negatively by the repressors Mig1p and Mig2p, and that Cat8p is needed for full derepression of the gene under non-fermentative growth conditions. Furthermore, we showed that Mig2p is also involved in the repression of CAT8 itself. The results presented in this study support a model in which Cat8p-dependent gene activation is not restricted to gluconeogenesis, but targets a wide variety of genes which are strongly derepressed under non-fermentative growth conditions.

Saturation Mutagenesis of the TATA Box and Upstream Activator Sequence in the Haloarchaeal bop Gene Promoter

Abstract: Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon Halobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum− bop) Halobacterium mutant for purple (Pum+ bop+) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5′-tyT(T/a)Ta-3′, corresponding to the promoter TATA box element 30 to 25 bp 5′ of the transcription start site. A putative UAS, 5′-ACCcnactagTTnG-3′, located 52 to 39 bp 5′ of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.

Multiple Elements Influence Transcriptional Regulation from the Human Testis-Specific PGK2 Promoter in Transgenic Mice

Abstract: The PGK2 gene is expressed in a strictly tissue-specific manner in meiotic spermatocytes and postmeiotic spermatids during spermatogenesis in eutherian mammals. Previous results indicate that this is regulated at the transcriptional level by core promoter sequences that bind ubiquitous transcription factors and by sequences in a 40-base pair (bp) upstream enhancer region (E1/E4) that bind tissue-specific transcription factors. Transgenic mice carrying different PGK2 promoter sequences linked to the chloramphenicol acetyltransferase (CAT) reporter gene, one containing only the 40-bp E1/E4 enhancer sequence plus the core promoter and two containing 515 bp of PGK2 promoter but with either the E1/E4 enhancer region or the Sp1-binding site in the core promoter disrupted by in vitro mutagenesis, all showed levels of expression reduced to less than half that of the wild-type 515 PGK2/CAT transgene. These results indicate that multiple factor-binding regions normally regulate initiation of transcription from the PGK2 promoter. The single disruption of any one of these binding activities reduced, but did not abolish, transgene expression. This is consistent with an "enhanceosome"-like function in this promoter involving multiple bound activator proteins that interact in a combinatorial manner to synergistically promote testis-specific transcription.

Specific components of the SAGA complex are required for Gcn4- and Gcr1-mediated activation of the his4-912delta promoter in Saccharomyces cerevisiae.

Abstract: Mutations selected as suppressors of Ty or solo delta insertion mutations in Saccharomyces cerevisiae have identified several genes, SPT3, SPT7, SPT8, and SPT20, that encode components of the SAGA complex. However, the mechanism by which SAGA activates transcription of specific RNA polymerase II-dependent genes is unknown. We have conducted a fine-structure mutagenesis of one widely used SAGA-dependent promoter, the delta element of his4-912delta, to identify sequence elements important for its promoter activity. Our analysis has characterized three delta regions necessary for full promoter activity and accurate start site selection: an upstream activating sequence, a TATA region, and an initiator region. In addition, we have shown that factors present at the adjacent UASHIS4 (Gcn4, Bas1, and Pho2) also activate the delta promoter in his4-912delta. Our results suggest a model in which the delta promoter in his4-912delta is primarily activated by two factors: Gcr1 acting at the UASdelta and Gcn4 acting at the UASHIS4. Finally, we tested whether activation by either of these factors is dependent on components of the SAGA complex. Our results demonstrate that Spt3 and Spt20 are required for full delta promoter activity, but that Gcn5, another member of SAGA, is not required. Spt3 appears to be partially required for activation of his4-912delta by both Gcr1 and Gcn4. Thus, our work suggests that SAGA exerts a large effect on delta promoter activity through a combination of smaller effects on multiple factors.

Paired Stat6 C-terminal transcription activation domains required both for inhibition of an IFN-responsive promoter and trans-activation.

Abstract: The cytokines IL-4 and IFN-γ exert biologically antagonistic effects that in part reflect opposing influences on gene transcription. While the molecular mechanisms for IL-4-mediated transcription activation have been extensively studied, little is known about molecular mechanisms required for IL-4 inhibition of IFN-γ signaling. We have investigated IL-4 inhibition of the IFN-γ-inducible promoter for IFN regulatory factor-1 (IRF-1). In a cell line with low endogenous Stat6, increasing levels of activated Stat6 at constant doses of IFN-γ and IL-4 leads to inhibition of the IRF-1 promoter. The Stat1-dependent IFN-γ activation sequence element of the IRF-1 promoter is a target for Stat6-mediated inhibition despite apparently normal Stat1 DNA binding. However, our data are inconsistent with competition between Stat1 and Stat6 for access to the IRF-1 IFN-γ activation sequence or for an essential coactivator as a mechanism for this Stat6-mediated inhibition. Instead, the data demonstrate that a threshold of Stat6 transcription activation domains is required for IL-4-dependent inhibition. The findings provide evidence of a novel mechanism in which the Stat6 transcription activation domains play a critical role in the IL-4-mediated inhibition of an IFN-γ-inducible promoter.

Different upstream transcriptional activators have distinct coactivator requirements.

Abstract: Activated transcription by RNA polymerase II (Pol II) requires coactivators, one of which is the SRB/mediator Whereas Srb4, an essential subunit of the SRB/mediator, is broadly required for Pol II transcription in yeast, we have shown that it is dispensable for the transcriptional activation of some genes Here, we show that transcriptional activation by different natural activators, and by artificial recruitment of various transcription factors, have very different degrees of Srb4 independence These data, and the analysis of an rgr1 mutant, point to an Rgr1 subcomplex of the SRB/mediator as the mechanistic route of activation by Srb4-independent activators in vivo

A dual specificity promoter system combining cell cycle-regulated and tissue-specific transcriptional control.

Abstract: The expression of both proliferation-associated and cell type-specific genes is a hallmark of both cancer cells and tumor endothelial cells. The possibility to combine both features in a single transcriptional control unit would greatly increase the selectivity of vectors used for cancer gene therapy. Previous studies by our laboratory have shown that the transcription of several cell cycle genes is regulated by a novel cell cycle-regulated repressor, termed CDF-1. This repressor functions by blocking in resting cells the transcriptional activation by specific factors binding to the upstream activating sequence (UAS), most notably the CCAAT-box binding factor NF-Y/CBF. Based on this work we have developed a dual specificity promoter system that combines cell type specificity with cell cycle regulation. A chimeric transcription factor (Gal4/NF-Y) consisting of the transactivation domain of NF-Y and the DNA-binding domain of Gal4 is expressed from a tissue-specific promoter. Gal4/NF-Y can bind to a second promoter consisting of a minimal cyclin A promoter with multiple Gal4 binding sites replacing the normal UAS. This leads to the tissue-specific expression of Gal4/NF-Y whose stimulatory activity on the promoter is restrained in resting cells by the recruitment of the CDF-1 repressor to the promoter. The functionality of this system is demonstrated for the specific transcriptional targeting of proliferating melanoma cells, where cell cycle regulation was >20-fold and cell type specificity was >50-fold.

An inactive open complex mediated by an UP element at Escherichia coli promoters

Abstract: A specific interaction between the α subunit of RNA polymerase and an A+T-rich upstream sequence (UP element) stimulates transcription at some promoters in Escherichia coli. We found that RNA polymerase formed a heparin-resistant nonproductive initiation complex at the malT promoter which has an A+T-rich upstream sequence that begins 9 bp upstream of the −35 region. Substitution of other sequences for the A+T-rich sequence eliminated both the formation of heparin-resistant complexes and α binding to the malT promoter. A 5-bp deletion between the A+T-rich sequence and the −35 region increased promoter activity. The UP element derived from the rrnB P1 promoter stimulated transcription of the malT core promoter when placed 4 bp upstream from the malT −35 region, but insertion of an additional 4 bp between the rrnB P1 UP element and the −35 element eliminated transcription activity without eliminating heparin-resistant complex formation. Similar UP element effects were observed in hybrids with the lac core promoter, even though the region around the transcription start site was melted in both productive and nonproductive complexes. We conclude that UP elements can mediate the formation of both productive and nonproductive open complexes, depending on their location with respect to the core promoter.

Cyclic AMP Can Decrease Expression of Genes Subject to Catabolite Repression in Saccharomyces cerevisiae

Abstract: External cyclic AMP (cAMP) hindered the derepression of gluconeogenic enzymes in a pde2 mutant of Saccharomyces cerevisiae, but it did not prevent invertase derepression. cAMP reduced nearly 20-fold the transcription driven by upstream activation sequence (UAS1FBP1) from FBP1, encoding fructose-1,6-bisphosphatase; it decreased 2-fold the activation of transcription by UAS2FBP1. Nuclear extracts from cells derepressed in the presence of cAMP were impaired in the formation of specific UASFBP1-protein complexes in band shift experiments. cAMP does not appear to act through the repressing protein Mig1. Control of FBP1 transcription through cAMP is redundant with other regulatory mechanisms.