Showing papers on "Upstream activating sequence published in 2006"

SAGA-associated Sgf73p facilitates formation of the preinitiation complex assembly at the promoters either in a HAT-dependent or independent manner in vivo

Abstract: Although Sgf73p, a yeast homologue of human Sca7p, has recently been implicated as a new component of Spt-Ada-Gcn5-acetyltransferase (SAGA), its association with SAGA and functional role in regulation of transcription remain unknown in vivo. Here, using a chromatin immunoprecipitation (ChIP) assay, we show in vivo that, like SAGA, Sgf73p is recruited to the upstream activating sequence (UAS) of a SAGA-dependent gene, GAL1, in an activator-dependent manner. Further, Sgf73p is required for recruitment of SAGA to the GAL1 UAS, and facilitates formation of the preinitiation complex (PIC) assembly at the GAL1 promoter. When PIC is not formed in Δsgf73, histone H3 is not evicted from the GAL1 promoter. Interestingly, PIC formation at GAL1 is not regulated by histone H3 acetylation or histone acetyltransferase (HAT) activity of SAGA. Similarly, Sgf73p facilitates PIC formation at another SAGA-dependent gene, ADH1, independent of histone H3 acetylation or HAT. In contrast, Sgf73p stimulates PIC formation at PHO84 (a SAGA-dependent gene), in a HAT-dependent-manner. Collectively, our data reveal that Sgf73p is required for SAGA recruitment, and stimulates PIC formation either in a HAT-dependent or -independent manner, providing significant information on how Sgf73p and possibly human Sca7p function physiologically.

Establishment of a patterned GAL4‐VP16 transactivation system for discovering gene function in rice

Abstract: A binary GAL4-VP16-UAS transactivation system has been established in rice (Oryza sativa L.) in this study for the discovery of gene functions. This binary system consists of two types of transgenic lines, pattern lines and target lines. The pattern lines were produced by transformation of Zhonghua 11, a japonica cultivar, with a construct consisting of the transactivator gene GAL4-VP16 controlled by a minimal promoter and the GUSplus reporter controlled by the upstream activation sequence (UAS; cis-element to GAL4). Target lines were generated by transformation of Zhonghua 11 with constructs carrying the EGFP reporter and target genes of interest, both controlled by the UAS but in opposite directions. Hybrid plants were obtained by crossing target lines of 10 putative transcription factor genes from rice with six pattern lines showing expression in anther, stigma, palea, lemma and leaves. The EGFP and target genes perfectly co-expressed in hybrid plants with the same expression patterns as in the pattern lines. Various phenotypic changes, such as delayed flowering, multiple pistils, dwarfism, narrow and droopy leaves, reduced tillers, growth retardation and sterility, were induced as a result of the expression of the target genes. It is concluded that this transactivation system can provide a useful tool in rice to unveil latent functions of unknown or known genes.

Functional domains of the BACE1 and BACE2 promoters and mechanisms of transcriptional suppression of the BACE2 promoter in normal neuronal cells.

Abstract: The β-amyloid (Aβ) protein present in the neuritic plaques of Alzheimer's disease is cleaved, from Aβ precursor protein (APP) by β-and γ-secretases. Following identification of β-APP cleaving enzyme (BACE1) as the β-secretase, a homologous β-secretase 2 (BACE2) was described. Our goal is to characterize the regulatory region of the BACE genes. We compare functional domains within the BACE1 and BACE2 regulatory regions. Both BACE genes lack canonical TATA and CAAT boxes, but they contain distinguishing transcription start sites and transcription factor-binding sites. The BACE1, sequence contains more repetitive elements than does BACE2 (no elements). Regulatory domains do not overlapstrongly between the two promoter regions. The BACE1 upstream sequence contains both negative and positive domains, separated from the transcription seat by a long neutral domain. The corresponding BACE2 sequence consists of a weakly positive domain directly upstream of a strongly positive domain, near a functionally active domain. DNA-protein interaction was corroborated by functional data. In primary rat cortical cultures, BACE1-driven reporter protein's expression was twice that of BACE2-driven reporter. The BACE2 gene promoter relatively reduced function in neuronal cells compared with BACE1. The BACE1 gene might operate through a single transcriptional control site. BACE2 operates through dual transcriptional control sites. Two (or more) regulatory pathways might control transcription in BACE2. Thus, BACE2 is partially suppressed in normal neuronal cells and likely to be a highly regulated gene expressed in a particularly tissue-specific fashion.

Distinct viral sequence elements are necessary for expression of Tomato golden mosaic virus complementary sense transcripts that direct AL2 and AL3 gene expression.

Abstract: Transient expression studies using Nicotiana benthamiana protoplasts and plants have identified sequences important for transcription of complementary sense RNAs derived from Tomato golden mosaic virus (TGMV) DNA component A that direct expression of AL2 and AL3. Transcription of two complementary sense RNAs, initiating at nucleotides 1,935 (AL1935) and 1,629 (AL1629), is directed by unique sequences located upstream of each transcription initiation site. One element is located between 28 and 124 nucleotides (nt) upstream of the AL1935 transcription start site, which differs from a second element located 150 nt downstream, between 129 and 184 nt upstream of the AL1629 transcription start site. Transcription initiation at nucleotide 1,935 is lower than that at nucleotide 1,629 as determined by run-on transcription assays, and the resulting transcript is only capable of expressing AL3. The transcript initiating at nucleotide 1,629 is capable of directing expression of both AL2 and AL3, although expression of AL3 is up to fourfold greater than that for AL2. Nuclear factors purified from tobacco suspension cells bind to sequences upstream of both AL1935 and AL1629, correlating with the ability of these sequences to direct gene expression. Thus, in tobacco, regulatory sequences direct transcription of two unique TGMV messenger RNAs that differentially express AL2 and AL3.

STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide.

Abstract: RNA-binding activity of IRP1 (iron regulatory protein 1) is regulated by the insertion/extrusion of a [4Fe-4S] cluster into/from the IRP1 molecule. NO (nitic oxide), whose ability to activate IRP1 by removing its [4Fe-4S] cluster is well known, has also been shown to down-regulate expression of the IRP1 gene. In the present study, we examine whether this regulation occurs at the transcriptional level. Analysis of the mouse IRP1 promoter sequence revealed two conserved putative binding sites for transcription factor(s) regulated by NO and/or changes in intracellular iron level: Sp1 (promoter-selective transcription factor 1) and MTF1 (metal transcription factor 1), plus GAS (interferon-γ-activated sequence), a binding site for STAT (signal transducer and activator of transcription) proteins. In order to define the functional activity of these sequences, reporter constructs were generated through the insertion of overlapping fragments of the mouse IRP1 promoter upstream of the luciferase gene. Transient expression assays following transfection of HuH7 cells with these plasmids revealed that while both the Sp1 and GAS sequences are involved in basal transcriptional activity of the IRP1 promoter, the role of the latter is predominant. Analysis of protein binding to these sequences in EMSAs (electrophoretic mobility-shift assays) using nuclear extracts from mouse RAW 264.7 macrophages stimulated to synthesize NO showed a significant decrease in the formation of Sp1–DNA and STAT–DNA complexes, compared with controls. We have also demonstrated that the GAS sequence is involved in NO-dependent down-regulation of IRP1 transcription. Further analysis revealed that levels of STAT5a and STAT5b in the nucleus and cytosol of NO-producing macrophages are substantially lower than in control cells. These findings provide evidence that STAT5 proteins play a role in NO-mediated down-regulation of IRP1 gene expression.

The role of an upstream promoter interaction in initiation of bacterial transcription

Abstract: The bacterial RNA polymerase (RNAP) recognizes promoters through sequence-specific contacts of its promoter-specificity components (sigma) with two DNA sequence motifs. Contacts with the upstream ('-35') promoter motif are made by sigma domain 4 attached to the flap domain of the RNAP beta subunit. Bacteriophage T4 late promoters consist solely of an extended downstream ('-10') motif specifically recognized by the T4 gene 55 protein (gp55). Low level basal transcription is sustained by gp55-RNAP holoenzyme. The late transcription coactivator gp33 binds to the beta flap and represses this basal transcription. Gp33 can also repress transcription by Escherichia coli sigma70-RNAP holoenzyme mutated to allow gp33 access to the beta flap. We propose that repression is due to gp33 blocking an upstream sequence-independent DNA-binding site on RNAP (as sigma70 domain 4 does) but, unlike sigma70 domain 4, providing no new DNA interaction. We show that this upstream interaction is essential only at an early step of transcription initiation, and discuss the role of this interaction in promoter recognition and transcriptional regulation.

The yeast CPC2/ASC1 gene is regulated by the transcription factors Fhl1p and Ifh1p

Abstract: CPC2/ASC1 is one of the most abundantly transcribed genes in Saccharomyces cerevisiae. It encodes a ribosome-associated Gβ-like WD protein, which is highly conserved from yeast to man. Here, we show that CPC2 transcription depends on the carbon source and is induced during utilization of the sugar glucose. CPC2 promoter deletion and insertion analyses identified two upstream activation sequence elements for CPC2, which are required for basal expression and regulation. One of these upstream activation sequence elements has an ATGTACGGATGT motif, which has previously been described as a putative binding site for the forkhead-like transcription factor Fhl1p. Deletion of FHL1 reduces CPC2 transcription significantly in presence of glucose, but has no effect when the non-fermentable carbon source ethanol is provided. Increased amounts of the Fhl1p co-regulator Ifh1p induce CPC2 transcription even when ethanol is utilized. These data suggest that the interaction between Fhl1p and Ifh1p is critical for the regulation of CPC2 transcription during utilization of different carbon sources.

Constitutive Expression of Murine Decay-Accelerating Factor 1 Is Controlled by the Transcription Factor Sp1

Abstract: The complement regulatory protein decay-accelerating factor (DAF or CD55) protects host tissue from complement-mediated injury by inhibiting the classical and alternative complement pathways. Besides its role in complement regulation, DAF has also been shown to be a key player in T cell immunity. Modulation of DAF expression could therefore represent a critical regulatory mechanism in both innate and adaptive immune responses. To identify and characterize key transcriptional regulatory elements controlling mouse Daf1 expression, a 2.5-kb fragment corresponding to the 5' flanking region of the mouse Daf1 gene was cloned. Sequence analysis showed that the mouse Daf1 promoter lacks conventional TATA and CCAAT boxes and displays a high guanine and cytosine content. RACE was used to identify one major and two minor transcription start sites 47, 20, and 17 bp upstream of the translational codon. Positive and negative regulatory regions were identified by transiently transfecting sequential 5'deletion constructs of the 5'flanking region into NIH/3T3, M12.4, and RAW264.7 cells. Mutational analyses of the promoter region combined with Sp1-specific ELISA showed that the transcription factor Sp1 is required for basal transcription and LPS-induced expression of the Daf1 gene. These findings provide new information on the regulation of the mouse Daf1 promoter and will facilitate further studies on the expression of Daf1 during immune responses.

Cell-specific molecule and method for importing DNA into osteoblast nuclei

Abstract: A plasmid, viral or linear DNA molecule containing a nucleic acid sequence derived from the promoter region of the hCol1α2 gene, which is selectively transported into the nuclei of cells in the osteoblast lineage. The sequence can be used independently as a nuclear entry sequence only, and/or as a nuclear entry sequence without regard to position, in a vector or linear DNA that directs gene expression and nuclear entry. The disclosure further includes a chimeric DNA sequence derived by the addition of osteoblast-specific enhancer sequences to the nuclear entry sequence/promoter sequence, to increase osteoblast-specific expression while retaining osteoblast-specific nuclear import. An enhancer sequence is derived from the promoter region of the human Core Binding Factor alpha 1 (Cbfa1/Runx2) gene. The Cbfa1/Runx2 promoter can be added to the sequence derived from, or alternatively, comprising the promoter region of the hCol1α2 gene. Also provided are methods of use of the novel sequences.

Isolation and characterization of the rat SND p102 gene promoter: putative role for nuclear factor-Y in regulation of transcription.

Abstract: In this work, we report the isolation and characterization of a 1,688-bp sequence corresponding to the promoter region of the rat endoplasmic reticulum (ER) cholesterol ester hydrolase gene, renamed as staphylococcal nuclease domain-containing protein of 102 kDa (SND p102) in GenBank database according to the structural properties and molecular weight of the protein. The transcription start site was located 216 bases upstream of the ATG start codon by RNA ligase mediated-rapid amplification of cDNA ends (RLM-RACE). Bioinformatic analysis of the isolated sequence revealed a lack of typical promoter TATA box and the presence of GC-rich motifs and CCAAT boxes recognized by Sp 1 and nuclear factor-Y among other putative binding sites for a number of transcription factors implicated in both basal and regulated processes. Electrophoretic mobility shift and supershift assays using nuclear extracts from human (HepG2) and rat (McA-RH7777) hepatoma cells demonstrated that nuclear factor-Y (NF-Y) transcription factor bound to the core sequences at (-257, -253), (-290, -286), and (-370, -366) upstream translation initiation site. The absence of TATA box and the location and reverse orientation of the CCAAT boxes in the promoter region strongly suggest a role for NF-Y in the regulation of transcription of SND p102 gene.

Mapping of a transcription promoter located inside the priA gene of the Bacillus subtilis chromosome

Abstract: The genome sequence of the Gram-positive soil bacterium Bacillus subtilis was completed in 1997 (Kunst et al., 1998) and the results included the identification of a putative transcription unit encompassing the yloI to yloS genes. Within this region of the B. subtilis chromosome 11 putative open reading frames were found with a wide diversity of probable functions. In this work we have analyzed transcription in the region of the priA-cpgA genes and we have mapped a promoter which is located inside the priA gene and its activity directs transcription of the def-yloM genes. Moreover, this transcript can be extended at low level to the prpC-priK-cpgA genes. Analysis of the sequence in proximity of the transcription start site revealed a sequence suitable for the housekeeping sigma(A) subunit of RNA polymerase. Analysis of the beta-glactosidase activity of transcription fusions revealed that the identified promoter is active at low level and its activity is increased during late exponential phase of growth.

Tripeptidyl-peptidase II : structure, function and gene regulation.

Abstract: Tripeptidyl-peptidase II (TPP II) is one of the many proteases involved in the important process of intracellular proteolysis. The widespread distribution and broad substrate specificity suggest that TPP II is encoded by a "house-keeping gene". However, both TPP II protein and mRNA levels vary in different cells. To investigate whether these variations are due to regulation on a genetic level, the promoter of the TPP2 gene has previously been identified. The promoter contains two inverted CCAAT-boxes and an E-box. By means of reporter assays and electrophoretic mobility shift assays the promoter has now been further characterized. It could be concluded that USF-1 (upstream stimulatory factor-1) binds to the E-box in the promoter. The transcription factors NF-Y and USF-1 are present in protein-DNA complexes of different sizes. Mutation of the E-box had no effect, indicating that only binding of NF-Y to the two CCAAT-boxes was important for activation of transcription. However, this does not exclude the possibility that USF-1 can play an important role in transcription in other types of cells. Furthermore, the region upstream of the promoter was investigated due to its ability to inhibit transcription. Several silencer elements were identified and we also showed that Oct-1 binds to one of these elements. Thus, this investigation reveals that TPP II expression could be regulated through both positive and negative regulatory elements. Further studies are required to establish the involvement of different genetic elements, and how the interplay between different transcription factors will affect the transcriptional rate in vivo.

Transcriptional Regulation: Evolution

Abstract: Changes in transcriptional regulation can result from mutations in cis (within regulatory sequences) or trans (in the structure or expression of transcription factors). Modifications in transcription can have a profound impact on gene function and constitute an important class of evolutionary change within genomes. Keywords: cis-regulatory sequence; enhancer; polymorphism; promoter; transcription factor