Search

The liver and pancreas are two major digestive organs, and among the different cell types in them, hepatocytes and the insulin-producing β cells have roles in both health and diseases. Accordingly, clinicians and researchers are very interested in the mechanisms underlying the development and regeneration of liver and pancreatic β cells. Gene and enhancer traps such as the Tol2 transposon-based system are useful for identifying genes potentially involved in developmental processes in the zebrafish model. Here, we developed a strategy that combines a Tol2-mediated enhancer trap and the Cre/loxP system by using loxP-flanked reporters driven by β cell– or hepatocyte-specific promoters and the upstream activating sequence (UAS)-driving Cre. Two double-transgenic reporter lines, Tg(ins:loxP-CFPNTR-loxP-DsRed; 10×UAS:Cre, cryaa:Venus) and Tg(fabp10:loxP-CFPNTR-loxP-DsRed; 10×UAS:Cre, cryaa:Venus), were established to label pancreatic β cells and hepatocytes, respectively. These two double-transgenic lines were each crossed with the Tol2-enhancer trap founder lines to screen for and identify genes expressed in the β cell and hepatocytes during development. This trap system coupled with application of nitroreductase (NTR)/metronidazole (Mtz)–mediated cell ablation could identify genes expressed during regeneration. Of note, pilot enhancer traps captured transiently and weakly expressed genes such as rab3da and ensab with higher efficiencies than traditional enhancer trap systems. In conclusion, through permanent genetic labeling by Cre/loxP, this improved Tol2-mediated enhancer trap system provides a promising method to identify transiently or weakly expressed, but potentially important, genes during development and regeneration.

RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.

Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter, but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter.

Bioluminescence microscopy is an area attracting considerable interest in the field of cell biology because it offers several advantages over fluorescence microscopy, including no requirement for excitation light and being phototoxicity free. This method requires brighter luciferase for imaging; however, suitable genetic resource material for this purpose is not available at present. To achieve brighter bioluminescence microscopy, we developed a new firefly luciferase. Using the brighter luciferase, a reporter strain of Drosophila Gal4-UAS (Upstream Activating Sequence) system was constructed. This system demonstrated the expression pattern of engrailed, which is a segment polarity gene, during Drosophila metamorphosis by bioluminescence microscopy, and revealed drastic spatiotemporal change in the engrailed expression pattern during head eversion in the early stage of pupation.

Reporter gene assays are widely used for the assessment of transcription factor activation following xenobiotic exposure of cells. A critical issue with such assays is the possibility of interference of test compounds with the test system, for example, by direct inhibition of the reporter enzyme. Here we show that the pyrrolizidine alkaloid heliotrine interferes with reporter signals derived from GAL4-based nuclear receptor transactivation assays by a mechanism independent of luciferase enzyme inhibition. These data highlight the necessity to conduct proper control experiments in order to avoid perturbation of reporter assays by test chemicals. (C) 2015 Elsevier Inc. All rights reserved.

35 p.-8 fig., The CD69 type II C-type lectin is one ofthe earliestindicators of leukocyte activation acting in lymphocyte migration and cytokine secretion. CD69 expression in hematopoietic lineage undergoes rapid changes depending on the cell-lineage,the activation state or the localization ofthe cell where itis expressed, suggesting a complex and tightly controlled regulation. Here we provide new insights on the transcriptional regulation of CD69 gene in mammal species. Through in silico studies, we analyzed several regulatory features of the 4 upstream conserved non-coding sequences (CNS 1–4) previously described, confirming a major function of CNS2 in the transcriptional regulation of CD69. In addition, multiple transcription binding sites are identified in the CNS2 region by DNA cross-species conservation analysis. By functional approaches we defined a core region of 226 bp located within CNS2 as the main enhancer element of CD69 transcription in the hematopoietic cells analyzed. By chromatin immunoprecipitation, binding of RUNX1 to the core-CNS2 was shown in a T cell line. In addition, we found an activating but not essential role of RUNX1 in CD69 gene transcription by site-directed mutagenesis and RNA silencing, probably through the interaction with this potent enhancer specifically in the hematopoietic lineage. In summary, in this study we contribute with new evidences to the landscape of the transcriptional regulation of the CD69 gene., This work was supported by the Spanish Ministry of Economics (SAF2010-15649) and the Carlos III National Health Institute (ISCIII) – RTICC (RD12/0036/0063)

α9β1 is the most recent addition to the integrin family of membrane receptors and consequently remains the one that is the least characterized. To better understand how transcription of the human gene encoding the α9 subunit is regulated, we cloned the α9 promoter and characterized the regulatory elements that are required to ensure its transcription. Transfection of α9 promoter/CAT plasmids in primary cultured human corneal epithelial cells (HCECs) and uveal melanoma cell lines demonstrated the presence of both negative and positive regulatory elements along the α9 promoter and positioned the basal α9 promoter to within 118 bp from the α9 mRNA start site. In vitro DNaseI footprinting and in vivo ChIP analyses demonstrated the binding of the transcription factors Sp1, c-Myb and NFI to the most upstream α9 negative regulatory element. The transcription factors Sp1 and NFI were found to bind the basal α9 promoter individually but Sp1 binding clearly predominates when both transcription factors are present in the same extract. Suppression of Sp1 expression through RNAi also caused a dramatic reduction in the expression of the α9 gene. Most of all, addition of tenascin-C (TNC), the ligand of α9β1, to the tissue culture plates prior to seeding HCECs increased α9 transcription whereas it simultaneously decreased expression of the α5 integrin subunit gene. This dual regulatory action of TNC on the transcription of the α9 and α5 genes suggests that both these integrins must work together to appropriately regulate cell adhesion, migration and differentiation that are hallmarks of tissue wound healing.

Summary Cellular signaling response pathways often exhibit a bow-tie topology [1,2]: multiple upstream stress signals converge on a single shared transcription factor, which is thought to induce different downstream gene expression programs (Figure 1A). However, if several different signals activate the same transcription factor, can each signal then induce a specific gene expression response? A growing body of literature supports a temporal coding theory where information about environmental signals can be encoded, at least partially, in the temporal dynamics of the shared transcription factor [1,2]. For example, in the case of the budding yeast transcription factor Msn2, different stresses induce distinct Msn2 activation dynamics: Msn2 shows pulsatile nuclear activation with dose-dependent frequency under glucose limitation, but sustained nuclear activation with dose-dependent amplitude under oxidative stress [3]. These dynamic patterns can then lead to differential gene expression responses [3–5], but it is not known how much specificity can be obtained. Thus, a major question of this temporal coding theory is how many gene response programs or cellular functions can be robustly encoded by dynamic control of a single transcription factor. Here we provide the first direct evidence that, simply by regulating the activation dynamics of a single transcription factor, it is possible to preferentially induce four distinct gene expression programs.

The Gal4-UAS regulatory system of yeast is widely used to modulate gene expression in Drosophila; however, there are limitations to its usefulness in transgenic zebrafish, owing to progressive methylation and silencing of the CpG-rich multicopy upstream activation sequence. Although a modified, less repetitive UAS construct may overcome this problem, it is highly desirable to have additional transcriptional regulatory systems that can be applied independently or in combination with the Gal4/UAS system for intersectional gene expression. The Q transcriptional regulatory system of Neurospora crassa functions similarly to Gal4/UAS. QF is a transcriptional activator that binds to the QUAS upstream regulatory sequence to drive reporter gene expression. Unlike Gal4, the QF binding site does not contain essential CpG dinucleotide sequences that are subject to DNA methylation. The QS protein is a repressor of QF mediated transcriptional activation akin to Gal80. The functionality of the Q system has been demonstrated in Drosophila and Caenorhabditis elegans and we now report its successful application to a vertebrate model, the zebrafish, Danio rerio. Several tissue-specific promoters were used to drive QF expression in stable transgenic lines, as assessed by activation of a QUAS:GFP transgene. The QS repressor was found to dramatically reduce QF activity in injected zebrafish embryos; however, a similar repression has not yet been achieved in transgenic animals expressing QS under the control of ubiquitous promoters. A dual reporter construct containing both QUAS and UAS, each upstream of different fluorescent proteins was also generated and tested in transient assays, demonstrating that the two systems can work in parallel within the same cell. The adoption of the Q system should greatly increase the versatility and power of transgenic approaches for regulating gene expression in zebrafish.

Pseudomonas chlororaphis strain PA23 is a plant-beneficial bacterium able to suppress disease caused by the pathogenic fungus Sclerotinia sclerotiorum. A number of compounds are believed to contribute to fun- gal antagonism including the antibiotics pyrrolnitrin and phenazine together with degradative enzymes. Overseeing production of these metabolites is a complex regulatory network that includes the GacS- GacA two-component system. In other bacteria, a second network, called Rsm, works in concert with Gac. The aim of the current study was to identify components of the Rsm system in PA23 and elucidate factors controlling their expression. Towards this end, genes encoding repressor proteins (RsmA and RsmE) and a regulatory RNA (RsmZ) were isolated. Through transcriptional fusion analysis, we discovered that rsmZ is positively regulated by GacA, PsrA, RpoS, the stringent response (SR) and the PhzI/PhzR quo- rum-sensing (QS) system. An upstream activating sequence (UAS) corresponding to the GacA recognition sequence was identified in the rsmZ promoter. Moreover, a PtrA box and a Phz box were present, suggest- ing that all three regulators activate rsmZ directly. rsmE was positively regulated by GacA, PsrA, RpoS, the SR and QS in PA23, but with the exception of RpoS, regulation was indirectly mediated. rsmA was nega- tively regulated by the SR and positively regulated by GacA. A UAS was not found in the promoter region of either rsmA or rsmE, suggesting indirect control by GacA. Identification of RsmZ, RsmA and RsmE and the discovery that these regulators are tightly controlled add to the increasingly complex network over- seeing biocontrol in P. chlororaphis PA23.

We developed an efficient chimeric promoter, MUASMSCP, with enhanced activity and salicylic acid (SA)/abscisic acid (ABA) inducibility, incorporating the upstream activation sequence (UAS) of Mirabilis mosaic virus full-length transcript (MUAS, -297 to -38) to the 5' end of Mirabilis mosaic virus sub-genomic transcript (MSCP, -306 to -125) promoter-fragment containing the TATA element. We compared the transient activity of the MUASMSCP promoter in tobacco/Arabidopsis protoplasts and in whole plant (Petunia hybrida) with the same that obtained from CaMV35S and MUAS35SCP promoters individually. The MUASMSCP promoter showed 1.1 and 1.5 times stronger GUS-activities over that obtained from MUAS35SCP and CaMV35S promoters respectively, in tobacco (Xanthi Brad) protoplasts. In transgenic tobacco (Nicotiana tabacum, var. Samsun NN), the MUASMSCP promoter showed 1.1 and 2.2 times stronger activities than MUAS35SCP and CaMV35S(2) promoters respectively. We observed a fair correlation between MUASMSCP-, MUAS35SCP- and CaMV35S(2)-driven GUS activities with the corresponding uidA-mRNA level in transgenic plants. X-gluc staining of transgenic germinating seed-sections and whole seedlings also support above findings. Protein-extracts made from tobacco protoplasts expressing GFP and human-IL-24 genes driven individually by the MUASMSCP promoter showed enhanced expression of the reporters compared to that obtained from the CaMV35S promoter. Furthermore, MUASMSCP-driven protoplast-derived human IL-24 showed enhanced cell inhibitory activity in DU-145 prostate cancer cells compared to that obtained from the CaMV35S promoter. We propose chimeric MUASMSCP promoter developed in the study could be useful for strong constitutive expression of transgenes in both plant/animal cells and it may become an efficient substitute for CaMV35S/CaMV35S(2) promoter.

Despite the prevalence of bidirectional promoters among the mammalian genomes, the majority of promoters are unidirectional. The mechanism through which unidirectional promoters are prevented from reverse transcription remains to be clarified. Here we investigate the transcriptional directionality of the mouse insulin-degrading enzyme (IDE) promoter, which contains a CpG island and has dispersed transcription initiation sites. Although IDE is unidirectionally transcribed according to its genomic context, the basic promoter region of mouse IDE has bidirectional transcriptional properties. The region between −219 and +133 of mouse IDE relative to its first transcription initiation site has bidirectional transcriptional activities, but the region between −350 and +133 can only be transcribed from the normal direction, implying that an upstream promoter element locating between −350 and −219 blocks the reverse transcription of mouse IDE. We further mapped this upstream promoter element to the region between −243 and −287. Promoter mutation analysis showed that the upstream promoter element contains two functional sub-regions. In conclusion, we identified an upstream promoter element which blocks the reverse transcription of mouse IDE. Our studies are suggestive for the transcriptional mechanism of bidirectional promoters in mammalian genomes.

Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.

Trichomonas vaginalis is a parasitic protozoan of both medical and biological relevance. Transcriptional studies in this organism have focused mainly on type II pol promoters, whereas the elements necessary for transcription by polI or polIII have not been investigated. Here, with the aid of a transient transcription system, we characterised the rDNA intergenic region, defining both the promoter and the terminator sequences required for transcription. We defined the promoter as a compact region of approximately 180 bp. We also identified a potential upstream control element (UCE) that was located 80 bp upstream of the transcription start point (TSP). A transcription termination element was identified within a 34 bp region that was located immediately downstream of the 28S coding sequence. The function of this element depends upon polarity and the presence of both a stretch of uridine residues (U's) and a hairpin structure in the transcript. Our observations provide a strong basis for the study of DNA recognition by the polI transcriptional machinery in this early divergent organism.

TATA-binding protein-like protein (TLP) is involved in development, checkpoint, and apoptosis through potentiation of gene expression. TLP-overexpressing human cells, especially p53-containing cells, exhibited a decreased growth rate and increased proportion of G1 phase cells. TLP stimulated expression of several growth-related genes including p21 (p21Waf1/Cip1). TLP-mediated activation of the p21 upstream promoter in cells was shown by a promoter-luciferase reporter assay. The p53-binding sequence located in the p21 upstream promoter and p53 itself are required for TLP-mediated transcriptional activation. TLP and p53 bound to each other and synergistically enhanced activity of the upstream promoter. TLP specifically activated transcription from the endogenous upstream promoter, and p53 was required for this activation. Etoposide treatment also resulted in activation of the upstream promoter as well as nuclear accumulation of TLP and p53. Moreover, the upstream promoter was associated with endogenous p53 and TLP, and the p53 recruitment was enhanced by TLP. The results of the present study suggest that TLP mediates p53-governed transcriptional activation of the p21 upstream promoter.

Helper virus-free Herpes Simplex Virus vector-mediated gene transfer has supported studies on neuronal physiology, and may support specific gene therapies. Long-term, neuron-specific expression is required for many of these applications. A neurofilament heavy gene (NFH) promoter does not support long-term expression. We previously developed a promoter that supports long-term expression by fusing 6.3 kb of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a NFH promoter, and this promoter has supported physiological studies. The TH promoter fragment contains an enhancer, as it has activity in both orientations and at a distance from the basal promoter. Identifying this enhancer may support further improvements in long-term expression. A previous deletion analysis identified two ~100 bp fragments that each support long-term expression, and are contained within an ~320 bp fragment located ~3 kb from the TH promoter transcription start site. As this analysis used overlapping fragments, the two ~100 bp fragments contained 44 or 23 bp of unique sequence. Here, we used mutagenesis to identify a short sequence that supports long-term expression. We studied a 42 bp sequence, centered on the 23 bp unique sequence. Analysis of the wt sequence, and five mutations containing clustered changes that spanned the sequence, identified two adjacent mutations that do not support long-term expression, which together defined a 16 bp maximum essential sequence. This 16 bp sequence contains a putative E2F-1/DP-1 transcription factor binding site, and this transcription factor is expressed in many brain areas.

The yeast Gal4/UAS transcriptional activation system is a powerful tool for regulating gene expression in Drosophila and has been increasing in popularity for developmental studies in zebrafish. It is also useful for studying the basis of de novo transcriptional silencing. Fluorescent reporter genes under the control of multiple tandem copies of the upstream activator sequence (UAS) often show evidence of variegated expression and DNA methylation in transgenic zebrafish embryos. To characterize this systematically, we monitored the progression of transcriptional silencing of UAS-regulated transgenes that differ in their integration sites and in the repetitive nature of the UAS. Transgenic larvae were examined in three generations for tissue-specific expression of a green fluorescent protein (GFP) reporter and DNA methylation at the UAS. Single insertions containing four distinct upstream activator sequences were far less susceptible to methylation than insertions containing fourteen copies of the same UAS. In addition, transgenes that integrated in or adjacent to transposon sequence exhibited silencing regardless of the number of UAS sites included in the transgene. Placement of promoter-driven Gal4 upstream of UAS-regulated responder genes in a single bicistronic construct also appeared to accelerate silencing and methylation. The results demonstrate the utility of the zebrafish for efficient tracking of gene silencing mechanisms across several generations, as well as provide useful guidelines for optimal Gal4-regulated gene expression in organisms subject to DNA methylation.

The Mediator complex interacts extensively with the RNA polymerase II enzyme and regulates its ability to express protein-coding genes. The mechanisms by which Mediator regulates gene expression remain poorly understood, in part because the structure of Mediator and even its composition can change, depending upon the promoter context. Combined with the sheer size of the human Mediator complex (26 subunits, 1.2 MDa), this structural adaptability bestows seemingly unlimited regulatory potential within the complex. Recent efforts to understand Mediator structure and function have identified expanded roles that include control of both pre- and post-initiation events; it is also evident that Mediator performs both general and gene-specific roles to regulate gene expression.

Triplex-forming oligonucleotides (TFOs) are attractive tools to control gene expression at the transcriptional level. This anti-gene approach has proven to be successful in various experimental settings. However, the mechanisms leading to transcriptional repression in cells have not been fully investigated yet. Here, we examined the consequence of triplex DNA formation on the binding of transcriptional activators, co-activators and RNA Polymerase II to the ets2 gene promoter using chromatin immunoprecipitation assays. The triplex target sequence was located approximately 40-bp upstream of the transcription start site (TSS) and overlapped an Sp1 binding site relevant for ets2 transcription. We found that the ets2-TFO prevented binding of Sp1, TAFII130 and TAFII250 to the ets2 promoter, while binding of RNA polymerase II and TBP were not affected. The effects were both sequence and target specific, since the TFO had no effect on the c-myc promoter and a mutated ets2 promoter construct. Thus, triplex DNA formation near a TSS leads to formation of a non-functional pre-initiation complex (PIC) by blocking binding of transcriptional activators and co-activator molecules. This is the first direct demonstration of interference with PIC assembly at the TSS by oligonucleotide-triplex DNA formation in cells.

The 26 S proteasome complex that comprises the 20 S core and 19 S regulatory (with six ATPases) particles is engaged in an ATP-dependent degradation of a variety of key regulatory proteins and, thus, controls important cellular processes. Interestingly, several recent studies have implicated the 19 S regulatory particle in controlling eukaryotic transcriptional initiation or activation independently of the 20 S core particle. However, the mechanism of action of the 19 S proteasome subcomplex in regulation of eukaryotic transcriptional activation is not clearly understood in vivo. Here, using a chromatin immunoprecipitation assay in conjunction with mutational and transcriptional analyses in Saccharomyces cerevisiae, we show that the 19 S proteasomal subcomplex establishes a specific protein interaction network at the upstream activating sequence of the promoter. Such an interaction network is essential for formation of the preinitiation complex at the core promoter to initiate transcription. Furthermore, we demonstrate that the formation of the transcription complex assembly at the promoter is dependent on 19 S ATPase activity. Intriguingly, 19 S ATPases appear to cross-talk for stimulation of the assembly of transcription factors at the promoter. Together, these results provide significant insights as to how the 19 S proteasome subcomplex regulates the formation of the active transcription complex assembly (and, hence, transcriptional initiation) at the promoter in vivo.

GLI2 (GLI-Kruppel family member 2), a zinc finger transcription factor that mediates Hedgehog signaling, is implicated in the progression of an ever-growing number of human malignancies, including prostate and pancreatic cancer, as well as basal cell carcinoma of the skin. Its expression is up-regulated by transforming growth factor-β (TGF-β) in a variety of cell types, both normal and transformed. We report herein that TGF-β-driven GLI2 expression is transcriptional and does not result from stabilization of GLI2 transcripts. We describe the characterization of the 5′-flanking sequence of human GLI2 mRNA, the identification of a transcription start site, the cloning of ∼1,600 bp of the regulatory promoter region and the identification and functional analysis of a TGF-β-responsive region mapped to a 91-bp sequence between nucleotides −119 and −29 of the promoter. This region harbors SMAD and lymphoid enhancer factor/T cell factor binding sites that allow functional cooperation between SMAD3 and β-catenin, recruited to the promoter in response to TGF-β to drive GLI2 gene transcription.

The advancement in Escherichia coli genome research has made the information regarding transcription start sites of many genes available. A study relying on the availability of transcription start locations was performed. The first question addressed was what an average DNA curvature profile upstream of genes would look like when these genes are aligned by transcription start sites in comparison to alignment by translation start sites. Since it was hypothesized that curvature plays a role in transcription regulation, the expectation was that curvature measurements relative to transcription starts, rather than translation, should strengthen the signal. Our study justified this expectation. The second question aimed to clarify the relation between DNA curvature and promoter strength. Through clustering based on DNA curvature profiles along promoter regions, a strong positive correlation between the promoter strength and the curved DNA was found. The third question dealt with dinucleotide periodicity in E. coli to see whether a periodicity pattern specific to promoter regions exists. Such unknown pattern might shed new light on transcription regulation mechanisms in E. coli. A sequence periodicity of about 11 bp is characteristic to the whole E. coli genome, and is especially well-expressed in intergenic regions. Here it was shown that regions of the size of about 100-150 bp centered 70-100 bp upstream to transcription starts carry hidden periodicity with a period of about 10.3 bp.

Glycerol-3-phosphate acyltransferase 1 (GPAT1) is a rate limiting enzyme in de novo glycerophospholipid synthesis. The murine GPAT1 promoter sequence (the "classical" sequence) was reported previously. However, the organization of this DNA sequence does not fully match the mouse genome sequences on NCBI/GenBank. Here we have identified net cis-acting promoter sequences for the mouse GPAT1 gene: promoter 1a which includes part of the classical sequence and the downstream promoter 1b. Promoter 1a facilitates transcription of two alternative GPAT1 transcript variants, GPAT1-V1 and V2, while promoter 1b produces a third transcript variant, GPAT1-V3. Upstream stimulating factor-1 (USF-1) controlled both promoters whereas sterol regulatory element-binding protein-1 (SREBP-1) exclusively regulated promoter 1a activity in vitro. Feeding increased GPAT1-V1 and V2, but not V3 mRNA levels in mouse liver. The obese condition of db/db mice did not alter the hepatic expression levels of any of the three GPAT1 variants. Feeding enhanced hepatic mRNA levels, intranuclear protein levels and promoter 1a-binding levels of SREBP-1, but not of USF-1. Thus, promoter 1a was exclusively activated by routine feeding in vivo. Our results indicate differential roles of the two promoters in the regulation of hepatic GPAT1 gene expression in mice.

Genes encoding peroxisomal proteins in the yeast Saccharomyces cereviasiae are induced in the presence of oleate in growth medium. This induction is known to be mediated by the binding of a heterodimer of transcription factors Oaf1 and Pip2 to an upstream activating sequence called ORE (oleate response element). By analyzing expression of nine ORE-containing genes we show that the presence of an ORE sequence is not sufficient to confer oleate inducibility, as three such genes were in fact expressed constitutively. Moreover, some of the oleate-inducible genes undergo activation even in the absence of Pip2. Using coimmunoprecipitation we show that, when Pip2 is missing, Oaf1 may form homodimers which apparently substitute for the Oaf1-Pip2 heterodimer.

MEF2 (myocyte enhancer factor 2) proteins are a small family of transcription factors that play pivotal roles in striated muscle differentiation, development, and metabolism, in neuron survival and synaptic formation, and in lymphocyte selection and activation. Products of the four mammalian MEF2 genes, MEF2A, MEF2B, MEF2C, and MEF2D, are expressed with overlapping but distinct temporospatial patterns. Toward analysis of MEF2A functions and the determinants of its regulated expression, we have mapped and begun studies of the transcriptional control regions of this gene. Heterogeneous 5′-untranslated regions of MEF2A mRNAs result from use of alternative promoters and splicing patterns. The two closely approximated TATA-less promoters are ∼65 kb upstream of the exon containing the sole initiation codon. Ribonuclease protection and primer extension assays show that each promoter is active in various adult tissues. A canonical MEF2 site overlies the major promoter 1 transcription start site. This element specifically binds MEF2 factors, including endogenous nuclear MEF2A according to chromatin immunoprecipitation studies, and is critical to MEF2A transcription in myocytes. The site exerts reciprocal control of the alternative promoters, silencing promoter 1 and activating promoter 2 under some conditions. Erk5 and p38 MAPK signaling stimulate MEF2A expression by activating both promoters from the MEF2 element. MEF2A transcription is therefore subject to positive or negative regulation by its protein products, depending on signaling activities that influence MEF2 factor trans-activity. The sole MEF2 gene of the cephalochordate amphioxus has a similar regulatory region structure, suggesting that this mode of autoregulatory control is conserved among higher metazoan MEF2 genes.

Mediator is an essential component of the RNA polymerase II-mediated transcription machinery. This component plays a key role both in the stimulation of basal transcription and in the regulation of eukaryotic mRNA synthesis. The Saccharomyces cerevisiae Mediator complex was the first to be studied and consists of at least 20 different subunits with multiple activities. Afterwards, its subunit composition was determined and related functions of C. elegans, Drosophila and mammalian complexes show a striking evolutionary conservation both of the structure and function from yeast to man. Recently, yeast studies strongly suggest additional roles for Mediator in coordinating transcription initiation with downstream transcriptional events in the coding region of genes; consequently, new models of recruitment-coupled regulation have been indicated. Further studies on transcription machinery should expand our knowledge of the pathways in which variant components of Mediator, or variant proteins interacting directly or in complexes, represent risk factors for complex inheritable disease.

Human and mouse show markedly different sensitivities toward bacterial endotoxins, and recent evidence suggests that a species-specific regulation of the lipopolysaccharide receptor Toll-like receptor 4 (Tlr4) may contribute to this phenomenon. To gain further insight into mechanisms of Tlr4 regulation, we conducted a detailed in vivo and in vitro study of the murine Tlr4 gene, including analysis of transcription start site location, transcription factor occupancy, and activities of its proximal regulatory sequences. Our analyses identified a PU.1-dependent myeloid promoter, which is conserved between humans and mouse. We also identified an additional, distal promoter, located approximately 200 bp upstream of the myeloid-specific promoter, which is a functional target of E-box binding factors. In contrast to humans, where non-myeloid cells utilize both promoters, the distal Tlr4 promoter initiates all Tlr4 transcripts in murine non-myeloid cells, indicating that species-specific differences in TLR4 mRNA regulation may primarily exist in non-myeloid cell types. Interestingly, PU.1 null murine myeloid progenitor cells predominantly use the distal promoter, and the conditional induction of PU.1 expression in these cells leads to the rapid switch of transcription initiation to the proximal myeloid promoter. This indicates a direct role for PU.1 in determining the transcriptional start site and in recruiting the basal transcription machinery to myeloid promoters.

We have identified novel over-represented and conserved motifs in the upstream regions of human and mouse miRNA stem-loop sequences by means of a new bioinformatic processing regimen. We observed sequence conservation -500 bp upstream in 189 human and mouse miRNAs declining with increasing distance from their putative miRNA stem-loop origin. We also found relatively GC-rich regions having more than 50% of guanine+cytosine (G+C) content at about -30 and -170 bp relative to human miRNA stem-loop sequence origin. To further identify specific sequence motifs that might be involved in the transcriptional regulation of miRNA precursors, we first searched 500 bp upstream sequences of 194 non-redundant human miRNA stem-loop sequences for frequently occurring motifs 5-15 bp long. We then found the comparable occurrences of the 20 most frequent motifs in the 2000 bp upstream regions of 242 human and 290 mouse miRNAs. The significantly reduced frequency of occurrence of all 20 motifs in the regions 2000 bp upstream of 23,570 human RefSeq genes demonstrated that these motifs were specific to the upstream miRNA sequences. The most frequently observed motif M1 (GTGCTTMTAGTGCAG), with a MEME E-value of 3.8e-57 was distributed within 500 bp upstream of stem-loop sequences and was also miRNA-specific. We suggest that these over-represented motif sites are good candidates for experimentally testing miRNA expression as well as possible interaction with regulatory factors.

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.

An in vivo study of the importance of the length and/or structures of sequences upstream of a mitochondrial promoter was undertaken in Saccharomyces cerevisiae . Short tandem mtDNA repeats were introduced upstream of the COX2 gene. Our data show that its expression is modulated by the sequence located over 200 bp upstream of the promoter. A deletion decreases the level of transcripts to about 50%. The initial level can be recovered by a fill-in AT-rich sequence or partially by the presence of a long repeat tract; on the contrary, a smaller number of copies tends to intensify the effect of the deletion. These results show that the length and base composition upstream of mitochondrial promoter are involved in vivo in the modulation of the gene expression.

The linear glycosaminoglycan hyaluronan (HA) is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2, and -3 and performs multiple functions as part of the vertebrate extracellular matrix. Up-regulation of HA synthesis in the renal corticointerstitium, and the resultant extracellular matrix expansion, is a common feature of renal fibrosis. However, the regulation of expression of these HAS isoforms at transcriptional and translational levels is poorly understood. We have recently described the genomic structures of the human HAS genes, thereby identifying putative promoter regions for each isoform. Further analysis of the HAS2 gene identified the transcription initiation site and showed that region F3, comprising the proximal 121 bp of promoter sequence, mediated full constitutive transcription. In the present study, we have analyzed this region in the human renal proximal tubular epithelial cell line HK-2. Electrophoretic mobility shift and promoter assay data demonstrated that transcription factors Sp1 and Sp3 bound to three sites immediately upstream of the HAS2 transcription initiation site and that mutation of the consensus recognition sequences within these sites ablated their transcriptional response. Furthermore, subsequent knockdown of Sp1 or Sp3 using small interfering RNAs decreased constitutive HAS2 mRNA synthesis. In contrast, significant binding of HK-2 nuclear proteins by putative upstream NF-Y, CCAAT, and NF-kappaB recognition sites was not observed. The identification of Sp1 and Sp3 as principal mediators of HAS2 constitutive transcription augments recent findings identifying upstream promoter elements and provides further insights into the mechanism of HAS2 transcriptional activation.

Although the m-xylene-responsive sigma54 promoter Pu of Pseudomonas putida mt-2, borne by the TOL plasmid pWWO, is one of the strongest known promoters in vivo, its base-line level in the absence of its aromatic inducer is below the limit of any detection procedure. This is unusual because regulatory networks (such as the one to which Pu belongs) can hardly escape the noise caused by intrinsic fluctuations in background transcription, including that transmitted from upstream promoters. This study provides genetic evidence that the upstream-activating sequences (UAS), which serve as the binding sites for the pWW0-encoded XylR protein (the m-xylene-responsive sigma54-dependent activator of Pu), isolate expression of the upper TOL genes from any adventitious transcriptional flow originating further upstream. An in vivo test system was developed in which different segments of the Pu promoter were examined for the inhibition of incoming transcription products from an upstream promoter in P. putida and Escherichia coli. Minimal transcription filter ability was located within a 105-bp fragment encompassing the UAS of Pu. Although S1 nuclease assays showed that the UAS prevented the buildup of downstream transcripts, the mechanism seems to diverge from a typical termination system. This was shown by the fact that the UAS did not halt transcription in vitro and that the filter effect could not be relieved by the anti-termination system of lambda phage. Because the Pu promoter lies adjacent to the edge of a transposon in pWW0, the preset transcriptional filter in the UAS may isolate the upper TOL operon from undue expression after random insertion of the mobile genetic element in a new replicon.

The yeast SPT10 gene encodes a putative histone acetyltransferase that binds specifically to pairs of upstream activating sequence (UAS) elements found only in the histone gene promoters. Here, we demonstrate that the DNA-binding domain of Spt10p is located between residues 283 and 396 and includes a His2-Cys2 zinc finger. The binding of Spt10p to the histone UAS is zinc-dependent and is disabled by a zinc finger mutation (C388S). The isolated DNA-binding domain binds to single histone UAS elements with high affinity. In contrast, full-length Spt10p binds with high affinity only to pairs of UAS elements with very strong positive cooperativity and is unable to bind to a single UAS element. This implies the presence of a “blocking” domain in full-length Spt10p, which forces it to search for a pair of UAS elements. Chromatin immunoprecipitation experiments indicate that, unlike wild-type Spt10p, the C388S protein does not bind to the promoter of the gene encoding histone H2A (HTA1) in vivo. The C388S mutant has a phenotype similar to that of the spt10Δ mutant: poor growth and global aberrations in gene expression. Thus, the C388S mutation disables the DNA-binding function of Spt10p in vitro and in vivo. The zinc finger of Spt10p is homologous to that of foamy virus integrase, perhaps suggesting that this integrase is also a sequence-specific DNA-binding protein.

In the yeast Saccharomyces cerevisiae, the mineral zinc is essential for growth and metabolism. Depletion of zinc from the growth medium of wild type cells results in changes in phospholipid metabolism, including an increase in phosphatidylinositol content (Iwanyshyn, W. M., Han, G.-S., and Carman, G. M. (2004) J. Biol. Chem. 279, 21976-21983). We examined the effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase, the enzyme that catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1Delta zrt2Delta mutant defective in plasma membrane zinc transport indicated that the cytoplasmic levels of zinc were responsible for the regulation of phosphatidylinositol synthase. PIS1 mRNA, its encoded protein Pis1p, and the beta-galactosidase activity driven by the P(PIS1)-lacZ reporter gene were elevated in zinc-depleted cells. This indicated that the increase in phosphatidylinositol synthase activity was the result of a transcriptional mechanism. The zinc-mediated induction of the P(PIS1)-lacZ reporter gene, Pis1p, and phosphatidylinositol synthase activity was lost in zap1Delta mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. Direct interaction between glutathione S-transferase (GST)-Zap1p(687-880) and a putative upstream activating sequence (UAS) zinc-responsive element in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UAS zinc-responsive element in the PIS1 promoter abolished the GST-Zap1p(687-880)-DNA interaction in vitro and abolished the zinc-mediated regulation of the PIS1 gene in vivo. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene.

The human SLC2A10 gene encodes the high-affinity glucose transporter 10 (GLUT10) and is widely expressed in adult tissues, including organs which play major roles in glucose homeostasis. Its function and genomic location in a region linked to Type 2 diabetes susceptibility are consistent with a potential role in Type 2 diabetes. Analysis of the CpG-rich promoter revealed the presence of two major transcription start points with differential use in tissues and cell lines. Mapping of transcriptionally active regions in the 5′ flanking sequence identified a region, located between nucleotides −70 and −14 (relative to the major transcription start point) as the SLC2A10 basal promoter. This sequence harbors consensus binding sites for Sp, AP2α, and other transcription factors. A juxtaposed Sp/AP2α motif located between −25 and −11 is critical for core promoter function. In cells expressing Sp and AP2 factors, the two motifs are required for maximal activation of the basal promoter. In cells lacking AP2α, transcription is dependent on the integrity of the Sp site. Using electrophoresis mobility shift assays, we demonstrate that Sp1 and Sp3 bind to the GC-box in site 5 forming specific complexes. In addition, a silencer region is present upstream of −696 which down-regulates SLC2A10 promoter activity independently of its distance to the transcript start site.

The vascular endothelial growth factor receptor-2 (VEGFR-2/KDR/flk-1) functions as the primary mediator of vascular endothelial growth factor activation in endothelial cells. Regulation of VEGFR-2 expression appears critical in mitogenesis, differentiation, and angiogenesis. Transcriptional regulation of the VEGFR-2 is complex and may involve multiple putative upstream regulatory elements including E boxes. Transcript initiation is dependent on an initiator (Inr) element flanking the transcriptional start site. The transcription factor, TFII-I, enhances VEGFR-2 transcription in an Inr-dependent fashion. TFII-I is unusual both structurally and functionally. The TFII-I transcription factor family members contain multiple putative DNA binding domains. Functionally, TFII-I acts at both the basal, Inr element as well as at several distinct upstream regulatory sites. It has been postulated that the structure of TFII-I might allow simultaneous interaction with both basal and regulatory sites in a given promoter. As TFII-I is known to act at regulatory sites including E boxes as well as at the basal Inr element, we evaluated the possibility of Inr-independent TFII-I activation of the VEGFR-2 promoter. We found that an Inr-mutated VEGFR-2 reporter construct retains TFII-I-stimulated activity. We demonstrated that TFII-I binds to both the Inr and to three regulatory E boxes in the human VEGFR-2 promoter. In addition, reduction in TFII-I expression by siRNA results in decreased VEGFR-2 expression. We also describe counter-regulation of the VEGFR-2 promoter by TFII-IRD1. We found that TFII-I is capable of acting at both basal and regulatory sites in one promoter and that the human VEGFR-2 promoter is functionally counter-regulated by TFII-I and TFII-IRD1.

Cyclic GMP-dependent protein kinase I plays a pivotal role in regulating smooth muscle cell relaxation, growth, and differentiation. Expression of the enzyme varies greatly in smooth muscle and in other tissues and cell types, yet little is known regarding the mechanisms regulating cGMP-dependent protein kinase gene expression. The present work was undertaken to characterize the mechanisms controlling kinase gene expression in vascular smooth muscle cells. A 2-kb human cGMP-dependent protein kinase I 5'-noncoding promoter sequence was characterized by serial deletion, and functional studies demonstrated that a 591-bp 5'-promoter construct possessed the highest activity compared with all other constructs generated from the larger promoter. Analysis of the sequence between -472 and -591 bp from the transcriptional start site revealed the existence of two E-like boxes known to bind upstream stimulatory factors. Electrophoretic mobility shift assays and functional studies using luciferase reporter gene assays identified upstream stimulatory factors as the transcription factors bound to the E-boxes in the 591-bp promoter. Site-directed mutagenesis of the E-boxes abolished the binding of upstream stimulatory factor proteins and decreased the activity of the cGMP-dependent protein kinase I 591-bp promoter, thus confirming the involvement of these transcription factors in mediating gene expression. Cotransfection experiments demonstrated that overexpression of upstream stimulatory factors 1 and 2 increased cGMP-dependent protein kinase I promoter activity. Collectively, these data suggest that the human proximal cGMP-dependent protein kinase I promoter is regulated by tandem E-boxes that bind upstream stimulatory factors.

The Pax6 transcription factor is a highly conserved regulator of eye and brain development. Its complex mode of regulation is shared between Drosophila melanogaster and the vertebrates, despite there being no significant sequence identity between their enhancer and promoter regions. In this article, a more detailed examination of the Pax6 genomic sequence reveals a common set of putative Pax6 and basic helix–loop–helix transcription factor binding sites.

Ribonucleotide reductase is essential for supplying a balanced pool of the four deoxyribonucleotides required for DNA synthesis and repair. The active enzyme consists of two non-identical subunits called proteins R1 and R2. There are multiple levels of regulation of ribonucleotide reductase activity, which is highest during the S and G(2) phases of an unperturbed cell cycle in mammalian cells. Previous reports in the literature have indicated that the S phase-specific transcription of the mammalian R2 gene is regulated by a transcriptional block, is dependent on the transcription factor E2F1, or is simply regulated by proteins that bind to promoter CCAAT boxes plus the TATA box. Here, we demonstrate that the S phase-specific transcription of the mouse R2 gene is dependent on an upstream promoter activating region (located at nucleotides (nt) -672 to -527 from the transcription start site) and one proximal promoter repressive element (located at nt -112 to -107) that binds E2F4. Binding to the E2F site is modulated by binding of nuclear factor-Y to an adjacent CCAAT element (nt -79 to -75). The upstream activating region is crucial for overall R2 promoter activity. Mutation of the E2F-binding site leads to premature promoter activation in G(1) and increases overall promoter activity but only when the upstream activating region is present and intact. Therefore, E2F-dependent repression is essential for cell cycle-specific R2 transcription.

The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is a basic helix-loop-helix transcription factor. The hepatic expression of SHARP-2 mRNA was investigated under various conditions. The level was decreased in the regenerating rat liver and malignant hepatoma cells. In contrast, the expression of SHARP-2 mRNA was induced in rat livers by feeding a high-carbohydrate diet. To analyze the molecular mechanism involved in the regulation of the rat SHARP-2 gene expression, the gene was cloned. It was approximately 6-kb in length and consists of five exons and four introns. To investigate the transcriptional regulatory region of this gene, SHARP-2/firefly luciferase reporter plasmids were transfected into hepatoma cells. A functional analysis of 5′-deletion constructs revealed that two E box sequences between –160 and –144 are mainly responsible for promoter activity. Although upstream stimulatory factors (USFs) bound to the element in vitro, USF2 failed to stimulate promoter activity from the element using the co-transfection experiment. Therefore, other E box-binding transcription factors differing from USF proteins or USF-associated proteins are necessary for transcriptional stimulation of the rat SHARP-2 gene.

Bone morphogenetic protein 4, BMP4, plays an important role in the development of various organs including the lungs. Little is known regarding the regulation of Bmp4 gene expression in any organ. In the lung, indirect evidence indicates that NKX2.1, a homeodomain transcriptional factor with a demonstrated role in lung morphogenesis, may be a potential upstream regulator of Bmp4 gene expression. In particular, Bmp4 mRNA is reduced or absent in Nkx2.1(-/-) lungs. The human Bmp4 gene has been reported to include two regions of promoter activity in an embryonal carcinoma cell line, Tera2EC. The hBmp4.1 promoter is located upstream of exon I, whereas the second promoter, hBmp4.2, is localized within intron 1 and upstream of exon II. In the current study, we used a co-transfection assay in lung epithelial cells to examine the response of the two hBmp4 promoters to transcriptional stimulation by NKX2.1. Two DNA sequences were identified on the hBmp4.1 promoter that bind NKX2.1 and serve as functional cis-active NKX2.1-responsive elements. Similarly, NKX2.1 stimulated transcription from the hBmp4.2 promoter through two consensus binding sites localized within 412 nucleotides from the site of transcriptional initiation. Thus, both hBmp4 promoters include specific cis-active elements that bind to and mediate transcriptional regulation by NKX2.1. These findings bear functional implications regarding the regulation of a key signaling molecule by a homeodomain transcriptional regulator of lung epithelial morphogenesis.

A mathematical model was developed to describe the physiological co-regulation of two Pseudomonas sigma54-dependent promoter/regulator systems, Pu/XylR and Po/DmpR of Pseudomonas strains mt2 and CF600, respectively. Five ordinary differential equations and six algebraic equations were developed to describe the following processes of transcription initiation: binding of the activator protein to the upstream activating sequence, union of the sigma factor with the core polymerase, formation of the open complex, and escape of the transcription machinery from the promoter region. In addition, growth-phase control of the integration host factor (IHF), sigma-70 regulation during stationary phase, and the contribution of (p)ppGpp to both sigma factor selectivity and promoter escape were hypothesized. By including any three of these four effects, the model predicted that expression from both promoters is repressed during exponential growth and sharply increases as the cells enter stationary phase. The difference in behavior of the two systems during overexpression of either sigma54 or (p)ppGpp could be explained by different values of two model parameters. To accurately represent the behavior of both promoters in (p)ppGpp null strains, an additional parameter must be varied. Although numerical data available for this system is scarce, the model has proved useful for helping to interpret the experimental observations and to evaluate four hypotheses that have been proposed to explain the phenomenon of exponential silencing.

The urokinase-type plasminogen activator receptor (u-PAR) contributes to cell migration and proteolysis in normal and cancerous tissues. Currently, there are no reports on the regulatory regions directing tissue-specific expression. Consequently, we undertook a study to identify novel promoter regions required for expression of this gene in transgenic mice bearing a LacZ reporter regulated by varying amounts (0.4, 1.5, and 8.5 kb) of upstream sequence. The 0.4-kb u-PAR upstream sequence directed weak and strong LacZ expression in the placenta and epididymis, respectively, both of which are tissues that express endogenous u-PAR. Conversely, transgene expression in the apical cells of the colon positive for endogenous u-PAR protein required 1.5 kb of upstream sequence for optimal expression. Furthermore, chromatin accessibility assays coupled with real-time polymerase chain reaction suggested a putative regulatory region spanning −1295/−1192 driving u-PAR expression in colonic cells. Interestingly, placental transgene expression was augmented with the 8.5-kb upstream fragment compared with the shorter 1.5-kb fragment indicating contributing element(s) between −1.5 and −8.5 kb. Thus, while 0.4 kb of upstream sequence directs u-PAR expression in the epididymis, sequences located between −0.4 and −1.5 kb and between −1.5 and −8.5 kb are required for optimal tissue-specific expression in the colon and the placenta, respectively.

Cytochrome P450 (CYP)1A2 is abundantly expressed in the liver of all vertebrate species. In most, its expression is restricted to the liver. Sequence analysis of the human CYP1A2 5′-flanking region from +3 to −3201 identified six E-box motifs within the 3-methylcholanthrene (MC) enhancer element (−1987 to −3201). The E-box motif is recognized by members of the basic helix-loop-helix (bHLH) family of transcription factors. Gel mobility shift and antibody supershift assays were used to examine each of the six upstream E-box motifs for their ability to bind nuclear proteins and to compete with the ubiquitously expressed bHLH protein, upstream stimulatory factor (USF), for binding. We found that USF-1 and USF-2 proteins bind to the upstream E-box motifs EB2, EB3, and EB4. Transient transfection assays in HepG2 cells were performed with different segments of the human CYP1A2 5′-flanking region linked to a luciferase reporter gene. Site-directed mutagenesis of one of the E-box motifs, EB2, resulted in a 60% reduction in basal reporter gene activity. Mutations in EB3 and EB4 had no effect. We found that transfection of expression vectors containing USF-1 or USF-2 cDNAs activated CYP1A2 reporter gene activity, while a dominant-negative USF-2 expression vector blocked such activity. Chromatin immunoprecipitation assays confirmed that the interaction of USF proteins with the CYP1A2 EB2 site occurs in vivo . These data support the role of USF as a constitutive transcriptional activator of human CYP1A2 .

A tRNA Leu -like sequence is located within a probable enhancer region of the RNA polymerase II-dependent gene encoding an RNA-binding protein, Atgrp7 , in Arabidopsis (Mol. Gen. Genet. 261 (1999) 811). To examine whether this sequence is transcribed, we used our in vitro transcription system from tobacco cell nuclei. In vitro assays demonstrated that this tRNA-like sequence is transcribed by RNA polymerase III and its transcript is processed into tRNA-size molecules. Transcription starts at the CAA motif, a transcription initiation site for many plant tRNA genes. Mutation analyses indicated that transcription of this sequence depends on promoter elements typical for plant tRNA genes. We therefore concluded that this is a transcriptionally active tRNA Leu (AAG) gene. Mutation of a basic promoter element of the tRNA gene exerted no influence on the transcription of the downstream protein-coding gene, suggesting that no apparent interference occurs between the two adjacent genes.

Expression of sodium-hydrogen exchanger isoform 3 (NHE3) in the intestinal and renal epithelium plays a critical role in sodium absorption and acid/base homeostasis. To decipher rat NHE3 gene regulation, its cis-acting regulatory elements and associated transcription factors were characterized by transient transfection of Caco-2, IEC-6, Qt6, and Drosophila SL2 cells. Deletion and mutational analyses demonstrated that the atypical TATA box located at bp -26/-31 was not necessary for promoter activity, and that a -20/+8-bp fragment represents a functional initiator. Within the 81-bp upstream region, three Sp transcription factor binding sites were critical because their mutation drastically reduced promoter activity. The roles of Sp1 and Sp3 were further demonstrated by electromobility shift assay and by transactivation of the NHE3 promoter in SL2 cells by forced expression of Sp1 and Sp3. Both of these transcription factors were found to act synergistically with GATA-5 bound to a GATA box in exon 1 (+20/+23 bp). These studies demonstrate that rat NHE3 promoter is initiator-driven and controlled mainly by Sp1 and Sp3, which functionally interact with GATA-5. This interaction represents a novel regulatory mechanism, which is likely to participate in a gradient of intestinal gene expression along the crypt-villus axis.

Constitutive and inducible expression of the gene encoding the modulator subunit of human glutamate-cysteine ligase (GCLM) is regulated by either of two regions of the promoter; an antioxidant response element (ARE) at −302:−291 and a 44-bp fragment (−346:−303) upstream of the ARE. This second region includes a consensus AP-1 site previously considered responsible for the enhancer activity of the upstream fragment. Deletion of a 165-bp fragment (−348:−183) including the ARE and upstream 44-bp fragment totally ablated t-butyl hydroquinone (tBHQ) inducibility of a GCLM promoter/luciferase transgene. Mutation analyses confirmed that both the ARE and the −346:−303 fragment could support induction following tBHQ exposure but demonstrated that induction in the latter case did not involve the AP-1 site at −341:−335. A region sharing significant homology with the consensus ARE sequence except for a single nucleotide mismatch at −330 (5′-TTACnnnGCA-3′ versus5′-TGACnnnGCA-3′) was identified at the 5′-end of the 44-bp fragment immediately adjacent to the AP-1 site. A G in this position has been considered an invariant requirement of functional ARE sequences. Mutation of T−330 to A (a substitution known to ablate ARE function) or C eliminated basal and inducible expression. Substitution of a G at −330 enhanced basal expression relative to the wild-type sequence, but induction following tBHQ exposure was comparable, indicating that either sequence (5′-TTACnnnGCA-3′ versus5′-TGACnnnGCA-3′) may function as an ARE, although the former sequence is less effective at directing basal expression. This possibility was confirmed by similar mutational analyses of the core sequence of hNQO1, a prototypic ARE. Electrophoretic mobility shift competition assays revealed that the 5′-TTACnnnGCA-3′ sequence could compete with the hNQO1 ARE for protein binding but was less effective than a similar probe containing the 5′-TGACnnnGCA-3′ motif. Probes including the T(−330)A or T(−330)C mutations were ineffective. These results reveal that the GCLM promoter includes two functional AREs, one having a variant sequence. The results indicate that the consensus ARE sequence should be revised to 5′-RTKAYnnnGCR-3′.

The general transcription factor TFIID is composed of the TATA box binding protein (TBP) and multiple TBP-associated factors (TAFs) [1–3]. In yeast, promoters can be grouped into two classes based on the involvement of TAFs [4, 5]. TAF-dependent (TAF dep ) promoters require TAFs for transcription, and TBP and TAFs are present at comparable levels on these promoters. TAF-independent (TAF ind ) promoters do not require TAFs for activity, and TAFs are either absent or present at levels far below those of TBP on these promoters. Here, we demonstrate that the upstream activating sequence (UAS) mediates the selective recruitment of TAFs to TAF dep promoters. A TAF ind UAS fails to recruit TAFs and to direct efficient transcription when inserted upstream of a TAF dep core promoter. This transcriptional defect can be overcome by a potent activator, indicating that a strong activation domain can compensate for the absence of TAFs on a TAF dep core promoter. Our results reveal a requirement for compatibility between the UAS and core promoter and thus help explain previous reports that only certain yeast UAS-core promoter combinations and mammalian enhancer-promoter combinations are efficiently transcribed [6–11]. The differential recruitment of TAFs by UASs provides strong evidence for the proposal that in vivo TAFs are the targets of some, but not all, activators.

The human PAX7 gene is a member of the paired box containing gene family of transcription factors implicated in development of the skeletal muscle of the trunk and limbs as well as elements of the central nervous system. To understand the molecular mechanisms involved in its expression, we have localized the transcription start sites in adult skeletal muscle and functionally characterized the 5'-flanking regulatory region responsible for PAX7 expression in this tissue. The major transcription start was identified 664 bp upstream from the ATG codon using primer extension and 5'-rapid amplification of cDNA ends (5'-RACE). Analysis of the 5'-flanking sequence revealed the absence of a TATA-box and the presence of an inverted CCAAT-box. Several consensus sites for common transcriptional regulators including Oct-1, NF1, AP2, AP4, CREB, Sp1, Nkx2.5, and MyoD are present in the promoter region. To determine the sites critical for the function of the PAX7 promoter, a series of deletion fragments of the 5'-flanking region were cloned adjacent to luciferase reporter gene and expressed in RD, Cos-7 and JAR cell lines. The maximal promoter activity was achieved by a fragment extending from the position -403 to +373. No strong positive or negative regulatory elements were discovered by adding of further sequences (up to 2.97 kb). A polymorphic (CCT)(n) repeat sequence was found 107 bp upstream of the transcription initiation site. PCR-based systematic screening for length variations in 227 unrelated individuals of a Caucasian population showed a bimodal distribution of three alleles containing 8, 10 or 11 repeat units. When different variants of this PAX7 gene-linked polymorphic region (PAX7-LPR) were fused to a luciferase reporter gene and transfected into RD cells, the variant with 11 repeat units revealed higher transcriptional efficiency compared to the 8 or 10 repeat alleles.

We used a biochemical genomics method of assaying Saccharomyces cerevisiae proteins, derived from a nearly complete set of glutathione S-transferase fusions, to develop an approach that is able to identify proteins that bind to a DNA element. Using the upstream activation sequence (UAS) of the promoter for the invertase gene, SUC2, we identified both specific and nonspecific binding activities, which could be classified based on whether they bound with equivalent affinity to a nonspecific DNA competitor. Three transcription factors, Mig1, Yer028c, and Rgt1, were found to be binding activities specific to the SUC2 UAS. Mig1 and Yer028c had been reported previously to bind to elements within the SUC2 UAS, validating the ability of the method to identify sequence-specific factors. The third activity, Rgt1, had not been reported previously to bind to SUC2. Additional gel shift assays narrowed the Rgt1 binding site to the SUC2-B element within the SUC2 UAS, which is similar to previously identified Rgt1 binding sites present in other genes. In vivo levels of invertase activity in an rgt1Delta strain were reduced relative to an isogenic RGT+ strain when these strains were grown under inducing (low glucose) conditions, suggesting that Rgt1 may have a role in the activated transcription of SUC2. This report demonstrates the feasibility of identifying DNA binding activities by rapidly assaying a large fraction of the predicted open reading frames of an organism for binding to a regulatory DNA motif.