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Results for “regulatory element transcripts”

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PubMed2003

Egr-1 induction in rat granulosa cells by follicle-stimulating hormone and luteinizing hormone: combinatorial regulation by transcription factors cyclic adenosine 3',5'-monophosphate regulatory element binding protein, serum response factor, sp1, and early growth response factor-1.

Early growth response factor (Egr-1) is an inducible zinc finger transcription factor that binds specific GC-rich enhancer elements and impacts female reproduction. These studies document for the first time that FSH rapidly induces Egr-1 expression in granulosa cells of small growing follicles. This response is transient but is reinitiated in preovulatory follicles exposed to the LH analog, human chorionic gonadotropin. Immunohistochemical analysis also showed gonadotropin induced Egr-1 in theca cells. The Egr-1 gene regulatory region responsive to gonadotropin signaling was localized within -164 bp of the transcription initiation site. Binding of Sp1/Sp3 to a proximal GC-box at -64/-46 bp was enhanced by FSH in immature granulosa cells but reduced after human chorionic gonadotropin stimulation of preovulatory follicles despite constant protein expression. This dynamic regulation of Sp1 binding was dependent on gonadotropin-regulated mechanisms that modulate Sp1/3-DNA binding activity. Serum response factor was active in granulosa cells and bound a consensus CArG-box/serum response element site, whereas two putative cAMP response elements within the -164-bp region bound cAMP regulatory element (CRE) binding protein (CREB) and a second cAMP-inducible protein immunologically related to CREB. Transient transfection analyses using Egr-1 promoter-luciferase constructs and site-specific mutations show that the serum response element, GC-box, and CRE-131 are involved in gonadotropin regulation of Egr-1 expression in granulosa cells. Specific kinase inhibitors of Erk or protein kinase A antagonized this induction while exogenously expressed Egr-1 enhanced reporter expression. These observations indicate that the Egr-1 gene is a target of both FSH and LH action that may mediate molecular programs of proliferation and/or differentiation during follicle growth, ovulation, and luteinization.

Animals↗
PubMed1998

Synergistic activation of transcription by nuclear factor Y and sterol regulatory element binding protein.

The current studies define the role of three distinct cis-elements in the proximal promoter of the rat farnesyl diphosphate (FPP) synthase gene. The three cis-elements, a sterol regulatory element 3 (SRE-3) flanked by an ATTGG motif (inverted CCAAT box), and a CCAAT box, form a sterol regulatory unit that is necessary and sufficient for sterol-regulated expression of FPP synthase promoter-reporter genes. FPP synthase promoter-reporter genes, that contain promoters with either wild-type nucleotide sequences or mutations in one or more of the three cis-elements, were transiently transfected into CV-1 cells. The activity of the wild-type promoter-reporter gene increased when the cells were incubated in sterol-depleted media or when the cells were co-transfected with a plasmid encoding the mature form of SRE binding protein (SREBP-1a). The results with the mutant promoter-reporter genes demonstrated that all three cis-elements were necessary for normal expression/regulation of the reporter gene by either sterols or by co-expressed SREBP-1a. Gel mobility shift assays demonstrated that the synergistic binding of SREBP-1a to SRE-3 was dependent on the binding of recombinant nuclear factor Y (NF-Y) to the DNA, consistent with the in vivo regulation studies.

Alkyl and Aryl Transferases↗
PubMed2004

Regression trees for regulatory element identification.

MOTIVATION: The transcription of a gene is largely determined by short sequence motifs that serve as binding sites for transcription factors. Recent findings suggest direct relationships between the motifs and gene expression levels. In this work, we present a method for identifying regulatory motifs. Our method makes use of tree-based techniques for recovering the relationships between motifs and gene expression levels. RESULTS: We treat regulatory motifs and gene expression levels as predictor variables and responses, respectively, and use a regression tree model to identify the structural relationships between them. The regression tree methodology is extended to handle responses from multiple experiments by modifying the split function. The significance of regulatory elements is determined by analyzing tree structures and using a variable importance measure. When applied to two data sets of the yeast Saccharomyces cerevisiae, the method successfully identifies most of the regulatory motifs that are known to control gene transcription under the given experimental conditions, and suggests several new putative motifs. Analysis of the tree structures also reconfirms several pairs of motifs that are known to regulate gene transcription in combination. AVAILABILITY: http://if.kaist.ac.kr/~phuong/RegTree

Algorithms↗
PubMed1996

Core promoters and transcriptional control.

Many biological processes are controlled, spatially and temporally, at the level of transcription. Thus, understanding the mechanisms of transcriptional regulation of gene expression is critical in deciphering the molecular modes of differentiation and development of a eukaryotic cell. Transcriptional control is mediated largely through interactions of regulatory transcription factors with their cognate enhancer elements. The regulatory signals generated at enhancer elements are communicated to the general transcription machinery formed at the core promoter elements of all genes. Recent observations suggest that the general transcription machinery can also generate regulatory signals independent of enhancer-generated interactions. Thus, the transcriptional regulation of gene expression, both in time and in space, may result from appropriate interfacing of independent signals generated at the core promoter and at the enhancer.

Animals↗
PubMed2005

Sterol Regulatory Element-Binding Protein-2 modulates human brain acyl-CoA hydrolase gene transcription.

The brain shows high catalyzing activity during hydrolysis of long-chain acyl-CoAs into fatty acids and CoA-SH. Brain acyl-CoA hydrolase (BACH) is responsible for most of the long-chain acyl-CoA hydrolyzing activity in the brain and is localized exclusively in neurons. We analyzed the human BACH gene promoter, focusing on transcriptional regulation by Sterol Regulatory Element-Binding Protein-2 (SREBP-2), which is a transcription factor that activates genes involved in cholesterol biosynthesis and uptake. When the nuclear form of SREBP-2 gene was transfected into human neuroblastoma cells, transcription of a BACH gene promoter-luciferase reporter gene was activated through a sterol regulatory element (SRE) motif. Moreover, a gel shift assay demonstrated that SREBP-2 specifically bound to the SRE motif. These results suggest that transcription of the BACH gene is activated by SREBP-2. This study also provides insights into BACH function in the interaction between the metabolism of acyl-CoAs and cholesterol in neurons.

Brain↗
PubMed1999

Fibroblast growth factor receptor 3 gene transcription is suppressed by cyclic adenosine 3',5'-monophosphate. Identification of a chondrocytic regulatory element.

Signaling through fibroblast growth factor receptors (FGFRs) is critical for the development and patterning of the vertebrate skeleton. Gain-of-function alleles of fgfr2 and fgfr3 have been linked to several dominant skeletal disorders in humans, while null mutations in fgfr3 result in the overgrowth of long bones in a mouse model system. Interestingly, the expression pattern of fgfr3 in growth plate chondrocytes overlaps that of the parathyroid hormone (PTH)-related peptide (PTHrP) receptor, a signaling molecule that also regulates endochondral ossification. The coincident expression of these two receptors suggests that their signaling pathways may also interact. To gain insight into the regulatory mechanism(s) that govern the expression of the fgfr3 gene in chondrocytes, we have identified a cell-specific transcriptional regulatory element (CSRh) by measuring the activity of various promoter fragments in FGFR3-expressing (CFK2) and nonexpressing (RCJ) chondrocyte-like cell lines. Furthermore, we demonstrate that activation of PTH/PTHrP receptors, either by stimulation with PTH or through the introduction of activating mutations, represses CSRh-mediated transcriptional activity. Finally, the transcriptional repression of the CSRh element was mimicked by treatment with forskolin, 8-bromo-cAMP, and 3-isobutyl-1-methylxanthine or by overexpression of the catalytic subunit of protein kinase A. Together, these data suggest that protein kinase A activity is a critical factor that regulates fgfr3 gene expression in the proliferative or prehypertrophic compartment of the epiphyseal growth plate. Furthermore, these results provide a possible link between PTHrP signaling and fgfr3 gene expression during the process of endochondral ossification.

1-Methyl-3-isobutylxanthine↗
PubMed2004

Transcriptional regulation of Msx2 in the AERs of developing limbs is dependent on multiple closely spaced regulatory elements.

In developing limb buds, Msx2 transcripts are expressed in the apical ectodermal ridge (AER) and in various regions of the limb mesenchyme. To identify DNA sequences responsible for Msx2 expression in the AER, we characterized the expression of LacZ reporter constructs driven by chicken Msx2 regulatory sequences in transgenic mice. We have identified a 55-bp enhancer that can direct AER-specific reporter gene expression. This 55-bp enhancer contains three elements that are evolutionary conserved among five vertebrate Msx2 genomic sequences. AER expression of reporter constructs in transgenic mice is lost or reduced when mutations are introduced into each of these three regions. Moreover, changing the relative orientation by reverse complementing one of the three elements also results in loss of expression, suggesting that the relative orientations of transcription factor binding is important. To identify the transcription factor(s) binding to these elements, we conducted one-hybrid screening and identified Dlx5 and Sox11. Both Dlx5 and Sox11 are expressed in the AER, and the proteins encoded by these genes bind to separate conserved elements, supporting their possible roles in regulating Msx2 expression.

Animals↗
PubMed2006

Unraveling transcription regulatory networks by protein-DNA and protein-protein interaction mapping.

Metazoan genomes contain thousands of protein-coding and noncoding RNA genes, most of which are differentially expressed, i.e., at different locations or at different times during development, function, or pathology of the organism. Differential gene expression is achieved in part by the action of regulatory transcription factors (TFs) that bind to cis-regulatory elements that are often located in or near their target genes. Each TF likely regulates many targets in the context of intricate transcription regulatory networks. Up to 10% of a genome may encode TFs, but only a handful of these have been studied in detail. Here, I will discuss the different steps involved in the mapping and analysis of transcription regulatory networks, including the identification of network nodes (TFs and their target sequences) and edges (TF-TF dimers and TF-DNA target interactions), integration with other data types, and network properties and emerging principles that provide insights into differential gene expression.

Animals↗
PubMed1992

Inducible expression bovine papillomavirus shuttle vectors containing ferritin translational regulatory elements.

The combination of transcriptional and translational control elements in an inducible expression vector suitable for use in stably transformed cell lines was explored. To this end, ferritin translational control elements have been inserted downstream from a mouse metallothionein (mMT-I) transcriptional promoter (PmMT-I), and upstream from various reporter protein-encoding open reading frames (ORFs), all carried on a bovine papillomavirus shuttle vector. Protocols which stimulate transcription (with zinc) and translation (with iron) were developed to optimize the induction of reporter protein synthesis. It was found that insertion of an iron regulatory element between the PmMT-I and a reporter ORF bestowed a sixfold inducibility of reporter protein synthesis with iron and a 90-fold inducibility with iron plus zinc in a classical superinduction protocol. Surprisingly, inclusion of other rabbit ferritin light chain sequences (rFL), including the ORF, enhanced reporter inducibilities to over 15- and 500-fold, respectively. These additional rFL sequences not only increased inducibility but also (i) increased the half-life of the mRNA and (ii) strongly inhibited translation of an ORF located downstream from the 5' proximal ORF. The maximum levels of reporter proteins attained in transformed cells after prolonged induction represented from 1% to 7% of total cellular protein. These inducible expression vectors should prove useful for the production and study of cytotoxic proteins.

Animals↗
PubMed1998

CBP is required for sterol-regulated and sterol regulatory element-binding protein-regulated transcription.

Cells were transfected with luciferase reporter genes, under the control of promoters derived from either the farnesyl diphosphate (FPP) synthase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, or low density lipoprotein receptor genes. The increase in luciferase activity that occurred when cells were either incubated in sterol-depleted medium or cotransfected with a cDNA encoding sterol regulatory element-binding protein (SREBP)-1a was prevented by coexpression of wild-type E1A or a Gal4-CBP (1-451) fusion protein. The inhibitory effect of E1A was overcome by coexpression of CBP. The increase in reporter gene activity noted above was not affected when the cells were cotransfected with cDNAs that encoded either a mutant E1A that is unable to interact with the transcriptional activator CBP or Gal4-CBP fusion proteins encoding separate fragments of CBP, which span the remainder of the CBP molecule. A preformed SREBP-1a:[32P]DNA complex bound specifically to membrane-immobilized GST-CBP fusion proteins that contained amino-terminal portions of CBP. In order to investigate the role of CBP in the regulation of endogenous genes, we isolated stable transformants that express Gal4-CBP(1-451) in response to added doxycycline. Induction of endogenous FPP synthase and HMG-CoA synthase mRNAs, in response to cellular cholesterol depletion, was prevented when cells expressed Gal4-CBP(1-451). We conclude that when cells are incubated in the absence of sterols, the transcriptional activation of the HMG-CoA synthase, HMG-CoA reductase, FPP synthase, and low density lipoprotein receptor genes is dependent on a specific interaction between SREBP, which is bound to the promoter DNA, and the amino-terminal domain (amino acids 1-451) of CBP.

Adenovirus E1A Proteins↗
PubMed2004

Functional characterization of tobacco transcription factor TGA2.1.

Activation sequence-1 (as-1)-like regulatory cis elements mediate transcriptional activation in response to increased levels of plant signalling molecules auxin and salicylic acid (SA). Our earlier work has shown that tobacco cellular as-1-binding complex SARP (salicylic acid responsive protein) is primarily comprised of bZIP protein TGA2.2 and of minor amounts of a protein that cross-reacts with an antibody directed against related bZIP factor TGA2.1. As this protein was significantly smaller than recombinant TGA2.1, the origin of this protein had remained unresolved. Here we demonstrate that it corresponds to a distinct cleavage product of TGA2.1 generated during extract preparation. Overexpression of TGA2.1 led to increased levels of the TGA2.1/TGA2.2 heterodimer which was as effective with regard to enhancing the SA-inducibility of as-1 containing target gene Nt103 as corresponding amounts of the TGA2.2 homodimer. Thus, the TGA2.1 specific N-terminal domain, which had revealed transcriptional activation potential in yeast, did not show enhanced transcriptional activation in planta. TGA2.1 even had a negative effect on the SA-induced expression of the truncated CaMV 35S (-90) promoter that contains an isolated as-1-element upstream of the TATA-box. Plants expressing a TGA mutant deficient in DNA binding (TGA2.1trd) showed reduced levels of SA-inducible Nt103 expression, thus resembling plants expressing the analogous TGA2.2 derivative TGA2.2trd. In contrast to TGA2.2trd, TGA2.1trd did not reduce auxin-induced expression of Nt103 and SA-induced expression of pathogenesis related protein PR-1a, indicating that TGA2.1trd and TGA2.2trd differ in their capacity to outcompete regulatory factors involved in these regulatory pathways.

Binding Sites↗
PubMed2000

Sterol regulatory element-binding protein-1 is regulated by glucose at the transcriptional level.

In vivo studies suggest that sterol regulatory element-binding protein (SREBP)-1 plays a key role in the up-regulation of lipogenic genes in the livers of animals that have consumed excess amounts of carbohydrates. In light of this, we sought to use an established mouse hepatocyte cell line, H2-35, to further define the mechanism by which glucose regulates nuclear SREBP-1 levels. First, we show that these cells transcribe high levels of SREBP-1c that are increased 4-fold upon differentiation from a prehepatocyte to a hepatocyte phenotype, making them an ideal cell culture model for the study of SREBP-1c induction. Second, we demonstrate that the presence of precursor and mature forms of SREBP-1 protein are positively regulated by medium glucose concentrations ranging from 5. 5 to 25 mm and are also regulated by insulin, with the amount of insulin in the fetal bovine serum being sufficient for maximal stimulation of SREBP-1 expression. Third, we show that the increase in SREBP-1 protein is due to an increase in SREBP-1 mRNA. Reporter gene analysis of the SREBP-1c promoter demonstrated a glucose-dependent induction of transcription. In contrast, expression of a fixed amount of the precursor form of SREBP-1c protein showed that glucose does not influence its cleavage. Fourth, we demonstrate that the glucose induction of SREBP could not be reproduced by fructose, xylose, or galactose nor by glucose analogs 2-deoxy glucose and 3-O-methyl glucopyranose. These data provide strong evidence for the induction of SREBP-1c mRNA by glucose leading to increased mature protein in the nucleus, thus providing a potential mechanism for the up-regulation of lipogenic genes by glucose in vivo.

Animals↗
PubMed1994

Identification of functional promoter elements in the rabbit smooth muscle myosin heavy chain gene.

Despite the importance of smooth muscle cell proliferation in vascular pathophysiological states, the mechanisms regulating smooth muscle cell growth and differentiation are poorly understood. Previous studies have shown that adult rabbit smooth muscles express two types of myosin heavy chain (MHC) isoforms, SM1 and SM2, which are generated through alternative RNA splicing from a single smooth muscle MHC (SMHC) gene. In the present study, we isolated and characterized the rabbit SMHC gene promoter. DNA sequence analysis of the upstream region of the SMHC gene revealed several putative cis-DNA regulatory elements proximal to the transcription start site. Most notably, cis-acting regulatory elements that closely resemble CC(A/T)6GG (CArG box) and myocyte enhancer binding factor 2 (MEF-2)-type sequence motifs were found in the SMHC 5'-flanking region. In addition, six E-box motifs were found in the 5'-flanking region of the SMHC gene between -374 and -2109 base pairs from the transcription start site. A series of transient transfection assays using SMHC promoter deletion constructs indicated that a promoter fragment extending to 2266 base pairs upstream of the transcription start site has the highest reporter activity in cultured rat aortic smooth muscle cells. Gel mobility shift analyses using the MEF-2-like sequence located at -1540 revealed a specific DNA protein complex, whereas the CArG-like element located at -1275 did not show protein binding. The SMHC promoter construct, p509-CAT, which included neither the CArG- nor MEF-2-type motifs, conferred 32% of chloramphenicol acetyltransferase activity in the same cells, whereas the construct p188-CAT, which contained the minimal promoter elements (TATA box), was significantly less active (7%; 2.0-fold over background). This is the first report describing the promoter elements of a gene whose expression is restricted to smooth muscle cells.

Alternative Splicing↗
PubMed1992

Transcriptional regulation of the human IgE receptor (Fc epsilon RII/CD23) by EBV. Identification of EBV-responsive regulatory elements in intron 1.

EBV infection of B cells induces the B cell activation Ag, CD23 (Fc epsilon RII). CD23 remains constitutively expressed at high levels in all EBV-immortalized B cells and likely plays an important role in the initiation and maintenance of immortalization by EBV. By utilizing an EBV-negative Burkitt's lymphoma line (BJAB) and EBV-positive sublines derived from it by in vitro infection, we have examined the molecular mechanisms involved in the regulation of CD23 by EBV. By nuclear runoff analysis, we have found that induction of CD23 is mediated by transcriptional activation that occurs in the presence of the transformation-competent B958 virus but not in the presence of the nontransforming P3HR-1 strain of EBV. To identify EBV-responsive transcriptional regulatory elements of CD23, we have performed reporter gene assays using plasmids containing fragments of the CD23 gene derived from its 5' terminus and adjacent flanking region transfected into EBV-positive and -negative BJAB lines. We have identified a 534-bp fragment of the gene which enhances transcription from a heterologous promoter (SV40) and reporter gene (chloramphenicol acetyltransferase) only in the presence of transformation-competent strains of EBV. Deletion of 144 bp of intron 1 from the 3' end of this fragment results in loss of EBV-responsive enhancer activity. The finding of an EBV-responsive enhancer element of CD23 is supported by mobility shift assays that demonstrated the formation of specific DNA-protein complexes between nuclear protein from transforming EBV-positive cells and the 144-bp intron sequence. These studies suggest that the transcriptional activation of CD23 by transforming strains of EBV involves regulatory elements that are located within the first intron of the gene.

Antigens, Differentiation, B-Lymphocyte↗
PubMed2003

A novel zinc finger transcriptional repressor, ZNF224, interacts with the negative regulatory element (AldA-NRE) and inhibits gene expression.

The interaction between the negative cis-element (AldA-NRE) and p97 repressor nuclear protein is a key step in modulating transcription of the human and mouse aldolase A (AldA) gene during the cell cycle and differentiation. In an attempt to clarify the role of transcriptional repression in regulating gene expression, we purified, from HeLa cells, the nuclear protein that specifically binds to the AldA negative regulatory element (NRE). Matrix-assisted laser desorption ionization-time of flight analysis and examination of protein profiles from the SwissProt database revealed that the previously defined p97 repressor is ZNF224, a zinc finger protein. We demonstrate that ZNF224, a Kruppel-like zinc finger transcription factor, is the repressor protein that specifically binds to the negative cis-element AldA-NRE and affects the AldA-NRE-mediated transcription.

Amino Acid Sequence↗
PubMed1999

Mechanism of intrinsic transcription termination and antitermination.

Gene expression is modulated by regulatory elements that influence transcription elongation by RNA polymerase: terminators that disrupt the elongation complex and release RNA, and regulators that overcome termination signals. RNA release from Escherichia coli RNA polymerase can be induced by a complementary oligonucleotide that replaces the upstream half of the RNA hairpin stem of intrinsic terminator transcripts, implying that RNA hairpins act by extracting RNA from the transcription complex. A transcription antiterminator inhibits this activity of oligonucleotides and therefore protects the elongation complex from destabilizing attacks on the emerging transcript. These effects illuminate the structure of the complex and the mechanism of transcription termination.

Base Pairing↗
PubMed1999

Fiber-type-specific transcription of the troponin I slow gene is regulated by multiple elements.

The regulatory elements that restrict transcription of genes encoding contractile proteins specifically to either slow- or fast-twitch skeletal muscles are unknown. As an initial step towards understanding the mechanisms that generate muscle diversity during development, we have identified a 128-bp troponin I slow upstream element (SURE) and a 144-bp troponin I fast intronic element (FIRE) that confer fiber type specificity in transgenic mice (M. Nakayama et al., Mol. Cell. Biol. 16:2408-2417, 1996). SURE and FIRE have maintained the spatial organization of four conserved motifs (3' to 5'): an E box, an AT-rich site (A/T2) that binds MEF-2, a CACC site, and a novel CAGG motif. Troponin I slow (TnIs) constructs harboring mutations in these motifs were analyzed in transiently and stably transfected Sol8 myocytes and in transgenic mice to assess their function. Mutations of the E-box, A/T2, and CAGG motifs completely abolish transcription from the TnI SURE. In contrast, mutation of the CACC motif had no significant effect in transfected myocytes or on the slow-specific transcription of the TnI SURE in transgenic mice. To assess the role of E boxes in fiber type specificity, a chimeric enhancer was constructed in which the E box of SURE was replaced with the E box from FIRE. This TnI E box chimera, which lacks the SURE NFAT site, confers essentially the same levels of transcription in transgenic mice as those conferred by wild-type SURE and is specifically expressed in slow-twitch muscles, indicating that the E box on its own cannot determine the fiber-type-specific expression of the TnI promoter. The importance of the 5' half of SURE, which bears little homology to the TnI FIRE, in muscle-specific expression was analyzed by deletion and linker scanning analyses. Removal of the 5' half of SURE (-846 to -811) results in the loss of expression in stably transfected but not in transiently expressing myocytes. Linker scanning mutations identified sequences in this region that are necessary for the function of SURE when integrated into chromatin. One of these sites (GTTAATCCG), which is highly homologous to a bicoid consensus site, binds to nuclear proteins from several mesodermal cells. These results show that multiple elements are involved in the muscle-specific activity of the TnIs promoter and that interactions between upstream and downstream regions of SURE are important for transcription in the context of native chromatin.

Animals↗