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Results for “regulatory element transcripts”

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PubMed1996

Distinct positive and negative regulatory elements control neuronal and hepatic transcription of the human transferrin gene.

Transferrin (Tf), the iron-transport protein, plays an essential role in the central nervous system development, plasticity, and aging. As a first step toward elucidating the role of each transcription factor involved in the regulation of Tf gene expression, we have recently shown that similar promoter elements direct cell-type specific transcription in oligodendrocytes, epithelial choroid plexus cells, and in the neuronal cell line B103. Here we have analyzed the regulatory elements that control the level of expression of the Tf gene in neuronal cells. Transient expression experiments in B103 cells revealed that the -164/+1 promoter region is stimulated by a position-dependent -1140/-1000 upstream region. DNase I footprinting, gel retardation assays, and antibody reactivity data allowed us to characterize the nuclear factors interacting with this region. The upstream region I-binding protein (URI-BP) belongs to the steroid/retinoid receptor family, while URII-BP is a member of the nuclear factor I (NF-I) family. Interestingly, no enhancer nor silencer activity is detected in B103 cells. This contrasts with our findings in hepatoma cells, where the activity of the -125/+1 promoter can be repressed by a -1000/-819 upstream negative-acting region and stimulated by the -3600/-3300 enhancer. We demonstrate that the negative-acting region presents the characteristics of a silencer that interacts with a nuclear protein present in liver and absent in B103 cells. Similarly, B103 cells lack a nuclear protein able to bind to an essential site of the enhancer. This shows that in B103 cells, the inactivity of the silencer and the enhancer regions results from the absence of at least one essential nuclear protein.

Base Sequence↗

PubMed1987

Regulatory elements in the first intron contribute to transcriptional control of the human alpha 1(I) collagen gene.

Several lines of evidence have suggested that the regulation of type I collagen gene transcription is complex and that important regulatory elements reside 5' to, and within, the first intron of the alpha 1(I) gene. We therefore sequenced a 2.3-kilobase HindIII fragment that encompasses 804 base pairs of 5' flanking sequence, the first exon, and most of the first intron of the alpha 1(I) human collagen gene. A 274-base-pair intronic sequence, flanked by Ava I sites (A274), contained a sequence identical to a high-affinity decanucleotide binding site for transcription factor Sp1 and a viral core enhancer sequence. DNase I protection experiments indicated zones of protection that corresponded to these motifs. When A274 was cloned 5' to the chloramphenicol acetyltransferase (CAT) gene, driven by an alpha 1(I) collagen promoter sequence, and expression was assessed by transfection, significant orientation-specific inhibition of CAT activity was observed. This effect was most apparent in chicken tendon fibroblasts, which modulate their level of collagen synthesis in culture. We propose that normal regulation of alpha 1(I) collagen gene transcription results from an interplay of positive and negative elements present in the promoter region and within the first intron.

Amino Acid Sequence↗

PubMed2004

Sterol and fatty acid regulatory pathways in a Giardia lamblia-derived promoter: evidence for SREBP as an ancient transcription factor.

The sterol regulatory element binding-proteins (SREBPs) are transcription factors that regulate the genes of lipid metabolism. Cholesterol and unsaturated fatty acids regulate SREBPs. Giardia lamblia (GL) is an intestinal parasite and one of the earliest derived members within the eukaryotic lineage. GLs exist as trophozoites and cysts. Growth in cholesterol depletion induces transcription of cyst-wall protein (CWP) genes that are upregulated during encystation. The hypothesis was investigated that SREBP-like pathways have a role in cwp gene transcription. Chinese hamster ovary cells were transfected with a cwp-2 promoter reporter construct. Incubation with cholesterol or oleate reduced cwp-2 mediated gene transcription to about half of the control. Incubation in sterol-depleted media, or in the presence of either an inhibitor of intracellular cholesterol movement or inhibitor of cholesterol synthesis, increased gene expression up to 3-fold. Overexpression of SREBPs increased reporter gene activity 2.5-fold. In the absence of functional SREBPs, cwp-2 was not regulated by cholesterol. Footprint analysis of cwp-2 reveals three novel binding sites for mammalian SREBPs with no homologies in other species or humans. The data show that SREBP binds to and can modulate transcription of a regulatory element from an ancient eukaryote and suggest the existence of an SREBP homolog in GL.

Animals↗

PubMed2006

Long-range downstream enhancers are essential for Pax6 expression.

Pax6 is a developmental control gene with an essential role in development of the eye, brain and pancreas. Pax6, as many other developmental regulators, depends on a substantial number of cis-regulatory elements in addition to its promoters for correct spatiotemporal and quantitative expression. Here we report on our analysis of a set of mice transgenic for a modified yeast artificial chromosome carrying the human PAX6 locus. In this 420 kb YAC a tauGFP-IRES-Neomycin reporter cassette has been inserted into the PAX6 translational start site in exon 4. The YAC has been further engineered to insert LoxP sites flanking a 35 kb long, distant downstream regulatory region (DRR) containing previously described DNaseI hypersensitive sites, to allow direct comparison between the presence or absence of this region in the same genomic context. Five independent transgenic lines were obtained that vary in the extent of downstream PAX6 locus that has integrated. Analysis of transgenic embryos carrying full-length and truncated versions of the YAC indicates the location and putative function of several novel tissue-specific enhancers. Absence of these distal regulatory elements abolishes expression in specific tissues despite the presence of more proximal enhancers with overlapping specificity, strongly suggesting interaction between these control elements. Using plasmid-based reporter transgenic analysis we provide detailed characterization of one of these enhancers in isolation. Furthermore, we show that overexpression of a short PAX6 isoform derived from an internal promoter in a multicopy YAC transgenic line results in a microphthalmia phenotype. Finally, direct comparison of a single-copy line with the floxed DRR before and after Cre-mediated deletion demonstrates unequivocally the essential role of these long-range control elements for PAX6 expression.

Animals↗

PubMed1999

Characterization of the human CR1 gene promoter.

The human CR1 is a single chain membrane glycoprotein that is a member of the group of regulators of the complement activation system. In order to clarify the regulatory mechanisms of human CR1 gene expression, the 5'-flanking region of the human CR1 gene was isolated and its promoter was characterized. The CR1 expression was found to be transcriptionally up-regulated in HL60 cells by stimulation with DMSO. The cloned CR1 gene promoter was sequenced and computer analyzed. The potential promoter region lacks a distinct TATA box sequence. The transcription initiation site was determined by primer extension and several possible regulatory elements for transcription were found in the promoter region.

Base Sequence↗

PubMed1993

cis-acting elements in the lytic origin of DNA replication of Epstein-Barr virus.

oriLyt, the cis-acting element of Epstein-Barr virus, mediates viral DNA replication in the lytic phase of the virus's life cycle. Oligonucleotide-directed in vitro mutagenesis of oriLyt plasmids allowed the identification of two noncontiguous components within the complex structure of oriLyt. Both components were indispensable for DNA replication of this origin. The upstream component colocalized with the promoter of the viral BHLF1-encoding gene, and mutants affecting DNA replication affected RNA transcription, too. The second component crucial for oriLyt function was determined to be 40 bp long and positioned approximately 530 bp downstream. It was dispensable for transcriptional transactivation but it was absolutely required for replication. Thus, the overall design of oriLyt has striking similarity to multipartite regulatory elements of transcription, consisting of proximal promoters and distal enhancers, but special elements are exclusively dedicated to DNA replication.

Base Sequence↗

PubMed2005

TRACTOR_DB: a database of regulatory networks in gamma-proteobacterial genomes.

Experimental data on the Escherichia coli transcriptional regulatory system has been used in the past years to predict new regulatory elements (promoters, transcription factors (TFs), TFs' binding sites and operons) within its genome. As more genomes of gamma-proteobacteria are being sequenced, the prediction of these elements in a growing number of organisms has become more feasible, as a step towards the study of how different bacteria respond to environmental changes at the level of transcriptional regulation. In this work, we present TRACTOR_DB (TRAnscription FaCTORs' predicted binding sites in prokaryotic genomes), a relational database that contains computational predictions of new members of 74 regulons in 17 gamma-proteobacterial genomes. For these predictions we used a comparative genomics approach regarding which several proof-of-principle articles for large regulons have been published. TRACTOR_DB may be currently accessed at http://www.bioinfo.cu/Tractor_DB, http://www.tractor.lncc.br/ or at http://www.cifn.unam.mx/Computational_Genomics/tractorDB. Contact Email id is tractor@cifn.unam.mx.

Base Sequence↗

PubMed2000

Polyunsaturated fatty acids suppress hepatic sterol regulatory element-binding protein-1 expression by accelerating transcript decay.

The reduction in hepatic abundance of sterol regulatory element binding protein-1 (SREBP-1) mRNA and protein associated with the ingestion of polyunsaturated fatty acids (PUFA) appears to be largely responsible for the PUFA-dependent inhibition of lipogenic gene transcription. Our initial studies indicated that the induction of SREBP-1 expression by insulin and glucose was blocked by PUFA. Nuclear run-on assays suggested PUFA reduced SREBP-1 mRNA by post-transcriptional mechanisms. In this report we demonstrate that PUFA enhance the decay of both SREBP-1a and -1c. When rat hepatocytes in monolayer culture were treated with albumin-bound 20:4(n-6) or 20:5(n-3) the half-life of total SREBP-1 mRNA was reduced by 50%. Ribonuclease protection assays revealed that the decay of SREBP-1c mRNA was more sensitive to PUFA than was SREBP-1a, i.e. the half-life of SREBP-1c and -1a was reduced from 10.0 to 4.6 h and 11.6 to 7.6 h, respectively. Interestingly, treating the hepatocytes with the translational inhibitor, cycloheximide, prevented the PUFA-dependent decay of SREBP-1. This suggests that SREBP-1 mRNA may need to undergo translation to enter the decay process, or that the decay process requires the synthesis of a rapidly turning over protein. Although the mechanism by which PUFA accelerate SREBP-1 mRNA decay remains to be determined, cloning and sequencing of the 3'-untranslated region for the rat SREBP-1 transcript revealed the presence of an A-U-rich region that is characteristic of a destablizing element.

3' Untranslated Regions↗

PubMed1999

Characterization of the lung Krüppel-like transcription factor gene and upstream regulatory elements.

We previously described the isolation and characterization of the cDNA for lung Krüppel-like factor (LKLF), a zinc finger transcription factor that is predominately expressed in the lung of adult mice. In this study, we report the complete structure and nucleotide sequence of the mouse LKLF gene, which is comprised of three exons and two small introns. Moreover, the identification of critical sequence elements required for expression is described using reporter constructs with the LKLF promoter transfected into LA-4 lung cells. Results from these constructs reveal an important region for transcriptional activity that lies between the -490/-72bp upstream sequence. This region contains two canonical Sp1 binding sites that affect expression levels in a non tissue-specific manner. In addition, using a base-pair mutagenesis strategy, a region from -157/-72bp was found to be necessary for upregulating expression. In transfection assays, mutations of the -138/-111bp region resulted in approximately 70-80% loss of promoter activity. This cis-element does not appear to correspond to any known transcription factor consensus sequence. Moreover, mutations within this cis-region disrupt the binding of a protein complex from nuclear extracts of various tissues.

Animals↗

PubMed2006

Nuclear factor-I regulates glial fibrillary acidic protein gene expression in astrocytes differentiated from cortical precursor cells.

The elucidation of the transcriptional mechanisms that regulate glial fibrillary acidic protein (GFAP) gene expression is important for the understanding of the molecular mechanisms that control astrocyte differentiation during brain development. We investigated regulatory elements located in a proximal region of the GFAP promoter, important for expression in cortical precursor cells differentiating into astrocytes. One of these elements recognizes transcription factors of the nuclear factor-I family (NFI). We found that, in primary cultures of cortical cells, NFI occupies the GFAP promoter prior to the induction of astrocyte differentiation. In the developing cerebral cortex, the onset of expression of NFI coincides chronologically with the beginning of astrocytogenesis. Mutational analysis of the GFAP gene and transfections in primary cortical precursors show that inhibition of binding of NFI to the GFAP promoter results in decreased levels of transcriptional activity and is required for the synergistic stimulation of the GFAP promoter by the astrogenic agents, pituitary adenylate cyclase-activating polypeptide and ciliary neurotrophic factor, which in combination enhance astrocyte differentiation to generate astrocytes with longer processes. Thus, NFI appears to be an important factor for the integration of astrogenic stimuli in the developing central nervous system.

Animals↗

PubMed1995

Yeast histone H3 and H4 N termini function through different GAL1 regulatory elements to repress and activate transcription.

Previous work has shown that N-terminal deletions of yeast histone H3 cause a 2- to 4-fold increase in the induction of GAL1 and a number of other genes involved in galactose metabolism. In contrast, deletions at the H4 N terminus cause a 10- to 20-fold decrease in the induction of these same GAL genes. However, H3 and H4 N-terminal deletions each decrease PHO5 induction only 2- to 4-fold. To define the GAL1 gene regulatory elements through which the histone N termini activate or repress transcription, fusions were made between GAL1 and PHO5 promoter elements attached to a beta-galactosidase reporter gene. We show here that GAL1 hyperactivation caused by the H3 N-terminal deletion delta 4-15 is linked to the upstream activation sequence. Conversely, the relative decrease in GAL1 induction caused by the H4N-terminal deletion delta 4-28 is linked to the downstream promoter which contains the TATA element. These data indicate that the H3 N terminus is required for the repression of the GAL1 upstream element, whereas the H4N terminus is required for the activation of the GAL1 downstream promoter element.

Base Sequence↗

PubMed1996

Transcriptional regulation of the lysozyme gene in airway gland serous cells.

Lysozyme is expressed in serous, but not mucous, cells of the tracheobronchial glands and thereby constitutes a marker of the serous cell lineage in these glands. To identify DNA regulatory elements and transcription factors mediating the commitment of progenitor cells to the serous cell lineage, we have characterized the regulatory activity and DNA-protein interactions of the 5'-flanking region of the bovine lysozyme gene lys 5a. Results obtained from these studies indicate that although approximately 94 bp of 5'flanking DNA are necessary for high level expression in transient transfection assays, an evolutionarily conserved promoter within 66 bp of the transcription start site is sufficient to confer serous cell-specific expression. Farther upstream, within 6.1 kb of the 5' flanking region, are 4 silencers. Analysis of the serous cell-specific lysozyme promoter by electrophoretic mobility shift assay (EMSA) revealed the presence of binding sites for 3 serous cell nuclear proteins, designated LSF1, LSF2 and LSF3. Binding of LSF2 and LSF3 was localized to a 20-mer subdomain (-50/-30) of the cell-specific promoter using binding competition assays. More accurate identification of the protein binding site(s) was achieved through the use of mutagenesis, which implicated the motif 5' AAGGAAT 3' (-46/-40) in both protein binding and serous cell-specific transcriptional activity. This motif has previously been identified as a binding site for ets protein transcription factors, suggesting that serous cell-specific regulation of lys 5a transcription is partly controlled by the binding of ets-like protein(s) to the motif 5'AGGAAGT3'.

Amino Acid Sequence↗

PubMed2004

Human E2F6 is alternatively spliced to generate multiple protein isoforms.

E2F6 protein belongs to the family of the E2F transcription factors. Here, we showed that the human E2F6 gene contains nine exons distributed along 20.4kbp of genomic DNA on chromosome 2 leading to the transcription of six alternatively spliced E2F6 mRNAs that encode four different E2F6 proteins. Moreover, we identified an E2F6 pseudogene localized on chromosome 22 completely spliced and devoid of exons 2, 3, and 4, and part of exons 1 and 5. Definition of the transcriptional initiation site and sequence analysis show that the gene contains a TATA less, CAAT less, GC-rich promoter with multiple transcription start sites. Regulatory elements necessary for basal transcription reside within a 134bp fragment as determined by transient transfection experiments.

Alternative Splicing↗

PubMed2006

Genome-wide location analysis: insights on transcriptional regulation.

Gene expression analysis of microarray data can provide a global view of the transcriptome of a cell or specific tissue type, revealing important information about the kinds of signaling pathways, genes and protein classifications that are active. However, transcript profiles alone do not reveal how expression levels are controlled or which transcription factors (TFs) are responsible. Establishing transcriptional regulatory networks requires knowledge of TFs bound to promoter, enhancer and repressor elements. Accessibility of these sites and an additional level of control are mediated by chromatin and DNA modifications. Genome-wide location analysis is a tool for identifying protein-DNA interaction sites on a genomic scale. Applications of this tool are proving invaluable in determining in vivo target genes of TFs, epigenetic marks and cis-regulatory elements. Here, we will discuss how advances have been made in each of these categories and how this has helped to elucidate regulatory networks and control mechanisms.

Animals↗

PubMed1994

Characterization of the proximal promoter and two silencer elements in the CYP2C11 gene expressed in rat liver.

The cytochrome P450 gene CYP2C11, expressed in the liver of male rats, is transcriptionally regulated in a dual fashion by the sexually dimorphic secretion pattern of growth hormone. To enable analysis of transcriptional regulatory DNA elements, rat genomic sequences were cloned. DNase I hypersensitivity analysis of rat liver nuclei revealed the existence of two hypersensitive sites whose presence in the vicinity of the transcription start site correlates to high transcriptional activity of the gene. Deletion mutants of the 5' flank were fused to reporter genes and transiently transfected into HepG2 cells or into primary adult rat hypatocytes. Transfection experiments in combination with DNase I footprinting analysis in vitro led to the identification of two negative regulatory regions spanning nucleotides -1,230 to -1,188 and -409 to -368 and designated (SIL1200) and (SIL400), respectively. When placed in front of the heterologous thymidine kinase promoter, SIL1200 and SIL400 reduced the activity of the chloramphenicol acetyl transferase reporter gene to 13% and 23% of the control value, respectively. No sex-dependent binding of liver nuclear extracts to the two silencers could be detected by in vitro footprinting or gel retardation assays. However, a sex-dependent footprint consistently stronger with male liver nuclear extracts than with female extracts was observed in the -320 to -294 region. A significant level of identity was found between the DNA sequence corresponding to this footprint and that of orphan steroid receptor elements as well as with that of a basal transcription element common to several CYP2C genes. However, the identity of a potential trans-acting factor binding between -320 and -294 or response of this element to growth hormone is as yet unknown. A sex- and GH secretory profile-dependent protein-DNA interaction in vitro was observed in the -107 to -95 region. In spite of the sequence similarity that exists between this region and the consensus binding site for HNF-1, this region does not bind HNF-1 alpha. This element acted as a repressor on the heterologous thymidine kinase promoter. To date, the two silencer elements and possibly also the HNF-1-like element are the only functional elements defined in the CYP2C11 gene, and it is conceivable that induction of the gene involves derepression of the silencer elements.

Animals↗

PubMed1996

Cloning the 5' flanking region of neuron-specific Hel-N1: evidence for positive regulatory elements governing cell-specific transcription.

A 5.4 kilobase-pair segment of DNA flanking the 5' end of Hel-N1 was isolated and characterized. Primer extension studies with normal human brain and neuroblastoma cells revealed a major and minor transcription-initiation site. Sequence analysis of the initial 536 bp upstream to the major start site revealed a core promoter (-1 to -181) which contained two CCAAT boxes, a weakly-conserved TATA box, and an SP1 site. This region was also moderately GC-rich (62%). Using a transient luciferase-reporter-gene assay, the core promoter was found to be essential for basal transcription both in neural (PC12) and non-neural (HeLa and glial) cell types. Two positive regulatory elements, however, were identified in the initial 536 bp (-1 to -181 and -182 to -350) which produced a five- to six-fold increase in transcriptional activity in PC12 cells vs. HeLa or glial cells. These elements, therefore, were sufficient to confer cell-specific enhanced transcription and likely contribute to the neuronal specificity of Hel-N1 mRNA expression.

Animals↗

PubMed1997

HIRF: a novel nuclear factor that binds to the human T-cell leukemia virus type I internal regulatory element (HIRE).

The transcription of human T-cell leukemia virus type I (HTLV-I) provirus starts from a promoter located in the 5' long terminal repeat (LTR). We have identified a second promoter at the 3' end of the pol gene. This internal promoter expresses the Tax transactivator protein, but does not require Tax for its activity. Furthermore, we have found the novel enhancer motif AGTTCTGCCC, which are located near the initiation site. We have named the sequence HIRE (HTLV-I internal regulatory element). The HIRE binding protein is a ubiquitous protein. We purified this protein from the HTLV-I producing cell line MT-2 cells by DNA affinity chromatography. SDS-PAGE analysis revealed four major bands (70, 85, 115 and more than 200 kDa) and some minor bands on the gel. We renatured each major protein and showed the 70 and 115 kDa proteins bind to DNA, although the 115 kDa protein seemed to bind nonspecifically. We have designated these components as HIRF (HTLV-I internal regulatory factor).

Base Sequence↗

PubMed2003

Mutation of a transcriptional motif of a distant regulatory element reduces the expression of embryonic and fetal globin genes.

High-level beta-globin gene expression is dependent on the presence of the locus control region (LCR), a powerful regulatory element physically characterized by five DNase I-hypersensitive sites (HS), designated HS1-HS5. Of these, HS3 contains seven GT motifs that are essential for its activity. One of the motifs, GT6, has been shown by in vivo footprinting to display the largest difference in signal between fetal and adult globin expressing cells. We assessed the contribution of GT6 on the downstream globin gene expression by mutating this motif in a 248 kb beta-globin locus yeast artificial chromosome and measuring the activity of beta-globin genes in GT6m beta-YAC transgenic mice. Seven transgenic lines were established, three of which contained at least one intact copy of the beta-globin locus and were further investigated. The mutation of the GT6 motif reduced the expression of epsilon- and gamma-globin genes during embryonic erythropoiesis. During definitive erythropoiesis, gamma-globin gene expression was significantly reduced while beta-globin gene expression was virtually indistinguishable from wild-type controls. We conclude that the GT6 motif of hypersensitive site 3 of the LCR is required for normal epsilon- and gamma-globin gene expression during embryonic erythropoiesis and for gamma-globin gene expression during definitive erythropoiesis in the fetal liver. Our results provide evidence that mutations of single transcriptional motifs of distant regulatory elements can have profound effects on gene expression.

Animals↗