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PubMed2003

Expression of the human oestrogen receptor-alpha gene is regulated by promoter F in MG-63 osteoblastic cells.

(O)estrogen receptor-alpha (ERalpha), a hormone-dependent transcription factor belonging to the steroid/thyroid-hormone-receptor superfamily, plays an essential role in the development and maintenance of the skeleton. Here we report the analysis of an unexplored sequence inside the bone-specific distal promoter F (PF) with respect to the regulation of ERalpha gene expression in bone. This sequence, 785 bp in size, is localized upstream of the assigned transcription start site of exon F, at -117140 bp from the originally described transcription start site +1. It contains a TA reach box, a conventional CAAT box and potential regulatory elements for many transcription factors, including Cbfa1 [OSE2 (osteoblast-specific element) core binding factor], GATA-1 [(A/T)GATA(A/G) binding protein], Sox5 [sex-determining region Y (SRY)-type HMG bOX protein, belonging to a subfamily of DNA-binding proteins with an HMG domain], Sry, AP1 (activator protein 1) and CP2 (activator of gamma-globin). It is able to strongly activate the luciferase reporter gene in MG-63 osteoblastic-like cells, but not in MCF7 breast-cancer cells. This is in agreement with different transcripts that we found in the two cell types. The footprinting and electrophoretic mobility-shift assays (EMSAs) showed that, inside the region analysed, there were some sequences that specifically reacted to nuclear proteins isolated from MG-63 cells. In particular, we identified two regions, named PF a and PF b, that do not present binding sites for known transcription factors and that are involved in a strong DNA-protein interaction in MG-63, but not in MCF7, cells. The analysis of three transcription factors (GATA-1, Sry and Sox) that might bind the identified footprinted areas suggested a possible indirect role of these proteins in the regulation of ERalpha gene expression in bone. These data provide evidence for different promoter usage of the ERalpha gene through the recruitment of tissue-specific transcription activators and co-regulators.

Base Sequence↗
PubMed1994

The ORD1 gene encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a gene that confers cisplatin sensitivity to Saccharomyces cerevisiae.

The yeast COX5a and COX5b genes encode isoforms of subunit Va of the mitochondrial inner membrane protein complex cytochrome c oxidase. These genes have been shown to be inversely regulated at the level of transcription by oxygen, which functions through the metabolic coeffector heme. In earlier studies we identified several regulatory elements that control transcriptional activation and aerobic repression of one of these genes, COX5b. Here, we report the isolation of trans-acting mutants that are defective in the aerobic repression of COX5b transcription. The mutants fall into two complementation groups. One group specifies ROX1, which encodes a product reported to be involved in transcriptional repression. The other group identified the gene we have designated ORD1. Mutations in ORD1 cause overexpression of COX5b aerobically but do not affect the expression of the hypoxic genes CYC7, HEM13, and ANB1. ORD1 mutations also do not affect the expression of the aerobic genes COX5a, CYC1, ROX1, ROX3, and TIF51A. The yeast genome contains a single ORD1 gene that resides on chromosome XI. Strains carrying chromosomal deletions of the ORD1 locus are viable and exhibit phenotypes similar to, but less severe than, that of the original mutant. The nucleotide sequence of ORD1 revealed that it is identical to IXR1, a yeast gene whose product contains two high mobility group boxes, binds to platinated DNA, and confers sensitivity to the antitumor drug cisplatin. Consistent with the latter observations, we found that the ORD1 product could bind to both the upstream region of COX5b and to DNA modified with cisplatin.

Amino Acid Sequence↗
PubMed1989

Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes.

Metallothionein (MT) gene promoters in higher eucaryotes contain multiple metal regulatory elements (MREs) that are responsible for the metal induction of MT gene transcription. We identified and purified to near homogeneity a 74-kilodalton mouse nuclear protein that specifically binds to certain MRE sequences. This protein, MBF-I, was purified employing as an affinity reagent a trout MRE that is shown to be functional in mouse cells but which lacks the G+C-rich and SP1-like sequences found in many mammalian MT gene promoters. Using point-mutated MREs, we showed that there is a strong correlation between DNA binding in vitro and MT gene regulation in vivo, suggesting a direct role of MBF-I in MT gene transcription. We also showed that MBF-I can induce MT gene transcription in vitro in a mouse extract and that this stimulation requires zinc.

Animals↗
PubMed1999

Transcriptional regulation in B cell differentiation.

B cell development is a multistage differentiation process that ultimately generates antibody-secreting plasma cells. This model developmental pathway is governed by a choreographed pattern of expression from genes encoding stage and lineage-specific proteins. This is achieved by the interaction of distinct transcription factors with regulatory elements, placing these proteins in a central position in B cell ontogeny. The importance of distinct transcription factors is also supported by the notion that B cell development is disrupted in mice bearing homozygous null alleles of certain factors involved in the control of B-cell-specific genes. In this review we compare and contrast the transcriptional regulation of well-studied B lymphoid restricted genes, on a gene by gene basis, focusing on the functional structure of transcription control regions and interacting transcription factors.

Animals↗
PubMed2004

The human involucrin gene is transcriptionally repressed through a tissue-specific silencer element recognized by Oct-2.

Involucrin is an important marker of epithelial differentiation which expression is upregulated just after basal cells are pushed into the suprabasal layer in stratified epithelia. Several transcription factors and regulatory elements had been described as responsible for turning on the gene. However, it is evident that in basal cell layer, additional mechanisms are involved in keeping the gene silent before the differentiation process starts. In this work, we located a potential transcriptional silencer in a 52bp sequence whose integrity is necessary for silencing the proximal enhancer promoter element (PEP) in multiplying keratinocytes. Octamer-binding sites were noticed in this fragment and the specific binding of Oct-2 transcription factor was detected. Oct-2 appears to be implicated in an epithelial-specific repression activity recorded only in keratinocytes and C33-A cell line. Overexpression of Oct-2 repressed the involucrin promoter activity in epithelial cells and in the presence of the silencer element.

Base Sequence↗
PubMed1998

Cis-regulatory elements controlling basal and inducible VIP gene transcription.

The cis-acting elements of the VIP gene important for basal and stimulated transcription have been studied by transfection of VIP-reporter gene constructs into distinct human neuroblastoma cell lines in which VIP transcription is constitutively high, or can be induced to high levels by protein kinase stimulation. The 5.2 kb flanking sequence of the VIP gene conferring correct basal and inducible VIP gene expression onto a reporter gene in these cell lines was systematically deleted to define its minimal components. A 425-bp fragment (-4656 to -4231) fused to the proximal 1.55 kb of the VIP promoter-enhancer was absolutely required for cell-specific basal and inducible transcription. Four additional components of the VIP gene were required for full cell-specific expression driven by the 425 bp TSE (region A). Sequences from -1.55 to -1.37 (region B), -1.37 to -1.28 (region C), -1.28 to -.094 (region D), and the CRE-containing proximal 94 bp (region E) were deleted in various combinations to demonstrate the specific contributions of each region to correct basal and inducible VIP gene expression. Deletion of region B, or mutational inactivation of the CRE in region E, resulted in constructs with low transcriptional activity in VIP-expressing cell lines. Deletion of regions B and C together resulted in a gain of transcriptional activity, but without cell specificity. All five domains of the VIP gene were also required for cell-specific induction of VIP gene expression with phorbol ester. Gelshift analysis of putative regulatory sequences in regions A-D suggests that both ubiquitous and neuron-specific trans-acting proteins participate in VIP gene regulation.

Animals↗
PubMed1997

A negative regulatory region of the murine Hox11 gene.

The HOX11 gene was isolated from the chromosomal breakpoint of human T cell acute lymphoblastic leukemias with a chromosomal translocation t(10;14). Expression of this proto-oncogene is strictly controlled in normal tissues. However, regulatory elements of the gene have never been studied. Since the HOX11 gene is well conserved between human and murine, we sequenced 5' flanking region of the murine Hox11 gene and analyzed the elements. We identified the transcription start site (+1) of the gene using mRNA from fetal spleens by primer extension analysis. The start site was determined at 795 bp upstream from the ATG site. A typical TATA box sequence was found at 35 bp upstream from the start site. Furthermore, promoter activity of the 5' flanking region of the start site was monitored by luciferase assay. The activity mainly located within a 540-bp fragment immediately upstream from the start site (-540 to +1). The (-1240 to -540) region contained a negative regulatory element of the transcription. The TATA box sequence and the nucleotide sequence around the transcription start site were conserved in the human HOX11 gene. The transcription start site of the human HOX11 gene in normal tissues is discussed.

Animals↗
PubMed2002

T cell-specific expression of the murine CD3delta promoter.

T cell-specific expression of human and mouse CD3delta is known to be governed by an enhancer element immediately downstream from the gene. Here we demonstrate by transgenic and in vitro studies that the murine CD3delta (mCD3delta) promoter prefers to be expressed in cells of the T lineage. Deletion analyses of a promoter segment (-401/+48 bp) followed by transient transfections indicate that upstream elements between -149 and -112 bp contribute to full expression of the gene. Furthermore, a core promoter region -37/+29 appears to contribute to a T cell specificity. Using substitution mutant scanning, four positive and one negative regulatory elements were found within the mCD3delta core promoter. The first two positive elements comprise two classical initiator-like sites, which recruit TFII-I, whereas a third contains a functional Ets binding site. Immediately adjacent to the observed transcription start site is a negative element that utilizes the transcription regulator YY1. The last positive regulatory element contains a potentially functional CREB binding site and the minor transcriptional start site. Because NERF-2, Elf-1, and Ets-1 are expressed preferentially in lymphocytes and because, in addition, YY1 represses the promoter activity strongly in non-T cells, we conclude that the combination of these transcription factors contributes to the T cell-specific expression pattern of mouse CD3delta.

Animals↗
PubMed2004

Deciphering regulatory patterns of inflammatory gene expression from interleukin-1-stimulated human endothelial cells.

OBJECTIVE: Endothelial cells comprise a key component of the inflammatory response. We set out to obtain a comprehensive overview of the immediate-early to early gene expression program of interleukin-1 (IL-1)-stimulated endothelial cells and to identify novel transcription factors and regulatory elements. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were stimulated with IL-1 for 0, 0.5, 1, 2.5, and 6 hours and analyzed using Affymetrix U133 microarrays. A total of 137 genes were found to be regulated >4-fold, including 18 transcription factors. The expression of selected genes was confirmed by real-time polymerase chain reaction. Cluster analysis was performed in order to group genes according to their expression profiles. To identify novel transcription factor-binding sites, the corresponding promoters were extracted from databases and analyzed for regulatory elements that were over-represented in specific clusters. Several potentially novel DNA binding sites were identified, and one was shown to specifically bind an IL-1-inducible protein from HUVEC. CONCLUSIONS: These results demonstrate that in the early phase after stimulation, IL-1 evokes a complex gene expression program that includes positive but also negative (feedback) regulators of diverse endothelial cell functions. Furthermore, the identification of a new promoter regulatory element demonstrates the feasibility of the bioinformatics-driven approach to discover novel regulatory mechanisms.

Binding Sites↗
PubMed1999

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

Recent developments in genome-wide transcript monitoring have led to a rapid accumulation of data from gene expression studies. Such projects highlight the need for methods to predict the molecular basis of transcriptional coregulation. A microarray project identified the 420 yeast transcripts whose synthesis displays cell cycle-dependent periodicity. We present here a statistical technique we developed to identify the sequence elements that may be responsible for this cell cycle regulation. Because most gene regulatory sites contain a short string of highly conserved nucleotides, any such strings that are involved in gene regulation will occur frequently in the upstream regions of the genes that they regulate, and rarely in the upstream regions of other genes. Our strategy therefore utilizes statistical procedures to identify short oligomers, five or six nucleotides in length, that are over-represented in upstream regions of genes whose expression peaks at the same phase of the cell cycle. We report, with a high level of confidence, that 9 hexamers and 12 pentamers are over-represented in the upstream regions of genes whose expression peaks at the early G(1), late G(1), S, G(2), or M phase of the cell cycle. Some of these sequence elements show a preference for a particular orientation, and others, through a separate statistical test, for a particular position upstream of the ATG start codon. The finding that the majority of the statistically significant sequence elements are located in late G(1) upstream regions correlates with other experiments that identified the late G(1)/early S boundary as a vital cell cycle control point. Our results highlight the importance of MCB, an element implicated previously in late G(1)/early S gene regulation, as most of the late G(1) oligomers contain the MCB sequence or variations thereof. It is striking that most MCB-like sequences localize to a specific region upstream of the ATG start codon. Additional sequences that we have identified may be important for regulation at other phases of the cell cycle.

5' Untranslated Regions↗
PubMed1996

Modulation of levels of a negative transcription factor for IL-2 by 12-O-tetradecanoyl phorbol-13-acetate and okadaic acid.

A negatively acting transcription factor, negative regulatory element-A (NREA) that suppresses the transcription of interleukin 2 (IL-2) mRNA, has been described previously. We found that treatment of primary bovine lymphocytes with 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, for at least 18 h both increased the levels of the factor and blocked concanavalin A (Con A)-induced proliferation. In contrast, treatments of less than 18 h with TPA decreased NREA levels and increased Con A-induced proliferation. NREA binding activity also increased over basal levels during the first 4 h of stimulation of lymphocytes with Con A in the absence of TPA; after 4 h, NREA levels fell. Phosphorylation of the NREA protein was required for binding to its DNA consensus sequence. Furthermore, incubation of lymphocytes with okadaic acid (OKA), a phosphatase inhibitor, led to increased levels of NREA binding activity and to decreased cell proliferation. Because exposure of lymphocytes to either OKA or TPA should lead to an increase in the phosphorylation and binding of the NREA protein, and a decrease in IL-2 production, proliferation should be decreased. Incubation of lymphocytes with either TPA or OKA inhibited proliferation. However, the mechanisms of action of OKA and TPA appeared to be different because exogenous IL-2 reversed the inhibition of proliferation caused by TPA but not by OKA.

Animals↗
PubMed1990

The same target sequences are differentially important for activation of the interleukin 2 receptor alpha-chain gene in two distinct T-cell lines.

High-affinity receptors for interleukin 2 (IL-2) are expressed on T cells following activation. These receptors are composed of both alpha and beta chains. Expression of alpha chains and, therefore, expression of high-affinity receptors are critically regulated at the level of transcription initiation. We have further dissected the regulatory elements involved in controlling transcription of the IL-2 receptor alpha-chain (IL-2R alpha) gene. The IL-2R alpha promoter contains a kappa B site and binding sites for additional nuclear factors within a 50-base-pair region (positions -290 to -240 relative to the major transcription start site). These include one upstream of the kappa B site and one similar to the c-fos serum response element (SRE), which is downstream of the kappa B site. Mutation of the kappa B site decreases IL-2R alpha promoter activity in MT-2 cells (a T-cell line that has been transformed with human T-cell lymphotropic virus type I), but not in Jurkat cells (a T-cell leukemia line) that have been activated by phorbol 12-myristate 13-acetate (PMA). In contrast, mutation of a region upstream of the kappa B site decreases activity in PMA-induced Jurkat cells but increases activity in MT-2 cells. Mutation of the SRE-like site decreases activity in both cell types but the effect in PMA-induced Jurkat is more pronounced. Thus, these distinct cis-acting elements play different physiological roles in IL-2R alpha gene activation in MT-2 cells and PMA-induced Jurkat T cells. These studies provide direct evidence for a functionally significant SRE-like sequence in a gene other than c-fos and the actin genes and identify other elements that are critical for IL-2R alpha gene expression.

Base Sequence↗
PubMed1996

Genomic structure and regulation of Kcn1, a cGMP-gated potassium channel.

We recently cloned a novel rabbit gene that encodes a 725-amino acid protein (Kcn1) (Y. Yao, A. S. Segal, P. Welling, X. Zhang, C. M. McNicholas, D. Engel, E. L. Boulpaep, and G. Desir. Proc. Natl. Acad. Sci. USA 92: 11711-11715, 1995). Kcn1 RNA injected in Xenopus oocytes leads to the expression of potassium channels that are specifically activated by guanosine 3',5'-cyclic monophosphate (cGMP). Northern blot and ribonuclease (RNase) protection analysis show that Kcn1 is differentially expressed in kidney, aorta, brain, and heart. The purpose of present study is to determine the structure of Kcn1 gene, analyze the promoter region, and identify cis-regulatory elements responsible for transcription. We find that the coding region of Kcn is intronless. The major transcription initiation site was identified by primer extension. Sequence analysis of the 5'-flanking region indicates that, although the gene lacks a typical TATA box, it does have a TATA-box-like region (-TAT-). Using luciferase reporter constructs transfected in the porcine kidney cell line (LLC-PK1), the promoter region and a 5' enhancer element were identified by deletion analysis. Phorbol esters (12-O-tetradecanoylphorbol-13-acetate) and forskolin stimulated Kcn1 gene expression 2.5- and 3.5-fold, respectively. In conclusion, we have identified the region of the novel potassium channel gene, Kcn1, that contains the promoter, a 5' enhancer, and several cis-regulatory elements and shown that gene transcription is stimulated by cAMP and phorbol esters.

Animals↗
PubMed1994

Kinetic and equilibrium binding studies of the human papillomavirus type-16 transcription regulatory protein E2 interacting with core enhancer elements.

The human papillomaviruses (HPVs) are a family of DNA viruses which cause benign tumours of the skin and mucosa that infrequently progress to malignant carcinoma. The E2 open reading frame of HPV is thought to encode a papillomavirus-specific transcription factor which also has a role in viral replication. The E2 proteins of all papillomaviruses studied to date have been shown to bind specifically to the common conserved sequence ACC(N)6GGT found at multiple locations in their genomes. In the case of HPV-16, a 'high risk' genital papillomavirus, the E2 protein is thought to negatively regulate expression of the major viral transforming genes E6 and E7, which have been directly implicated in the oncogenic process. However, little information exists concerning the relative or absolute affinities of the native HPV-16 protein for its palindromic recognition sequences; moreover, interpretation of any transcription or replication phenomena attributed to this protein is more complicated in the absence of such data. Here we describe the overexpression, purification and characterisation of the C-terminal 89 amino acids of the protein encompassing the DNA binding/dimerisation domain. We show that the recombinant protein purified from E.coli by a combination of non-group-specific chromatography steps retains high biological activity and is able to bind to all sites in the HPV-16 genome with high affinity (approximately 8 x 10(-11) M). In addition, kinetic studies show that the E2-DNA complexes are very stable, with half-lives ranging from 2.15 to greater than 240 min, and that nucleotides internal and external to the conserved palindrome appear to influence stability.

Base Sequence↗
PubMed1997

Differential regulation of major surface promoter in hepatitis B virus.

The major surface promoter of human hepatitis B virus can produce three distinct groups of S transcripts. The initiation sites of these transcripts are in close proximity. Encompassing the ATG for the middle surface protein, the largest S transcript (+1) encodes the middle surface protein whereas the other two (+20 and +31) can only code for small surface protein. Sequence analysis does not reveal any TATA element. In this study, we employ deletion, linker scanning, and linker insertion analyses to study systematically the sequence requirements for the initiations of all three transcripts and their upstream regulatory sequences. Our study reveals that the sequence downstream of -16 is sufficient for precise initiation of all three groups of S transcripts. The 3' boundary of minimal promoter element is +15 for the +1 transcript, whereas it is +39 for both +20 and +31 transcripts. Furthermore, there are distinct sequence requirements for the initiations of three groups of S transcripts. The sequences from -17 to -10 and from -1 to +7 are required for the initiation of +1 transcript, the sequence from +16 to +39 is essential for the +20 transcript, and the sequences from -17 to -10 and from +24 to +39 are required for the + 31 transcript. Our results also suggest that the transcription initiations of major surface promoter may be mediated in part by initiators. The initiations of these three groups of S transcripts are under differential regulation. The region from -39 to -16 containing both negative and positive regulatory elements selectively regulates the transcription levels of the two major S transcripts. Most notably, mutation of the sequence from -17 to -10, which contains a Sp1 site, leads to an increase in the imprecise initiation at +1 site and depresses the initiation of +20 and, to a greater extent, +31 transcript. The relevance of differential regulation of major surface promoter to the varied production of different surface protein isoforms in viral life cycle is discussed.

Gene Deletion↗
PubMed1993

Deletional analysis of the human renin promoter: transcriptional activation by the SV40 enhancer enables identification of promoter regulatory elements in non-renin-expressing cells.

Identification of regulatory elements in the human renin gene promoter has been hindered by the lack of suitable renin-expressing cell lines. In this paper, the SV40 viral enhancer is coupled to a human renin promoter/chloramphenicol acetyltransferase (CAT) reporter gene construct, to determine whether promoter regulatory elements can be identified in the context of enhanced transcription in the normally non-renin-expressing HeLa cell line. The present results indicate that the SV40 enhancer can overcome tissue-specificity of the human renin promoter, and confer correctly initiated transcriptional activity to HeLa cells. Analysis of a series of 5'-end deletions of the human renin promoter linked to the CAT gene and SV40 enhancer identified negative regulatory elements between positions-275 and -225, and between-142 and -102 of the human renin promoter, as well as a positive regulatory element between -225 and -142. Thus, studies of renin promoter regulatory elements need not be limited to renin-expressing cells, but can be performed in non-renin-expressing cells with the addition of an enhancer. This strategy can be generally applicable to the study of tissue-specific gene regulation in cases where there are no specific cell lines available.

Chloramphenicol O-Acetyltransferase↗
PubMed1994

Signaling by the cytoplasmic domain of hematopoietin receptors involves two distinguishable mechanisms in hepatic cells.

The receptor for granulocyte colony stimulating factor (G-CSFR) and chimeric receptors consisting of the extracellular domain of G-CSFR and the transmembrane and cytoplasmic domain of the leukemia inhibitory factor receptor, gp130, or c-mpl function as homodimeric complexes. These receptors mediate a similar stimulation of gene transcription via separate regulatory elements of acute phase plasma protein genes. To identify the receptor regions within the cytoplasmic domains necessary for transcriptional regulation, the receptors were transiently expressed in rat hepatoma cells. Each receptor form reconstituted G-CSF-induced expression of a chloramphenicol acetyltransferase gene construct containing the cytokine response element of the rat alpha 1-acid glycoprotein gene. This regulation required the presence of two conserved sequence motifs (referred to as box 1 and box 2) in the cytoplasmic domains of each receptor. With the exception of G-CSFR-MPL chimera, the receptors also mediated a similarly high stimulation via the IL-6 response element of the rat beta-fibrinogen and hemopexin genes. Regulation of the IL-6 response element required, however, in addition to boxes 1 and 2, a third sequence motif (box 3). This motif is absent in the cytoplasmic domain of c-mpl, possibly explaining its inability to activate the IL-6 response element. When cells which express receptor forms with prominent box 3 function were treated with suramin, a ligand-independent gene stimulation via the IL-6 response element was observed. The suramin effect probably involves a receptor dimerization mediated by the extracellular G-CSFR domain and by the intracellular regions that include box 3.

Acute-Phase Proteins↗