Levels Of Reporter Gene Activity Research Articles

Combinatorial interaction between two human serotonin transporter gene variable number tandem repeats and their regulation by CTCF

Two distinct variable number tandem repeats (VNTRs) within the human serotonin transporter gene (SLC6A4) have been implicated as predisposing factors for CNS disorders. The linked polymorphic region in the 5′-promoter exists as short (s) and long (l) alleles of a 22 or 23 bp elements. The second within intron 2 (Stin2) exists as three variants containing 9, 10 or 12 copies of a 16 or 17 bp element. These VNTRs, individually or in combination, supported differential reporter gene expression in rat neonate prefrontal cortical cultures. The level of reporter gene activity from the dual VNTR constructs indicated combinatorial action between the two domains. Chromatin immunoprecipitation demonstrated that both these VNTR domains can bind the CCCTC-binding factor and this correlated with the ability of exogenously supplied CCCTC-binding factor to modulate the expression supported by these reporter gene constructs. We suggest that the potential for interaction between multiple polymorphic domains should be incorporated into genetic association studies.J. Neurochem. (2010) 112, 296–306.

Epigenetic Control of C/EBPa by Distant Synergic Regulatory Elements.

Abstract 1470Poster Board I-493The transcription factor C/EBPa plays a pivotal role in hematopoietic stem cell (HSC) commitment and differentiation. Expression of the C/EBPa gene is tightly regulated during normal hematopoietic development, and dysregulation of C/EBPa expression can lead to lung cancer and leukemia. However, little is known about how the C/EBPa gene is regulated in vivo.In this study, we demonstrate synergetic regulation of C/EBPa by two distant cis-elemets located 5' and 3' to the gene and their effect on chromatin architecture.Previous studies have indicated that as much as 4.8 kb of 5' upstream C/EBPa regulatory sequences were unable to express significant levels of reporter gene activity in transgenic mice. Therefore, we initiated a search for important distal elements in the C/EBPa locus. We have applied a combination of 1) comparative analysis of human and mouse genomic sequences; 2) DNase I hypersensitive studies; 3) chromosome conformation capture (3C); 4) analysis of reporter constructs in stable cells lines; and 5) generation and analysis of transgenic mouse lines. This let us to identify the regulatory role of two distal conserved homology elements located at ∼38 kb 5' of the transcription start site (TSS) of murine C/EBPa (corresponding to ∼45 kb 5' of the TSS of human C/EBPa) and at ∼33 kb 3' to TSS of both murine and human C/EBPa. We show that the constructs lacking both distal elements were unable to express C/EBPa mRNA, while addition of each region resulted in detectable (by Northern blot analysis) expression in transgenic animals.We have observed a cooperative effect of these two regions on C/EBPa expression, a construct carrying both elements expresses ∼2.5-fold level over constructs carrying either one element alone.We have investigated the mechanism for the increased expression by these distal elements by using deletion constructs. Our results suggest that lack of these elements results in aberrant gene expression due to proximal promoter DNA hypermethylation and point to a novel mechanism in establishment of critical epigenetic marks. Disclosures:No relevant conflicts of interest to declare.

Function of Spodoptera exigua nucleopolyhedrovirus late gene expression factors in the insect cell line SF-21

We used a well established transient expression assay to test the ability of the baculovirus Spodoptera exigua M nucleopolyhedrovirus (Se MNPV) homologs of Autographa californica MNPV (Ac MNPV) late expression factors ( lefs) to activate a late promoter–reporter gene cassette in SF-21 cells. This insect-derived cell line is fully permissive for Ac MNPV infection but not for Se MNPV. In the assay, 19 Ac MNPV lefs stimulate optimal levels of late gene promoter activity. Se MNPV lef-5 successfully replaced the corresponding Ac MNPV gene in the context of the remaining set of Ac MNPV lefs, whereas Se MNPV dnapol and 39k exhibited partial activity. When all the Se MNPV lefs were assayed together or in the presence of four lefs encoded only in Ac MNPV, it resulted in background levels of late promoter-driven reporter gene activity. However, Se MNPV genomic DNA and the four Ac MNPV-specific lefs stimulated low levels of reporter gene activity. Moreover, Se MNPV IE-1, but not Ac MNPV IE-1, further stimulated late gene expression in the presence of Se MNPV DNA. Ac MNPV IE-1 was able to mediate early gene expression cis-linked to homologous regions ( hrs) derived from Ac MNPV and Se MNPV. In contrast, Se MNPV IE-1 was more specific for Se MNPV-derived hr elements.

Functional Characterization of SDF-1 Proximal Promoter

Stromal-cell derived factor 1 (SDF1) is a CXC chemokine that binds and signals through the CXCR4 receptor, playing an essential role in embryonic B lymphopoiesis, myelopoiesis and organogenesis. The CXCR4/SDF1 pathway is associated with several pathologies. CXCR4 serves as a fusion cofactor for lymphotropic strains of human immunodeficiency virus type 1 and SDF1 inhibits viral entry. Moreover, recent works suggest an important role for SDF1 in metastasis progression and autoimmune diseases such as rheumatoid arthritis. To understand the molecular mechanisms that regulate SDF1 expression, we have cloned and functionally analysed its 5' flanking regulatory region. An SDF1-promoter luciferase construct showed high levels of reporter gene activity in transient transfection experiments. DNase I footprinting analysis revealed that the proximal promoter was occupied by six putative Sp1-binding motifs. Binding of Sp1 to the promoter was confirmed by electrophoretic mobility shift assay, and its importance in SDF1 gene expression verified by in vitro mutagenesis. Particularly, mutation of an Sp1 motif located between -57 and -39 upstream of the main transcription start-site resulted in a marked reduction in promoter activity. It has been shown that the SDF1 expression could be induced by mitogenic stimuli, X-ray radiation or treatment with IL1beta, depending on cell environment. We have analysed the effect of these stimuli on SDF1 promoter transactivation in three different cell lines. Phorbol myristated acetate plus ionomycin increased promoter activity in U373 and LC5 but repressed it in MS5 cells. On the contrary, gamma irradiation promoted SDF1 transcription in MS5 cells but not in the other cell lines. Interferon-gamma acted as a transcriptional repressor in U373 and LC5 but not in MS5 cells. Finally, IL1beta functions as mild activator only in U373 cells. The present study demonstrates that these stimuli mediate SDF1 production through promoter activation in a cell-specific manner.

Improved ecdysone receptor-based inducible gene regulation system.

To develop an ecdysone receptor (EcR)-based inducible gene regulation system, several constructs were prepared by fusing DEF domains of Choristoneura fumiferana EcR (CfEcR), C. fumiferana ultraspiracle (CfUSP), Mus musculus retinoid X receptor (MmRXR) to either GAL4 DNA binding domain (DBD) or VP16 activation domain. These constructs were tested in mammalian cells to evaluate their ability to transactivate luciferase gene placed under the control of GAL4 response elements and synthetic TATAA promoter. A two-hybrid format switch, where GAL4 DBD was fused to CfEcR (DEF) and VP16 AD was fused to MmRXR (EF) was found to be the best combination. It had the lowest background levels of reporter gene activity in the absence of a ligand and the highest level of reporter gene activity in the presence of a ligand. Both induction and turn-off responses were fast. A 16-fold induction was observed within 3 h of ligand addition and increased to 8942-fold by 48 h after the addition of ligand. Withdrawal of the ligand resulted in 50% and 80% reduction in reporter gene activity by 12 h and 24 h, respectively.

Nucleotide Variation Regulates the Level of Enhancement by Hypersensitive Site 2 of the β-Globin Locus Control Region

ABSTRACTThe β-globin locus control region hypersensitive site 2 (HS2) enhancer possesses a unique property for stimulating high-level globin gene expression. Although the deletion of cis-acting motifs influences the level of enhancement conferred by HS2, there is controversy on whether polymorphism of the same elements contributes to variation of the fetal hemoglobin (HbF) level among patients with sickle cell anemia. We analyzed reporter gene activity of constructs containing variant HS2 enhancers derived from βS chromosomes to directly test the effect of polymorphism on enhancer activity. Constructs containing four enhancer variants linked to an identical γ-globin promoter showed markedly different levels of reporter gene activity. Juxtaposition of HS2 derived from the Asian and Senegal chromosomes, which are associated with similarly high levels of HbF, to cognate sequence extending to −1500 of the Gγ globin gene showed significantly different levels of reporter gene activity. Our findings indicate that nucleotide variation regulates the level of enhancement conferred by HS2; however, the reporter activities showed no correlation with the level of Hb F associated with the common βS chromosomes.

Structural and Functional Characterizations of the 5′-Flanking Region of the Mouse Glucagon Receptor Gene: Comparison with the Rat Gene

A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5′-rapid amplification of cDNA ends from mouse liver tissue. The 5′-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5′ untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5′ region of the rat GR gene. The mouse distal promoter region, extending up to −1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5′-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.

Selective Binding of Steroid Hormone Receptors to Octamer Transcription Factors Determines Transcriptional Synergism at the Mouse Mammary Tumor Virus Promoter

Transcriptional synergism between glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1 and Oct-2) in the induction of mouse mammary tumor virus (MMTV) transcription has been proposed to be mediated through directed recruitment of the octamer factors to their binding sites in the viral long terminal repeat. This recruitment correlates with direct binding between the GR DNA binding domain and the POU domain of the octamer factors. In present study, in vitro experiments identified several nuclear hormone receptors to have the potential to bind to the POU domains of Oct-1 and Oct-2 through their DNA binding domains, suggesting that POU domain binding may be a property shared by many nuclear hormone receptors. However, physiologically relevant binding to the POU domain appeared to be a property restricted to only a few nuclear receptors as only GR, progesterone receptor (PR), and androgen receptor (AR), were found to interact physically and functionally with Oct-1 and Oct-2 in transfected cells. Thus GR, PR, and AR efficiently promoted the recruitment of Oct-2 to adjacent octamer motifs in the cell, whereas mineralocorticoid receptor (MR), estrogen receptor alpha, and retinoid X receptor failed to facilitate octamer factor DNA binding. For MMTV, although GR and MR both induced transcription efficiently, mutation of the promoter proximal octamer motifs strongly decreased GR-induced transcription without affecting the total level of reporter gene activity in response to MR. These results suggest that the configuration of the hormone response element within the MMTV long terminal repeat may promote a dependence for the glucocorticoid response upon the recruitment of octamer transcription factors to their response elements within the viral promoter.

Switch from antagonist to agonist of the androgen receptor bicalutamide is associated with prostate tumour progression in a new model system.

Advanced prostate cancer is treated by androgen ablation and/or androgen receptor (AR) antagonists. In order to investigate the mechanisms relevant to the development of therapy-resistant tumours, we established a new tumour model which closely resembles the situation in patients who receive androgen ablation therapy. Androgen-sensitive LNCaP cells were kept in androgen-depleted medium for 87 passages. The new LNCaP cell subline established in this manner, LNCaP-abl, displayed a hypersensitive biphasic proliferative response to androgen until passage 75. Maximal proliferation of LNCaP-abl cells was achieved at 0.001 nM of the synthetic androgen methyltrienolone (R1881), whereas 0.01 nM of this compound induced the same effect in parental cells. At later passages (> 75), androgen exerted an inhibitory effect on growth of LNCaP-abl cells. The non-steroidal anti-androgen bicalutamide stimulated proliferation of LNCaP-abl cells. AR protein expression in LNCaP-abl cells increased approximately fourfold. The basal AR transcriptional activity was 30-fold higher in LNCaP-abl than in LNCaP cells. R1881 stimulated reporter gene activity in LNCaP-abl cells even at 0.01 nM, whereas 0.1 nM of R1881 was needed for induction of the same level of reporter gene activity in LNCaP cells. Bicalutamide that acts as a pure antagonist in parental LNCaP cells showed agonistic effects on AR transactivation activity in LNCaP-abl cells and was not able to block the effects of androgen in these cells. The non-steroidal AR blocker hydroxyflutamide exerted stimulatory effects on AR activity in both LNCaP and LNCaP-abl cells; however, the induction of reporter gene activity by hydroxyflutamide was 2.4- to 4-fold higher in the LNCaP-abl subline. The changes in AR activity were associated neither with a new alteration in AR cDNA sequence nor with amplification of the AR gene. Growth of LNCaP-abl xenografts in nude mice was stimulated by bicalutamide and repressed by testosterone. In conclusion, our results show for the first time that the non-steroidal anti-androgen bicalutamide acquires agonistic properties during long-term androgen ablation. These findings may have repercussions on the natural course of prostate cancer with androgen deprivation and on strategies of therapeutic intervention. © 1999 Cancer Research Campaign

Structure and hormone responsiveness of the gene encoding the alpha-subunit of the rat amiloride-sensitive epithelial sodium channel.

The rat amiloride-sensitive epithelial sodium channel (rENaC) is the rate-limiting step for vectorial transport of Na+ across tight epithelia. The complex is composed of three subunits, alpha, beta, and gamma. Expression of the subunits has been shown to be tissue-specific and developmentally and hormonally regulated. To study mechanisms involved in transcriptional regulation of alpharENaC, we determined the genomic organization of the alpharENaC gene. By 5' rapid amplification of cDNA ends and primer extension, two transcriptional start sites were detected 453 base pairs (bp) apart, resulting in alternative 5' untranslated region (UTR) lengths of 515 or 62 bp. The longer 5' UTR is more prevalent in fetal lung than in adult lung or kidney. The 5' untranslated and coding regions are contained within 12 exons, with the translation start site located within the first exon. Sequence analysis of approximately 1,500 bp of 5' flanking DNA identified putative binding sites for transcription factors PEA3, SP1, AP-1, nuclear factor-kappaB, and thyroid and glucocorticoid receptors. alpharENaC promoter-reporter gene constructs produced low levels of reporter gene activity in transiently transfected cells, which could be increased by dexamethasone (DEX) treatment. Tri-iodothyronine treatment alone had no effect but potentiated stimulation by DEX.

Isolation and Characterization of a Genomic Region Upstream from the Ovine Na+/d-Glucose Cotransporter (SGLT1) cDNA

d-Glucose and non-metabolisable analogues of D-glucose regulate the expression of intestinal SGLT1 at both transcriptional and post-transcriptional levels. In order to investigate the molecular mechanisms involved in the transcriptional regulation of the ovine intestinal SGLT1 gene, we have isolated an upstream element of about 1 kb in size. This DNA fragment contains a TATA box motif, 48 bp upstream of the transcriptional start site and includes transcription factor binding sites for HNF-1 and AP-2. We have shown that the ovine SGLT1 promoter fragment can drive the transcription of a reporter gene when transfected into the epithelial cell lines STC-1 and LLC-PK1, which endogenously express SGLT1. Deletion analyses of the promoter indicate that −66/+21 bp proximal sequence directs the highest level of reporter gene activity. There are one and possibly two sites of transcriptional suppression.

The varicella-zoster virus (VZV) open-reading frame 29 protein acts as a modulator of a late VZV gene promoter.

Transient expression assays have shown that the varicella-zoster virus (VZV) open-reading frame (ORF) 29 gene product can act as a modulator of VZV gene expression. The ORF 29 protein alone does not appear to have any effect on transcription; however, in its presence, changes in the level of reporter gene activity mediated by the VZV immediate early (IE) 62 major transactivator are seen. Increased expression was observed in human fibroblasts, MeWo cells, HeLa cells, and T cells. In contrast, the presence of the ORF 29 protein results in a down-regulation of IE62 activation in PC-12 rat neuronal cells. Competition filter binding assays indicate that the ORF 29 protein binds specifically to the glycoprotein I promoter. Since transcripts for ORF 29 and ORF 62 have been detected in latently infected ganglia, the gene regulatory properties of the ORF 29 protein may be relevant to maintenance or establishment of VZV latency.

The CArG boxes in the promoter of the Arabidopsis floral organ identity gene APETALA3 mediate diverse regulatory effects.

APETALA3 is a MADS box gene required for normal development of the petals and stamens in the Arabidopsis flower. Studies in yeast, mammals and plants demonstrate that MADS domain transcription factors bind with high affinity to a consensus sequence called the CArG box. The APETALA3 promoter contains three close matches to the consensus CArG box sequence. To gain insights into the APETALA3 regulatory circuitry, we have analyzed the APETALA3 promoter using AP3::uidA(GUS) fusions. 496 base pairs of APETALA3 promoter sequence 5' to the transcriptional start directs GUS activity in the same temporal and spatial expression pattern as the APETALA3 RNA and protein in wild-type flowers. A synthetic promoter consisting of three tandem repeats of a 143 base pair sequence directs reporter gene activity exclusively to petals and stamens in the flower. We have analyzed the role of the CArG boxes by site-specific mutagenesis and find that the three CArG boxes mediate discrete regulatory effects. Mutations in CArG1 result in a decrease in reporter expression suggesting that CArG1 is the binding site for a positively acting factor or factors. Mutations in CArG2 result in a decrease in reporter expression in petals, but the expression pattern in stamens is unchanged. By contrast, mutations in CArG3 result in an increase in the level of reporter gene activity during early floral stages suggesting that CArG3 is the binding site for a negatively acting factor.

Human microsomal epoxide hydrolase: 5'-flanking region genetic polymorphisms.

Microsomal epoxide hydrolase (mEH) catalyses the hydrolysis of xenobiotic epoxides, including various epoxide derivatives of the procarcinogenic polyaromatic hydrocarbons. Levels of mEH enzymatic activity among different cell types and between individuals within the population vary considerably. Genetic polymorphisms within the structural region of the human mEH gene exist and appear to contribute to the population variance in functional expression. In this study, we used single strand conformational polymorphism analysis and direct DNA sequencing approaches to identify seven additional polymorphic sites within the upstream region of the mEH gene, spanning -743 to +185 bp, relative to the transcription initiation site. Allelic frequencies and linkages of the polymorphic nucleotides were determined in 51 individuals using restriction fragment length polymorphism or competitive oligonucleotide priming assays. To determine the functional significance of the individual nucleotide substitutions, DNA fragments representing the variant alleles were cloned into the heterologous pBRAMScat2 reporter vector, transfected into HepG2 cells and assessed for reporter gene expression. Results indicated that certain of these polymorphic loci might differentially regulate transcription, with the maximum contribution of any of the variants modifying levels of reporter gene activity by approximately 30%. These observations establish that genetic variation in the 5' flanking sequence of mEH gene is likely an additional contributing factor to the range of functional mEH expression existing in human populations.

The Macrosialin Promoter Directs High Levels of Transcriptional Activity in Macrophages Dependent on Combinatorial Interactions between PU.1 and c-Jun

Macrosialin is a transmembrane glycoprotein that is highly expressed in macrophages. In the present studies, macrosialin mRNA levels are shown to be markedly up-regulated during macrophage differentiation of bone marrow progenitor cells in response to macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. To investigate the mechanisms responsible for regulation of macrosialin expression, we have isolated the macrosialin gene and performed an initial analysis of its transcriptional regulatory elements. The macrosialin promoter and 7.0 kilobase pairs of 5'-flanking information direct high levels of reporter gene activity in monocyte/macrophage-like cells, but little or no expression in nonmyeloid cells. This pattern of expression is dependent on regulatory elements located between -7.0 and -2.5 kilobase pairs from the transcriptional start site that exhibit strong enhancer activity in macrophages and repressor activity in nonmyeloid cells. Analysis of the proximal macrosialin promoter indicates that combinatorial interactions between at least four classes of transcriptional activators, including PU.1/Spi-1 and members of the AP-1 family are required for basal promoter function. PU.1/Spi-1 and c-Jun act synergistically to activate the macrosialin promoter in a nonmyeloid cell line, suggesting that combinatorial interactions between these proteins are involved in regulating macrosialin expression during macrophage differentiation.

In vivo footprinting and mutational analysis of the proximal CD19 promoter reveal important roles for an SP1/Egr-1 binding site and a novel site termed the PyG box.

Abstract CD19 expression begins at the pro-B cell stage of B cell development. As such it serves as a good prototype for B cell-specific genes whose expression begins shortly after lineage commitment. To understand the molecular mechanisms controlling CD19 gene expression, we isolated and functionally characterized the CD19 promoter using in vivo footprinting, gel shift assays, and transfection studies. Reporter constructs spanning portions of the promoter identified a region between -85 and -200 that produced high levels of reporter gene activity in lymphoid cells. In vivo footprinting identified protected regions over the known high affinity B cell lineage-specific activator protein (BSAP) site, the low affinity BSAP site, a SP1/Egr-1 site termed the CD19 GC box, and two novel sites named the AT box and PyG box. Phorbol ester treatment of a pre-B cell line up-regulated CD19 expression, induced Egr-1, and enhanced the footprint over the GC box. Gel shift assays demonstrated SP1 and Egr-1 binding to the CD19 GC box, while unknown nuclear proteins bound the PyG and AT boxes. Mutations in the AT box or in the BSAP sites did not affect CD19 reporter construct activity, while a mutation of the GC box reduced it modestly, and a PyG box mutation reduced it dramatically. BSAP failed to trans-activate CD19 promoter constructs in B cells or non-B cells, suggesting that cis elements such as the PyG and GC boxes are also necessary for high level CD19 promoter expression.

A putative binding site for Sp1 is involved in transcriptional regulation of CYP17 gene expression in bovine ovary.

In the bovine ovary, thecal cells are the only cell type capable of expressing the CYP17 gene in response to LH. With the onset of ovulation and luteinization in the cow, there is complete loss of P450c17alpha expression. To characterize the molecular mechanisms involved in tissue-specific regulation of the CYP17 gene in the bovine ovary, deletion mutations of the bovine CYP17 promoter were ligated into a promoterless luciferase expression vector, and reporter constructs were transiently transfected into primary cultures of bovine thecal and luteal cells. Deletion of the promoter sequences between -191 and 101 bp dramatically decreased the levels of reporter gene activity in both thecal and luteal cells. Computer-assisted analysis revealed the presence of a putative inverted Sp1-like binding site at -188/-180 bp. Deletion or mutation of this sequence caused a decrease in both basal and forskolin-stimulated reporter gene activity. In addition, mutation or deletion of this sequence also decreased reporter gene expression induced by overexpression of the protein kinase A catalytic subunit. Electrophoretic mobility shift assays showed that this sequence binds to a nuclear protein(s) from both thecal and luteal cells that is related to Sp1, as suggested by the results of gel mobility supershift assay employing an antibody raised against Sp1. DNA-binding activity was not increased by the addition of forskolin to thecal or luteal cells. We conclude that this inverted Sp1-like binding sequence is involved in constitutive as well as cAMP-dependent expression of the CYP17 gene in the bovine ovary.

Analysis of gene expression in Streptococcus mutans in biofilms in vitro.

The purpose of this study was to develop methods for the consistent production of biofilms of S. mutans containing reporter gene fusions, and to examine the expression of genes involved in sucrose metabolism in adherent populations of this organism. Three strains of S. mutans harboring reporter gene fusions to the gene promoter regions of the gtfBC genes, ftf, and scrA were grown in a Rototorque biofilm fermenter in a tryptone-yeast extract-sucrose medium. Quasi-steady-state levels of reporter gene activity were measured after the biofilms were grown for either 48 hrs of 7 days. Also, induction of gene expression by the addition of sucrose to biofilm cells was monitored. Reporter gene activity was measurable from all gene fusion strains. This study (i) establishes the feasibility of doing detailed molecular and physiologic studies on immobilized populations of S. mutans, (ii) demonstrates that the polysaccharide synthesis machinery of S. mutans is differentially expressed in biofilms, and (iii) opens the way for a more detailed analysis of the environmental signals and signal transduction pathways governing the regulation of gene expression by S. mutans cells that are immobilized on a solid surface.

Genomic organization and transcriptional regulation of the RANTES chemokine gene.

RANTES is a member of a large supergene family of pro-inflammatory cytokines called CC chemokines that appear to play a fundamental role in inflammatory processes. The RANTES protein causes release of histamine from basophils and is a chemoattractant for CD45RO/CD4+ "memory" T lymphocytes, monocytes, and eosinophils. Although expression of RANTES was first thought to be limited to activated T cells, recent data have shown that it is produced by a variety of tissue types in response to specific stimuli. RANTES mRNA is expressed late (3 to 5 days) after activation of resting T cells whereas in fibroblasts, renal epithelial and mesangial cells, RANTES mRNA is quickly up-regulated by TNF-alpha stimulation. In order to gain a better understanding of the molecular mechanisms that regulate expression of the RANTES locus, we have characterized the RANTES gene and determined a putative promoter region. The RANTES gene spans approximately 7.1 kb and is composed of three exons of 133, 112 and 1075 bases and two introns of approximately 1.4 and 4.4 kb with the position of intron/exon boundaries conserved relative to the other CC chemokine family members. Approximately 1 kb of DNA from the immediate 5' upstream region of RANTES was sequenced and found to contain a large number of potential consensus elements for specific T cell/hemopoietic, myeloid, muscle, and ubiquitously expressed DNA-binding factors. RANTES-promoter-luciferase gene fusion assays demonstrate high levels of reporter gene activity in a "mature" T cell line Hut78, the erythroleukemic cell line HEL, and the rhabdomyosarcoma cell line RD, with little or no activity in the "early" T cell line Jurkat, the gamma delta T cell line PEER, the thymic tumor Molt4, or the pre-erythroid cell line K562. Deletion analysis of the promoter region indicates that different transcriptional mechanisms control expression of RANTES in the various tissues studied.

Cell-specific transcription of the peripherin gene in neuronal cell lines involves a cis-acting element surrounding the TATA box.

Peripherin is a neurone-specific intermediate filament protein expressed mostly in the peripheral nervous system. To localize sequences that are important for the regulation of peripherin gene transcription, we have functionally dissected its promoter. Transfection into different cell lines and deletion mapping of peripherin-lacZ hybrid constructs indicated that the first 98 bp preceding the transcription start site of the gene were sufficient to confer cell-type specific expression. DNase I footprinting experiments revealed three protected sequences in this region, that were named PER1, PER2 and PER3. The PER2 and PER3 elements, localized between -98 to -46, interact with proteins that seem widely distributed. Deletion of these elements severely decreased the level of reporter gene activity. The PER1 element, which overlaps the TATA box, interacts with a DNA-binding protein prevailing in peripherin expressing cell lines. However, the core promoter, which contains the PER1 element, was inefficient in driving gene expression. Experiments designed to test the contribution of each element showed that PER2 and PER3 were important in determining the level of expression, while PER1 was important for cell-type specificity. In fact the polyoma virus enhancer linked to the peripherin gene core promoter was found to limit reporter gene activity to peripherin expressing cell lines. Together, these experiments indicate that co-operative interactions between different regions of the promoter are necessary for efficient and cell-type specific transcription of the peripherin gene in a subset of neuronal cells.