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PubMed2006

MoD Tools: regulatory motif discovery in nucleotide sequences from co-regulated or homologous genes.

Understanding the complex mechanisms regulating gene expression at the transcriptional and post-transcriptional levels is one of the greatest challenges of the post-genomic era. The MoD (MOtif Discovery) Tools web server comprises a set of tools for the discovery of novel conserved sequence and structure motifs in nucleotide sequences, motifs that in turn are good candidates for regulatory activity. The server includes the following programs: Weeder, for the discovery of conserved transcription factor binding sites (TFBSs) in nucleotide sequences from co-regulated genes; WeederH, for the discovery of conserved TFBSs and distal regulatory modules in sequences from homologous genes; RNAProfile, for the discovery of conserved secondary structure motifs in unaligned RNA sequences whose secondary structure is not known. In this way, a given gene can be compared with other co-regulated genes or with its homologs, or its mRNA can be analyzed for conserved motifs regulating its post-transcriptional fate. The web server thus provides researchers with different strategies and methods to investigate the regulation of gene expression, at both the transcriptional and post-transcriptional levels. Available at http://www.pesolelab.it/modtools/ and http://www.beacon.unimi.it/modtools/.

Binding Sites↗

PubMed1991

Enhancement of expression of exogenous genes by 2-aminopurine. Regulation at the post-transcriptional level.

In human and mouse cell lines, expression of exogenous genes was enhanced by treatment with 2-aminopurine (2-AP). Chloramphenicol acetyltransferase (CAT) and neomycin phosphotransferase activities were increased by up to 50-fold upon 2-AP treatment of cells permanently transfected with genes encoding these enzymes. Neomycin phosphotransferase activity was also increased by this treatment in cells infected with a retroviral vector carrying the neomycin phosphotransferase gene. 2-AP-mediated increase in CAT activity was observed in various cell lines which had been permanently transfected with chimeric CAT genes containing transcriptional regulatory elements of the interferon-inducible genes 6-16 and 561, SV40 early genes, mouse mammary tumor viral gene, or metallothionein II gene. The increase in the cellular CAT enzymatic activity was due to an elevated level of CAT protein. The 2-AP-mediated enhancement of CAT expression was operative at the translational level; the rate of transcription of CAT mRNA or its steady-state level was affected only marginally. The translational up-regulation by 2-AP was restricted to the genes introduced from outside; there was no gross change in the rate of synthesis of other cellular proteins.

2-Aminopurine↗

PubMed1993

In vivo expression of the Bacillus subtilis spoVE gene.

In vivo expression of the Bacillus subtilis spoVE gene was studied by S1 nuclease mapping and spoVE gene fusion analysis. Transcription of spoVE is induced at about the second hour of sporulation from two closely spaced promoters designated P1 and P2. Examination of the precise transcription initiation site by high-resolution primer extension mapping indicated that the nucleotide sequences of the -10 and -35 regions of both P1 and P2 were similar to those of promoters recognized by E sigma E. Moreover, S1 nuclease mapping and translational spoVE-lacZ fusion studies with various spo mutants suggest that the expression of spoVE P2 requires the spoIIG gene product, sigma E. The sporulation of a wild-type strain was inhibited severely in the presence of a multicopy plasmid, pKBVE, carrying the spoVE promoter, indicating the possible titration of a transcriptional regulatory element(s).

Amino Acid Sequence↗

PubMed1986

A specific DNA sequence controls termination of transcription in the gastrin gene.

We located and characterized a downstream transcriptional regulatory element in the human gastrin gene by transferring the gastrin gene 3' fragment, from which the polyadenylation signal sequence was deleted, into the shuttle vector pSCAT10 at a site located immediately downstream from the chloramphenicol acetyltransferase (CAT) gene and upstream from the simian virus 40 polyadenylation region. Study of CAT RNA derived from the hybrid plasmids, indicated regulation of transcription on the gastrin gene fragment. Analysis of deletion mutants generated from the 5' region of the fragment by CAT assay and by S1 nuclease mapping of mRNAs indicated the possible involvement of an oligothymidylate-rich sequence in transcription regulation. Mapping of gastrin gene RNA 3' ends to the 5' side proximal to the oligothymidylate-rich sequence clearly demonstrated that this sequence is a transcriptional terminator element. This unique sequence, interspersed with one or two adenines, which also functions in an orientation-dependent manner, is located 192 nucleotides downstream from the gastrin gene polyadenylation site, and serves as a transcriptional termination signal.

Acetyltransferases↗

PubMed2003

Impact of infection with recombinant adenovirus carrying melittin gene on CD54 expression in HepG2 cells.

OBJECTIVE: To observe the effects of melittin gene transfection on the biological behavior of hepatocarcinoma cells. METHODS: Melittin gene was recombined into a shuttle plasmid under the control by alpha-fetoprotein (AFP) transcription regulatory element (rAFP), and the gene of interest was then recombined into an adenoviral backbone plasmid through a simplified efficient bacterial homologous recombination system. The recombinant adenoviral plasmids were subsequently linearized with Pac I and transfected into 293 cells mediated by lipofectin to generate the desired recombinant adenoviruses. After infections of the hepatocarcinoma cells with the resultant viruses carrying melittin gene was achieved, melittin gene transcription was verified by way of RT-PCR, and CD54 expression was measured by flow cytometry. RESULTS: Recombinant adenoviral vectors containing melittin gene were successfully constructed, and melittin gene transcription was approved by RT-PCR. Flow cytometry results showed that melittin gene transfection inhibited CD54 expression in the hepatocarcinoma cells. CONCLUSION: Melittin gene transfection may result in changes of the biological behavior of hepatocarcinoma cells, thus decreasing the malignancy of the tumor cells.

Adenoviridae↗

PubMed2001

The hcKrox gene family regulates multiple extracellular matrix genes.

The transcription factor cKrox was originally identified as a protein that bound to a negative transcription regulatory element in the murine alpha1(I) collagen promoter. We recently reported the cloning and characterization of human cKrox (hcKrox). Overexpression of hcKrox in NIH3T3 fibroblasts efficiently repressed the promoters of the fibronectin and alpha1(I) collagen genes (70-90%) in transient transfection assays and suppressed the endogenous genes in hcKrox expressing permanent cell lines. We have now isolated genomic clones and cDNAs encoding two novel transcription factors related to hcKrox termed hcKrox-beta and hcKrox-gamma (the original clone is now referred to as hcKrox-alpha). Both contain three kruppel-like zinc-finger DNA binding motifs that are 71-78% identical to those of hcKrox-alpha. The NH(2)-terminus of all three proteins contains a POZ domain, a conserved 120 amino acid motif involved in transcriptional repression and protein dimerization. RT-PCR experiments demonstrate that all three hcKrox family members are expressed in foreskin and dermal fibroblasts. Transient transfection studies in NIH3T3 fibroblasts demonstrate that hcKrox-alpha -beta and -gamma, as well as the murine cKrox-beta homologue, LRF, suppress transcription driven by promoters for the alpha1(I) and alpha2(I) collagen, fibronectin and elastin genes. Electrophoretic mobility shift assays and coimmunoprecipitation studies suggest that homo- and heterodimerization occurs between cKrox family members. Dimer formation is influenced by amino acids in the NH(2)-terminal POZ domain and the Zn(+2)-finger region. Immunoprecipitation studies indicate that cKrox can form heterodimers in solution in the absence of DNA. Thus, a multi-gene family exists that can coordinately regulate several extracellular matrix genes and has the potential to form many heterodimeric transcription factors.

3T3 Cells↗

PubMed2001

Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy.

Gene transfer with adenoviral vectors is an attractive approach for the treatment of atherosclerosis and restenosis. However, because expression of a therapeutic gene in nontarget tissues may have deleterious effects, artery-specific expression is desirable. Although expression vectors containing transcriptional regulatory elements of genes expressed solely in smooth muscle cells (SMCs) have proved efficient to restrict expression of the transgene, their use in the clinical setting can be limited by their reduced strength. In the present study, we show that low levels of transgene expression are obtained with the smooth muscle (SM)-specific SM22alpha promoter compared with the viral cytomegalovirus (CMV) enhancer/promoter. We have generated chimeric transcriptional cassettes containing either a SM (SM-myosin heavy chain) or a skeletal muscle (creatine kinase) enhancer combined with the SM22alpha promoter. With both constructs we observed significantly stronger expression that remains SM-specific. In vivo, reporter gene expression was restricted to arterial SMCs with no detectable signal at remote sites. Moreover, when interferon-gamma expression was driven by one of these two chimeras, SMC growth was inhibited as efficiently as with the CMV promoter. Finally, we demonstrate that neointima formation in the rat carotid balloon injury model was reduced to the same extent by adenoviral gene transfer of interferon-gamma driven either by the SM-myosin heavy chain enhancer/SM22alpha promoter or the CMV promoter. These results indicate that such vectors can be useful for the treatment of hyperproliferative vascular disorders.

Adenoviridae↗

PubMed1989

The gypsy retrotransposon of Drosophila melanogaster: mechanisms of mutagenesis and interaction with the suppressor of Hairy-wing locus.

We have used the yellow gene of Drosophila melanogaster as a model system in which to study the molecular mechanisms by which the gypsy retrotransposon causes mutant phenotypes that can be reversed by nonallelic mutations at the suppressor of Hairy-wing locus. This gene encodes a 109,000 dalton protein that contains an acidic domain and 12 copies of the Zn finger motif, which are characteristic of some transcription factors and DNA binding proteins. The suppressible y2 allele is caused by the insertion of the gypsy element at -700 bp from the start of transcription of the yellow gene, resulting in a phenotype characterized by mouth parts and denticle belts in the larvae, and by bristles in the adults, that show wildtype coloration, but mutant wings and body cuticle in the adult flies. This phenotype is the result of the interaction of gypsy sequences homologous to mammalian enhancers with tissue-specific yellow transcriptional regulatory elements located upstream from the gypsy insertion site and responsible for the expression of the yellow gene in the mutated tissues. This interaction is dependent on the binding of the su(Hw) protein to the specific gypsy sequences involved in the induction of the mutant phenotype.

Amino Acid Sequence↗

PubMed1997

Identification of the transcription termination site of the mouse nkx-1.2 gene: involvement of sequence-specific factors.

We have identified a transcription termination site in the 3' flanking region of the mouse nkx-1.2 gene. A downstream transcription regulatory element in the mouse nkx-1.2 gene was characterized by transferring its 3'-fragment into a chloramphenicol acetyl transferase (CAT) expression vector. Analysis of recombinant plasmids transfected into mouse NIH3T3 cells by CAT assay showed the possible region of regulation. There were two direct repeat structures containing poly(dG-dT) x poly(dC-dA) sequences (GT repeats) in this region. The precise location of transcription termination was mapped by nuclease S1 analysis of the transcripts from recombinant plasmids transfected into COSM6 cells. It was approximately 20 nucleotides upstream of the first GT repeat within the 5' sequences of the first element of the two direct repeats. Gel mobility shift assay and footprinting analysis demonstrated that nuclear DNA binding proteins bound specifically to the sequences where the termination occurred as well as the other sequences in the second element of the direct repeats. Southwestern analysis showed that 90-, 54-, 36- and 15-kDa nuclear proteins bound to the region of the termination. It is possible that one or more of those proteins are involved in blocking the elongation of the mouse nkx-1.2 gene transcript and then result in termination.

3T3 Cells↗

PubMed2005

Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors.

Although adeno-associated virus (AAV)-mediated gene therapy has been hindered by the small viral packaging capacity of the vector, trans-splicing AAV vectors are able to package twice the size of the vector genome. Unfortunately, the efficiency of current trans-splicing vectors is very low. Here we show that rational design of the gene splitting site has a profound influence on trans-splicing vector-mediated gene expression. Using mRNA accumulation as a guide, we generated a set of efficient trans-splicing vectors and achieved widespread expression of the 6-kb DeltaH2-R19 mini-dystrophin gene in skeletal muscle of mdx mice, a model for Duchenne muscular dystrophy. The dystrophic phenotype was ameliorated in both adult and aged mice. This demonstrates the use of trans-splicing vectors to efficiently express a large therapeutic structural protein. This strategy should be applicable to other large therapeutic genes or large transcription regulatory elements.

Animals↗

PubMed1994

Structure, genomic organization, and expression of the human interleukin-8 receptor B gene.

Two distinct receptors for the chemoattractant interleukin-8 (designated IL-8RA and -B) have been cloned recently. The receptors are expressed almost exclusively on neutrophils and myelomonocytic cell lines. In an attempt to understand the tissue-specific expression and to identify transcriptional regulatory elements we have cloned, sequenced, and characterized the human IL-8RB gene. The gene consists of 3 exons, interrupted by two introns of 3 and 5.4 kilobases (kb). A 1065-base pair open reading frame is encoded entirely in the third exon. A 1.4-kb 3'-untranslated region contains clustered AU-rich elements, similar to those described for genes regulated by altering mRNA stability. The start site of transcription was mapped by a modified rapid amplification of cDNA ends technique and revealed an unexpectedly long 5'-untranslated region of 423 base pairs. A TATA box equivalent was found in the 5'-flanking region 20 nucleotides upstream of the start of the first exon. The promoter was separated from the ATG-initiation codon by 8.75 kb. Comparison of the IL-8RB promoter with the promoter region of the receptor for another chemoattractant ligand, the bacterial peptide f-Met-Leu-Phe, revealed 3 novel but conserved motifs occupying similar positions. The immediate 5'-flanking region was GC-rich with 3 SP-1-like and 2 AP-2 sites identified in close proximity to the transcription start site. This essential promoter region was found to be responsible for constitutive expression, inducible by granulocyte colony-stimulating factor and controlled by silencer elements located further upstream between positions -779 and -118.

Amino Acid Sequence↗

PubMed1998

Cloning of the promoter region of human endoglin, the target gene for hereditary hemorrhagic telangiectasia type 1.

Endoglin (CD105) is a cell surface component of the transforming growth factor-beta (TGF-beta) receptor complex highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for the hereditary hemorrhagic telangiectasia type 1 (HHT1), also known as Osler-Weber-Rendu syndrome (OMIM 187300). This is an autosomal dominant vascular disorder probably caused by a haploinsufficiency mechanism displaying low levels of the normal protein. To understand the mechanisms underlying the regulated expression of endoglin, a genomic DNA clone containing 3.3 kb of the 5'-flanking sequence of the human endoglin gene has been isolated. The 5'-flanking region of the endoglin gene lacks consensus TATA and CAAT boxes, but contains two GC-rich regions and consensus motifs for Sp1, ets, GATA, AP-2, NFkappaB, and Mad, as well as TGF-beta-, glucocorticoid-, vitamin D-, and estrogen-responsive elements. As determined by primer extension and 5' RACE experiments, a cluster of transcriptional start sites was found to be located 350 bp upstream from the translation initiation codon. To analyze the endoglin promoter activity, the upstream -400/+341 fragment was fused to the luciferase gene and transient transfections were conducted in several cell types. This construct displayed a tissue-specific activity in human and bovine endothelial cells. Analysis of various deletion constructs showed the existence of a basal promoter region within the -81/+350 fragment as well as major transcriptional regulatory elements within the -400/-141 fragment. Electrophoretic mobility shift assays demonstrated the specific interaction of a member of the ets family with a consensus motif located at position -68. A promoter construct mutated at this ets sequence showed a much reduced activity as compared with the wild-type construct, supporting the involvement of this ets motif in the basal activity of the promoter. The endoglin promoter exhibited inducibility in the presence of TGF-beta1, suggesting possible therapeutic treatments in HHT1 patients, in which the expression level of the normal endoglin allele might not reach the threshold required for its function. Isolation and characterization of the human endoglin promoter represents an initial step in elucidating the controlled expression of the endoglin gene.

Amino Acid Sequence↗

PubMed2000

Interactions between regulatory proteins that bind to the nicotinic receptor beta4 subunit gene promoter.

The genes encoding the alpha3, alpha5 and beta4 subunits of nicotinic acetylcholine receptors are tightly clustered within the genome. As these three subunits constitute the predominant acetylcholine receptor subtype expressed in the peripheral nervous system, their genomic proximity suggests a regulatory mechanism ensuring their coordinate expression. We previously identified two transcriptional regulatory elements within the beta4 promoter. One of these elements, a CT box, interacts with the regulatory factors heterogeneous nuclear ribonucleoprotein K and Puralpha. Another element, a CA box, interacts with Sp1 and Sp3. The binding site for a fifth factor, Sox10, overlaps the CT and CA boxes. As the CT and CA boxes are adjacent, we postulated that the proteins that bind to the elements interact. Here we report that the CT box-binding factors interact with each other as do the CA box-binding factors. However, there are no direct associations between the two pairs of proteins. Interestingly though, Sox10 directly interacts with all four proteins, suggesting a central role in beta4 gene expression for this member of the Sox family of regulatory factors.

Cyclic AMP Response Element-Binding Protein↗

PubMed1996

Transcriptional regulation of neuronal nicotinic acetylcholine receptor genes. Functional interactions between Sp1 and the rat beta4 subunit gene promoter.

To date, 11 members (alpha2-alpha9 and beta2-beta4) of the neuronal nicotinic acetylcholine receptor gene family have been identified. These genes encode subunits that form distinct receptors with different pharmacological and physiological profiles in temporally and spatially restricted patterns within the nervous system. Distinct molecular mechanisms probably orchestrate the expression of various receptor subtypes, yet little is known of specific transcriptional regulatory elements and their associated factors that are responsible for this segregated pattern of expression. Here we report the identification of an element, in the 5'-flanking region of the rat beta4 subunit gene, containing a CA box that is necessary for beta4 promoter activity in a transiently transfected cholinergic cell line, SN17. This element was shown to interact with a protein(s) in SN17 nuclear extracts that is antigenically related to the transcriptional activator Sp1. Furthermore, co-transfection experiments confirmed that Sp1 can transactivate a beta4 promoter-reporter gene construct, indicating that Sp1 is necessary, at least in part, for transcriptional activation of the beta4 subunit gene.

Animals↗

PubMed1998

Insertion of the IgH locus 3' regulatory palindrome in expression vectors warrants sure and efficient expression in stable B cell transfectants.

We have explored the effect of inserting 3' immunoglobulin heavy chain (IgH) locus transcriptional regulatory elements in stable expression vectors driven by a heavy chain variable gene promoter (pVH). A cassette was constructed, associating three enhancer elements from the palindromic part of the 3' IgH regulatory region, namely Calpha3'/hs3 reverse, alpha3'E/hs1-2, and hs3. As regard to stable expression, this cassette carried some features of a locus control region (LCR) and conferred expression to an associated cat reporter gene in the majority of B cells having integrated the transgene. The palindromic cassette was inserted in an expression vector carrying Ig light chain coding sequences. In this construct, transcription driven by a pVH promoter/Emu cassette upstream of the transcription initiation site was boosted by the palindromic cassette located downstream of the coding sequence. This potent expression plasmid mimicking the architecture of endogenous Ig loci, definitely manifested a potent stimulatory activity for stable transcription, outscoring conventional ubiquitous or B-cell specific expression vectors.

Animals↗

PubMed2003

Optimization of human erythropoietin secretion from MLV-infected human primary fibroblasts used for encapsulated cell therapy.

BACKGROUND: The transplantation of encapsulated cells genetically engineered to secrete human erythropoietin (hEpo) represents an alternative to repeated injections of the recombinant hormone for the treatment of Epo-responsive anemia. In the present study, the ability of primary human foreskin fibroblasts to secrete high levels of hEpo and the importance of cis-acting elements and infection conditions on transgene expression level were assessed. METHODS: The transduction efficiency was first evaluated with beta-galactosidase (LacZ)-encoding retroviral vectors derived from the murine leukemia retrovirus (MLV) pseudotyped either with an amphotropic envelope or with the G glycoprotein of vesicular stomatitis virus (VSV-G). Human fibroblasts were then infected with an amphotropic hEpo-expressing retroviral vector, which was modified by insertion of a post-transcriptional regulatory element from the woodchuck hepatitis virus (WPRE) and a Kozak consensus sequence (KZ). Human Epo production was further optimized by increasing the multiplicity of infection and by selecting high producer cells. The survival and the transgene expression of these fibroblasts were finally evaluated in vivo. The cells were encapsulated into microporous hollow fibers and subcutaneously implanted in nude mice. RESULTS: A secretion level of approximately 5 IU hEpo/10(6) cells/day was obtained with the basal vector. A 7.5-fold increase in transgene expression was observed with the insertion of WPRE and KZ elements. Finally, according to the optimization of infection conditions, we obtained a 40-fold increase in hEpo secretion, reaching approximately 200 IU hEpo/10(6) cells/day. The in vivo experiments showed an increase in the hematocrit during the first 2 weeks and elevated levels exceeding 60% were maintained over a 6-week period. CONCLUSIONS: These results indicate that primary human fibroblasts represent a promising source for encapsulated cell therapy.

Animals↗

PubMed1991

Testis-specific expression of the human MYCL2 gene.

We have characterized the expression of MYCL2, an intronless X-linked gene related to MYCL1. RNase protection analysis of a panel of human normal and tumor tissues has revealed that MYCL2 is expressed almost exclusively in human adult normal testis; much lower levels of transcript were detected in one human lung adenocarcinoma. No MYCL2 transcript was found in human testis RNA obtained from second trimester fetuses. This observation suggests a germ cell rather than somatic cell origin of the transcript and possible developmental regulation of MYCL2. Northern blot analysis of poly(A)+ RNA from adult human normal testis with an antisense riboprobe revealed a transcript of approximately 4.8-kb, which is in agreement with the size predicted from the MYCL2 nucleotide sequence. Antisense transcripts were found spanning regions of MYCL2 corresponding to all three exons of MYCL1. No sizable open reading frame was seen for the MYCL2 antisense transcripts suggesting that they may represent either regulatory sequences or an intron of a gene encoded by the complementary strand. RNase protection assays and the 5' RACE protocol (Rapid Amplification of cDNA Ends) were used to address the localization of the transcription start site of the MYCL2 sense transcript and different putative promoters and transcription regulatory elements have been identified.

Amino Acid Sequence↗

PubMed2003

Biallelic transcriptional activation of oncogenic transcription factors in T-cell acute lymphoblastic leukemia.

Aberrant expression of transcription factor oncogenes such as HOX11, HOX11L2, TAL1/SCL, LYL1, LMO1, and LMO2 can be detected in lymphoblasts from up to 80% of patients with acute T-cell lymphoblastic leukemia (T-ALL). Transcriptional activation of these oncogenes in leukemic cells typically results from chromosomal rearrangements that place them next to highly active cis-acting transcriptional regulatory elements. However, biallelic activation of TAL1 in some T-ALL cases has been previously proposed. We have used allele-specific mRNA analysis to show that trans-acting mechanisms leading to biallelic overexpression of TAL1 are involved in 10 (42%) of 24 TAL1+ informative T-ALL cases, 2 (17%) of 12 HOX11+ informative cases, and 7 (64%) of 11 LMO2+ informative cases. We propose that aberrant expression of oncogenic transcription factors in a significant fraction of T-ALLs may result from loss of the upstream transcriptional mechanisms that normally down-regulate the expression of these oncogenes during T-cell development.

Adaptor Proteins, Signal Transducing↗