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PubMed1996

Identification of transmembrane domain residues determinant in the structure-function relationship of the human platelet-activating factor receptor by site-directed mutagenesis.

Platelet-activating factor (PAF) is a potent phospholipid mediator that produces a wide range of biological responses. The PAF receptor is a member of the seven-transmembrane GTP-binding regulatory protein-coupled receptor superfamily. This receptor binds PAF with high affinity and couples to multiple signaling pathways, leading to physiological responses that can be inhibited by various structurally distinct PAF antagonists. We have used site-directed mutagenesis and functional expression studies to examine the role of the Phe97 and Phe98 residues located in the third transmembrane helix and Asn285 and Asp289 of the seventh transmembrane helix in ligand binding and activation of the human PAF receptor in transiently transfected COS-7 cells. The double mutant FFGG (Phe97 and Phe98 mutated into Gly residues) showed a 3-4-fold decrease in affinity for PAF, but not for the specific antagonist WEB2086, when compared with the wild-type (WT) receptor. The FFGG mutant receptor, however, displayed normal agonist activation, suggesting that these two adjacent Phe residues maintain the native PAF receptor conformation rather than interacting with the ligand. On the other hand, substitution of Ala for Asp289 increased the receptor affinity for PAF but abolished PAF-dependent inositol phosphate accumulation; it did not affect WEB2086 binding. Substitution of Asn for Asp289, however, resulted in a mutant receptor with normal binding and activation characteristics. When Asn285 was mutated to Ala, the resulting receptor was undistinguishable from the WT receptor. Surprisingly, substitution of Ile for Asn285 led to a loss of ligand binding despite normal cell surface expression levels of this mutant, as verified by flow cytometric analysis. Our data suggest that residues 285 and 289 are determinant in the structure and activation of the PAF receptor but not in direct ligand binding, as had been recently proposed in a PAF receptor molecular model.

Amino Acid Sequence↗

PubMed1997

Characterization of Skn-1a/i POU domain factors and linkage to papillomavirus gene expression.

Tissue-restricted POU domain transcription factors, which bind octamer or octamer-like gene sequences, play roles in cellular differentiation and the development of several organs. We have previously identified a POU domain gene, Skn-1a/i, expressed primarily in epidermis, that encodes at least two products through alternative splicing. One of these, Skn-1a, acts as a transcriptional activator, and the other, Skn-1i, contains an inhibitory domain in the NH2 terminus, which prevents DNA-binding in vitro and transcriptional activation in vivo. We now demonstrate that when Skn-1i is expressed in eukaryotic cells it can bind to an octamer site, suggesting that in vivo cellular factors modulate the activity of the inhibitory domain to permit DNA-binding. Yet the inhibitory domain does not allow transactivation by Skn-1i or by a heterologous transactivator containing this domain in cis. Furthermore, we demonstrate that Skn-1a, Tst-1, and Oct-1 are the major octamer-binding proteins in epidermis. Since Skn-1a is primarily expressed in suprabasal cells of the epidermis, we have tested its possible role in the regulation of epidermal papillomaviruses. In transient transfection assays, Skn-1a and Tst-1 can activate the long control region of the epidermis-specific human papillomavirus 1A (HPV-1A). Consistent with these in vivo transcription data, in vitro DNA binding studies identify three octamer-like sites, which are capable of binding Skn-1a, in the HPV-1A long control region. Mutations of all three octamer-like sites prevent transactivation by Skn-1a in transient transfection assays. Taken together, these results provide evidence that Skn-1a and Tst-1 may provide a molecular link between HPV gene expression and epidermal differentiation.

Base Sequence↗

PubMed1999

Interferon-gamma-mediated inhibition of cyclin A gene transcription is independent of individual cis-acting elements in the cyclin A promoter.

Interferons (IFNs) affect cellular functions by altering gene expression. The eukaryotic cell cycle is governed in part by the periodic transcription of cyclin genes, whose protein products associate with and positively regulate the cyclin-dependent kinases. To understand better the growth inhibitory effect of IFN-gamma on vascular smooth muscle cells (VSMCs), we compared the expression and activity of G1 and S phase cyclins in control and IFN-gamma-treated VSMCs. IFN-gamma treatment did not inhibit the G1 cyclins but did decrease cyclin A protein, mRNA, and associated kinase activity by 85, 90, and 90%, respectively. Nuclear run-on and mRNA stability determinations indicated that this decrease was the result of transcriptional inhibition. To investigate the molecular basis of this inhibition, we examined protein-DNA interactions involving the cyclin A promoter. Electromobility shift assays showed little change with IFN-gamma treatment in the binding of nuclear proteins to isolated ATF, NF-Y, and CDE elements. In vivo genomic footprinting indicated that IFN-gamma treatment changed the occupancy of chromosomal NF-Y and CDE sites slightly and did not affect occupancy of the ATF site. In a previous study of transforming growth factor-beta1-mediated inhibition of the cyclin A promoter, we mapped the inhibitory effect to the ATF site; in the present study of IFN-gamma treatment, functional analysis by transient transfection showed that inhibition of the cyclin A promoter persisted despite mutation of the ATF, NF-Y, or CDE elements. We hypothesize that IFN-gamma inhibits cyclin A transcription by modifying co-activators or general transcription factors within the complex that drives transcription of the cyclin A gene.

Animals↗

PubMed2003

Determinants of inhibition of transiently expressed voltage-gated calcium channels by omega-conotoxins GVIA and MVIIA.

The Conus magus peptide toxin omega-conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and is now in clinical trials as an intrathecal analgesic. Here, we have examined the action of MVIIA on mutant and wild type calcium channels transiently expressed in tsA-201 cells. Although we have shown previously that mutations in a putative external EF-hand motif in the domain IIIS5-H5 region alters block by both omega-conotoxin GVIA and MVIIA (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), the introduction of five point mutations known to affect GVIA blocking (and located downstream of the EF-hand) affected MVIIA block to a smaller degree compared with GVIA. These data suggest that despite some overlap, MVIIA and GVIA block does not share identical channel structural determinants. At higher concentrations (approximately 3 microm), MVIIA reversibly blocked L-, P/Q-, and R-type, but not T-type channels, indicating that the overall architecture of the MVIIA site is conserved in all types of high voltage-activated calcium channels. A kinetic analysis of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Although the development of MVIIA block did not appear to be voltage-, nor frequency-dependent, the degree of recovery from block strongly depended on the potential applied during washout. Interestingly, the degree of washout was highly variable and appeared to weakly depend on the holding potential applied during toxin application. We propose a model in which N-type calcium channels can form both reversible and irreversible complexes with MVIIA.

Amino Acid Sequence↗

PubMed2006

Fulvestrant (ICI 182,780)-dependent interacting proteins mediate immobilization and degradation of estrogen receptor-alpha.

The antiestrogen fulvestrant (ICI 182,780) causes immobilization of estrogen receptor-alpha (ERalpha) in the nuclear matrix accompanied by rapid degradation by the ubiquitin-proteasome pathway. In this study we tested the hypothesis that fulvestrant induces specific nuclear matrix protein-ERalpha interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)-ERalpha-activating function-2 (AF2) fusion protein was used to isolate and purify receptor-interacting proteins in cell lysates prepared from human MCF-7 breast cancer cells. After SDS-PAGE and gel excision, mass spectrometry was used to identify two major ERalpha-interacting proteins, cytokeratins 8 and 18 (CK8.CK18). We determined, using ERalpha-activating function-2 mutants, that helix 12 (H12) of ERalpha, but not its F domain, is essential for fulvestrant-induced ERalpha-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ERalpha immobilization/degradation, transient transfection assays were performed using wild type ERalpha,ERalpha with a mutated H12, and ERalpha with a deleted F domain. Of those, only the ERalpha H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ERalpha degradation in CK8.CK18-positive human breast cancer cells, and CK8 and CK18 depletion by small interference RNAs partially blocked fulvestrant-induced receptor degradation. Furthermore, fulvestrant-induced ERalpha degradation was not observed in CK8 or CK18-negative cancer cells, suggesting that these two intermediate filament proteins are necessary for fulvestrant-induced receptor turnover. Using an ERalpha-green fluorescent protein construct in fluorescence microscopy revealed that fulvestrant-induced cytoplasmic localization of newly synthesized receptor is mediated by its interaction with CK8 and CK18. In summary, this study provides the first direct evidence linking ERalpha immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ERalpha to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ERalpha turnover.

Breast Neoplasms↗

PubMed2006

The inner interhelix loop 4-5 of the melibiose permease from Escherichia coli takes part in conformational changes after sugar binding.

Cytoplasmic loop 4-5 of the melibiose permease from Escherichia coli is essential for the process of Na+-sugar translocation (Abdel-Dayem, M., Basquin, C., Pourcher, T., Cordat, E., and Leblanc, G. (2003) J. Biol. Chem. 278, 1518-1524). In the present report, we analyze functional consequences of mutating each of the three acidic amino acids in this loop into cysteines. Among the mutants, only the E142C substitution impairs selectively Na+-sugar translocation. Because R141C has a similar defect, we investigated these two mutants in more detail. Liposomes containing purified mutated melibiose permease were adsorbed onto a solid supported lipid membrane, and transient electrical currents resulting from different substrate concentration jumps were recorded. The currents evoked by a melibiose concentration jump in the presence of Na+, previously assigned to an electrogenic conformational transition (Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606-12613), were much smaller for the two mutants than the corresponding signals in cysteineless MelB. Furthermore, in R141C the stimulating effect of melibiose on Na+ affinity was lost. Finally, whereas tryptophan fluorescence spectroscopy revealed impaired conformational changes upon melibiose binding in the mutants, fluorescence resonance energy transfer measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that: 1) loop 4-5 contributes to the coordinated interactions between the ion and sugar binding sites; 2) it participates in an electrogenic conformational transition after melibiose binding that is essential for the subsequent obligatory coupled translocation of substrates. A two-step mechanism for substrate translocation in the melibiose permease is suggested.

Cysteine↗

PubMed2006

Identification of a novel apical sorting motif and mechanism of targeting of the M2 muscarinic acetylcholine receptor.

Previous studies have shown that the M2 receptor is localized at steady state to the apical domain in Madin-Darby canine kidney (MDCK) epithelial cells. In this study, we identify the molecular determinants governing the localization and the route of apical delivery of the M2 receptor. First, by confocal analysis of a transiently transfected glycosylation mutant in which the three putative glycosylation sites were mutated, we determined that N-glycans are not necessary for the apical targeting of the M2 receptor. Next, using a chimeric receptor strategy, we found that two independent sequences within the M2 third intracellular loop can confer apical targeting to the basolaterally targeted M4 receptor, Val270-Lys280 and Lys280-Ser350. Experiments using Triton X-100 extraction followed by OptiPrep density gradient centrifugation and cholera toxin beta-subunit-induced patching demonstrate that apical targeting is not because of association with lipid rafts. 35S-Metabolic labeling experiments with domain-specific surface biotinylation as well as immunocytochemical analysis of the time course of surface appearance of newly transfected confluent MDCK cells expressing FLAG-M2-GFP demonstrate that the M2 receptor achieves its apical localization after first appearing on the basolateral domain. Domain-specific application of tannic acid of newly transfected cells indicates that initial basolateral plasma membrane expression is required for subsequent apical localization. This is the first demonstration that a G-protein-coupled receptor achieves its apical localization in MDCK cells via transcytosis.

Amino Acid Motifs↗

PubMed1993

Molecular regulation of the human IL-3 gene: inducible T cell-restricted expression requires intact AP-1 and Elf-1 nuclear protein binding sites.

Interleukin 3 (IL-3) is a hematopoietic stem-cell growth and differentiation factor that is expressed solely in activated T and NK cells. Studies to date have identified elements 5' to the IL-3 coding sequences that regulate its transcription, but the sequences that confer T cell-specific expression remain to be clearly defined. We have now identified DNA sequences that are required for T cell-restricted IL-3 gene transcription. A series of transient transfections performed with human IL-3-chloramphenicol acetyltransferase (CAT) reporter plasmids in T and non-T cells revealed that a plasmid containing 319 bp of 5' flanking sequences was active exclusively in T cells. Deletion analysis revealed that T cell specificity was conferred by a 49-bp fragment (bp -319 to -270) that included a potential binding site for AP-1 transcription factors 6 bp upstream of a binding site for Elf-1, a member of the Ets family of transcription factors. DNaseI footprint and electrophoretic mobility shift assay analyses performed with MLA-144 T cell nuclear extracts demonstrated that this 49-bp region contains a nuclear protein binding region that includes consensus AP-1 and Elf-1 binding sites. In addition, extracts prepared from purified human T cells contained proteins that bound to synthetic oligonucleotides corresponding to the AP-1 and Elf-1 binding sites. In vitro-transcribed and -translated Elf-1 protein bound specifically to the Elf-1 site, and Elf-1 antisera competed and super shifted nuclear protein complexes present in MLA-144 nuclear extracts. Moreover, addition of anti-Jun family antiserum in electrophoretic mobility shift assay reactions completely blocked formation of the AP-1-related complexes. Transient transfection studies in MLA-144 T cells revealed that constructs containing mutations in the AP-1 site almost completely abolished CAT activity while mutation of the Elf-1 site or the NF-IL-3 site, a previously described nuclear protein binding site (bp. -155 to -148) in the IL-3 promoter, reduced CAT activity to < 25% of the activity given by wild-type constructs. We conclude that expression of the human IL-3 gene requires the AP-1 and Elf-1 binding sites; however, unlike other previously characterized cytokine genes such as IL-2, the AP-1 and Elf-1 factors can bind independently in the IL-3 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

A conserved region in intron 1 negatively regulates the expression of the PCNA gene.

The Proliferating Cell Nuclear Antigen (PCNA) gene is a growth-regulated gene, whose expression is under the control of both transcriptional and posttranscriptional mechanisms. In previous work, it was shown that the 73 bp immediately upstream of the CAP site and intron 4 are major regulatory elements. We show here that intron 1 also plays a role in determining the levels of PCNA mRNA. Specifically, we show: 1) deletion of intron 1 increases the expression of PCNA mRNA in serum-deprived cells; 2) a 35 bp sequence in intron 1, containing a reverse CCAAT element specifically binds proteins from nuclear extracts; 3) this intron 1 sequence inhibits the expression of a co-tranfected human PCNA gene in transient expression assays suggesting that it competes for positive transcription factors; 4) mutations in the CCAAT region of the 35bp intron 1 probe abrogate both its protein-binding capacity and its ability to inhibit the expression of a co-transfected wt PCNA gene; and 5) the CCAAT region of human intron 1 is highly conserved in the mouse gene. We conclude that the reverse CCAAT region of intron 1 is a negative regulatory element of PCNA gene expression, and hypothesize that its inhibitory effect is abolished when certain protein(s) bind to it and that inhibition is restored if these proteins are competed out by an homologous sequence.

Antigens, Neoplasm↗

PubMed1995

The influence of a metastable structure in plasmid primer RNA on antisense RNA binding kinetics.

Replication of the ColE1 group plasmids is kinetically regulated by the interaction between plasmid-encoded primer RNA II and antisense RNA I. The binding is dependent on alternative RNA II conformations, formed during the transcription, and effectively inhibits the primer function within some time interval. In this paper, the folding pathways for the wild type and copy number mutants of ColE1 RNA II are studied using simulations by a genetic algorithm. The simulated pathways reveal a transient formation of a metastable structure, which is stabilized by copy number mutations. The folding kinetics of the proposed conformational transitions is calculated using a model of a multistep refolding process with elementary steps of double-helical stem formation or disruption. The approximation shows that the lifetime of the metastable structure is relatively long and is considerably increased in the mutants, resulting in a delay of the formation of the stable RNA II structure, which is the most sensitive to the inhibition by the antisense RNA I. Thus the effect of copy number mutations can be interpreted as a compression of the time window of effective inhibition due to an increased time spent by the RNA II in the metastable state. The implications of metastable foldings in RNA functioning are discussed.

Algorithms↗

PubMed2005

The PSEA promoter element of the Drosophila U1 snRNA gene is sufficient to bring DmSNAPc into contact with 20 base pairs of downstream DNA.

Most of the major spliceosomal small nuclear RNAs (snRNAs) (i.e. U1, U2, U4 and U5) are synthesized by RNA polymerase II (pol II). In Drosophila melanogaster, the 5'-flanking DNA of these genes contains two conserved elements: the proximal sequence element A (PSEA) and the proximal sequence element B (PSEB). The PSEA is essential for transcription and is recognized by DmSNAPc, a multi-subunit protein complex. Previous site-specific protein-DNA photo-cross-linking assays demonstrated that one of the subunits of DmSNAPc, DmSNAP43, remains in close contact with the DNA for 20 bp beyond the 3' end of the PSEA, a region that contains the PSEB. The current work demonstrates that mutation of the PSEB does not abolish the cross-linking of DmSNAP43 to the PSEB. Thus the U1 PSEA alone is capable of bringing DmSNAP43 into close contact with this downstream DNA. However, mutation of the PSEB perturbs the cross-linking pattern. In concordance with these findings, PSEB mutations result in a 2- to 4-fold reduction in U1 promoter activity when assayed by transient transfection.

Animals↗

PubMed2004

Neurotrophin-3 and a CREB-mediated signaling pathway regulate Bcl-2 expression in oligodendrocyte progenitor cells.

Our previous results suggested that the transcription factor CREB mediates the actions of neuroligands and growth factor signals that coupled to different signaling pathways may play different roles along oligodendrocyte (OLG) development. We showed before that CREB phosphorylation in OLG progenitors is up-regulated by neurotrophin-3 (NT-3); and moreover CREB is required for NT-3 to stimulate the proliferation of these cells. We now show that treatment of OLG progenitors with NT-3 is also accompanied by an increase in the levels of the anti-apoptotic protein Bcl-2. Interestingly, the presence of a putative CREB binding site (CRE) in the Bcl-2 gene raised the possibility that CREB could also be involved in regulating Bcl-2 expression in the OLGs. Supporting this hypothesis, the NT-3 dependent increase in Bcl-2 levels is abolished by inhibition of CREB expression. In addition, transient transfection experiments using various regions of the Bcl-2 promoter and mutation of the CRE site indicate a direct role of CREB in regulating Bcl-2 gene activity in response to NT-3. Furthermore, protein-DNA binding assays show that the CREB protein from freshly isolated OLGs indeed binds to the Bcl-2 promoter CRE. Together with our previous results, these observations suggest that CREB may play an important role in linking proliferation and survival pathways in the OLG progenitors.

Activating Transcription Factor 1↗

PubMed2004

Molecular determinants of frequency dependence and Ca2+ potentiation of verapamil block in the pore region of Cav1.2.

Verapamil block of Ca(v)1.2 is frequency-dependent and potentiated by Ca(2+). We examined the molecular determinants of these characteristics using mutations that effect Ca(2+) interactions with Ca(v)1.2. Mutant and wild-type Ca(v)1.2 channels were transiently expressed in tsA 201 cells with beta(1b) and alpha(2)delta subunits. The four conserved glutamates that compose the Ca(2+) selectivity filter in Ca(v)1.2 were mutated to Gln (E363Q, E709Q, E1118Q, E1419Q) and the adjacent conserved threonine in each domain was mutated to Ala (T361A, T707A, T1116A, T1417A). The L-type-specific residues in the domain III pore region (F1117G) and the C-terminal tail (I1627A) were also mutated and assayed for block by verapamil using whole-cell voltage-clamp recordings in 10 mM Ba(2+) or 10 mM Ca(2+). In Ba(2+), none of the pore-region mutations reduced the fraction of current blocked by 30 microM verapamil at 0.05 Hz stimulation. However, all of the pore-region mutations abolished Ca(2+) potentiation of verapamil block at 0.05 Hz. The T1116A, F1117G, E1118Q, and E1419Q mutations all significantly reduced frequency-dependent verapamil block (1-Hz stimulation) in both Ba(2+) and Ca(2+). The I1627A mutation, which disrupts Ca(2+)-dependent inactivation, increased the fraction of closed channels blocked by 30 microM verapamil in Ba(2+) but did not affect frequency-dependent block in Ba(2+) or Ca(2+). Our data suggest that the pore region of domain III may contribute to a high affinity verapamil binding site accessed during 1-Hz stimulation and that Ca(2+) binding to multiple sites may be required for potentiation of verapamil block of closed channels.

Alanine↗

PubMed1988

Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR.

In studies of the genetics of human immunodeficiency virus type 1 (HIV-1), the product of the nef gene, formerly known as F, 3'-orf, or B-ORF, was a negative regulator of HIV-1 replication. Proviruses with mutations in the nef gene replicated better than their standard counterparts during transient expression, and the mutant virus maintained its enhanced replication even after serial passages in T lymphocytes. The nef protein trans-suppressed, in a dose-dependent manner, the replication of wild-type and nef mutant proviruses and the expression of reporter genes linked to the HIV-1 long terminal repeat (LTR). The repression induced by the nef protein was mediated by inhibition of transcription from the HIV-1 LTR, which contains a far upstream cis element (previously recognized to be a negative regulatory element) between 340 and 156 nucleotides upstream of the RNA initiation site.

Gene Expression Regulation↗

PubMed1992

Role of the Bacillus subtilis gsiA gene in regulation of early sporulation gene expression.

The Bacillus subtilis gsiA operon was induced rapidly, but transiently, as cells entered the stationary phase in nutrient broth medium. A mutation at the gsiC locus caused sporulation to be defective and expression of gsiA to be elevated and prolonged. The sporulation defect in this strain was apparently due to persistent expression of gsiA, since a gsiA null mutation restored sporulation to wild-type levels. Detailed mapping experiments revealed that the gsiC82 mutation lies within the kinA gene, which encodes the histidine protein kinase member of a two-component regulatory system. Since mutations in this gene caused a substantial blockage in expression of spoIIA, spoIIG, and spoIID genes, it seems that accumulation of a product of the gsiA operon interferes with sporulation by blocking the completion of stage II. It apparently does so by inhibiting or counteracting the activity of KinA.

Alleles↗

PubMed1992

Role of the apical stem in maintaining the structure and function of adenovirus virus-associated RNA.

Adenovirus virus-associated (VA) RNAI maintains efficient protein synthesis during the late phase of infection by preventing the activation of the double-stranded-RNA-dependent protein kinase, DAI. A secondary structure model for VA RNAI predicts the existence of two stems joined by a complex stem-loop structure, the central domain. The structural consequences of mutations and compensating mutations introduced into the apical stem lend support to this model. In transient expression assays for VA RNA function, foreign sequences inserted into the apical stem were fully tolerated provided that the stem remained intact. Mutants in which the base of the apical stem was disrupted retained partial activity, but truncation of the apical stem abolished the ability of the RNA to block DAI activation in vitro, suggesting that the length and position of the stem are both important for VA RNA function. These results imply that VA RNAI activity depends on secondary structure at the top of the apical stem as well as in the central domain and are consistent with a two-step mechanism involving DAI interactions with both the apical stem and the central domain.

Adenoviruses, Human↗

PubMed1985

Promoter domains required for expression of plasmid-borne copies of the herpes simplex virus thymidine kinase gene in virus-infected mouse fibroblasts and microinjected frog oocytes.

A transient expression assay was used to measure the relative template activities of mutated tk genes in mouse L cells induced in trans by herpes simplex virus (HSV). In this assay, expression of the wild-type HSV type 1 tk gene is induced at least 200-fold by the superinfecting virus. Genetic lesions that were assayed include 5' deletions, clustered base substitutions, single base substitutions, intrapromoter inversions, and intrapromoter recombinants with the HSV type 2 tk gene. Roughly half of the mutations that were tested were found to weaken tk expression efficiency, and the remaining mutations did not alter expression. The spatial distribution of mutations that reduce expression efficiency in trans-induced mouse fibroblasts facilitated the construction of a map of promoter domains. The most gene-proximal promoter domain is located between 16 and 32 base pairs (bp) upstream of the tk mRNA cap site and contains a TATA homology. Two more distally located promoter domains were mapped to discrete locations upstream from the TATA homology. One of these distal domains is located between 47 and 79 bp upstream from the mRNA cap site, and the other is located between 84 and 105 bp upstream from the tk gene. The boundaries of these three promoter domains, with one exception, coincided with the set of domains delineated previously in a frog oocyte microinjection assay. The concordant behavior of tk promoter mutants in microinjected frog oocytes and trans-induced mouse fibroblasts leads us to propose that recognition and activation of the HSV tk promoter is mediated by cellular transcription factors that are common to frogs and mice.

Animals↗

PubMed2004

Down-regulation of the homeodomain factor Cdx2 in colorectal cancer by collagen type I: an active role for the tumor environment in malignant tumor progression.

The homeobox transcription factor Cdx2 specifies intestinal development and homeostasis and is considered a tumor suppressor in colorectal carcinogenesis. However, Cdx2 mutations are rarely found. Invasion of colorectal cancer is characterized by a transient loss of differentiation and nuclear accumulation of the oncoprotein beta-catenin in budding tumor cells. Strikingly, this is reversed in growing metastases, indicating that tumor progression is a dynamic process that is not only driven by genetic alterations but also regulated by the tumor environment. Here we describe a transient loss of Cdx2 in budding tumor cells at the tumor host interface, and reexpression of Cdx2 in metastases. Cell culture experiments show that collagen type I, through beta(1) integrin signaling, triggers a transient transcriptional down-regulation of Cdx2 and its intestine-specific target gene sucrase isomaltase, associated with a loss of differentiation. These data indicate an active role for the tumor environment in malignant tumor progression.

Adenocarcinoma↗