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PubMed2002

Obligatory role of cyclic adenosine monophosphate response element in cyclooxygenase-2 promoter induction and feedback regulation by inflammatory mediators.

BACKGROUND: Cyclooxygenase-2 (COX-2) plays a key role in human inflammatory disorders such as vascular inflammation. COX-2 promoter activity is induced by proinflammatory mediators, but the role of cyclic adenosine monophosphate response element (CRE) in promoter stimulation remains unclear. METHODS AND RESULTS: Transient transfection of a 0.9-kb COX-2 promoter fragment bearing CRE mutation abrogated COX-2 promoter activity induced by proinflammatory mediators in human endothelial cells and fibroblasts. Dual mutations of CRE and an upstream CCAAT/enhancer binding protein (C/EBP) site did not have an additional effect. Binding of CREB-2, ATF-2, USF-2, and c-Jun transactivators to a wild-type and CRE-mutated oligonucleotide was analyzed by a novel DNA-binding assay. CREB-2 and ATF-2 in nuclear extracts of unstimulated endothelial cells bound to CRE, whereas USF-2 and c-Jun or c-Fos bound to non-CRE sites. CREB-2 and c-Fos binding was increased by phorbol 12-myristate 13-acetate but not tumor necrosis factor-alpha. The binding assay and chromatin immunoprecipitation revealed binding of P300 coactivator to the COX-2 promoter region. CONCLUSIONS: CRE plays an obligatory role in COX-2 promoter activation by diverse stimuli. CREB-2 and ATF-2 bound to CRE serve as an anchor for P300 interaction with upstream transactivators and downstream transcription machinery.

Activating Transcription Factor 4↗
PubMed2004

Functional importance of Myc-associated zinc finger protein for the human parathyroid hormone (PTH)/PTH-related peptide receptor-1 P2 promoter constitutive activity.

The aim of the present study was to analyze the functional importance for the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) gene P2 promoter activity of the putative proximal Myc-associated zinc finger protein (MAZ) site localized at position bp -45 to -39 bp, taking advantage of a G/A mutation identified at position -40 in the human sequence. Wild-type 'full-length' (1285P2) and truncated (760P2) promoter sequences were inserted upstream to the luciferase basic (pLucB) and enhancer (pLucE) reporter gene expression vectors. Transient transfections in osteoblast-like SaOS-2 cells and renal cells (RC.SV3A2) showed that the -40 G/A mutation significantly impaired transcriptional activity of wild-type 1285P2-pLucB and 760P2-pLucE promoter constructs. Further truncation of the P2 sequence demonstrated that the sequence -109/-37 bp was essential for promoter activity. Co-transfection with a MAZ expression vector did not modify the wild-type 1285P2-pLucB construct reporter activity but significantly increased 2-fold the mutated construction activity (P<0.05). Electrophoretic mobility shift assays using SaOS-2 nuclear extracts and a double-stranded DNA fragment encompassing the -45 to -39 putative MAZ site (ds-MAZ-oligo) disclosed two specific DNA-protein complexes. Complex II (fast moving) had a lower affinity for the mutated MAZ motif than for the wild-type MAZ motif while complex I (slow moving) had the same affinity for both wild-type or mutated MAZ sequences. Competition studies with Sp1 consensus oligonucleotide (ds-Sp1-oligo) markedly reduced complex I intensity, with a concomitant increase in that of complex II. Finally, ribonuclease protection assays showed that P2-specific PTHR1 mRNA transcript expression was significantly decreased in SaOS-2 cells transfected with ds-MAZ-oligo as compared with that for control (P<0.001) and ds-Sp1-oligo (P<0.05). Taken together, our studies suggest that the putative -45 to -39 MAZ-binding site regulates the constitutive activity of human PTHR1 P2 promoter.

Cell Nucleus↗
PubMed1999

The viral protein Apoptin induces apoptosis in UV-C-irradiated cells from individuals with various hereditary cancer-prone syndromes.

Apoptin, a protein derived from chicken anemia virus, has previously been shown to induce apoptosis in a p53-independent and Bcl-2-stimulated manner in transformed and tumorigenic human cells but not in normal diploid human cells, suggesting that it is a potential agent for tumor therapy. Here we report that Apoptin can induce apoptosis in UV-C-irradiated diploid skin fibroblasts from individuals with various hereditary cancer-prone syndromes that are characterized by a germ-line mutation in a tumor suppressor gene. The same effect is found when these cells are irradiated with X-rays. In contrast, diploid skin fibroblasts from healthy donors or from individuals with DNA repair disorders are not responsive to Apoptin-induced apoptosis upon UV-C or X-ray irradiation. After transfection of untreated cells, Apoptin is found predominantly in the cytoplasm, whereas in UV-C-exposed Apoptin-responsive cancer-prone cells, it migrates to the nucleus, where it causes rapid apoptosis. Apoptin remains localized in the cytoplasm after UV-C treatment of diploid cells from healthy individuals. The induction of apoptosis by Apoptin in cancer-prone cells with a germ-line mutation in a tumor suppressor gene is UV dose-dependent and transient, just like many other UV-induced processes. These results suggest that Apoptin may be used as a diagnostic tool for detection of individuals with an increased risk for hereditary cancer and premalignant lesions.

Apoptosis↗
PubMed1999

Functional synergism of STAT6 with either NF-kappa B or PU.1 to mediate IL-4-induced activation of IgE germline gene transcription.

Ig heavy chain class switching to IgE is directed by IL-4 and IL-13 by inducing transcription from the IgE germline promoter. A crucial transcription factor in this process is STAT6, which binds to a specific DNA element upon cytokine activation. In this paper it is shown that the B cell- and monocyte-specific factor PU.1 interacts with a closely spaced sequence in the human IgE germline promoter that overlaps with a previously described binding site for NF kappa B/rel. The authenticity of PU.1 was demonstrated by specific competition and supershifts in EMSA experiments. In addition, in vitro translated PU.1 could interact with an oligonucleotide derived from the IgE germline promoter containing the PU.1 binding site and migrated with the same mobility compared with the complex formed with nuclear extracts. Transient transfection experiments using IgE germline promoter reporter gene constructs demonstrated that mutations affecting DNA binding of PU.1 or NF kappa B/rel had no or little effect on IL-4 inducibility of these plasmids. However, point mutations that abolished binding of both factors abrogated cytokine inducibility. No strict spacing of the STAT6 and the composite PU. 1/NF-kappa B elements is required for IL-4 induction. IL-4-induced STAT6 DNA binding was retained in PU.1-/NF kappa B/rel- double mutants. The data demonstrate that cooperation of STAT6 with at least PU.1 or NF kappa B/rel is necessary for IL-4-induced activation of IgE germline gene transcription.

Adjuvants, Immunologic↗
PubMed1998

[YY1 and its repressive effect on human papillomavirus 16 early promoter P97 existed widely among human epithelial cell lines].

Cellular transcription factor YY1 represses the activity of early promoter P97 of HPV 16. Removal of the specific binding sites for YY1 in HPV 16 LCR can increase the P97 activity. To analyse the expression of YY1 and the existence of the activation effect on P97 due to removing YY1 binding sites in the LCR region, we extracted nuclear proteins from four cervical cancer cell lines and two human keratinocytes. EMSA assays were carried out in vitro. At the same time, we transiently transfected the luciferase reporter plasmids, which contain HPV 16 reference and mutated LCR sequences, respectively, into the different cells and determined the luciferase expressions. The results showed that all the analysed cell lines contained biologically functional YY1 proteins. No remarkable difference in the concentration of native YY1 proteins could be detected among the tested cells. Removal of YY1 binding sites in HPV 16 LCR could elevate P97 activity in several analysed cell lines, including normal human primary kerationcytes from foreskin. It suggests that YY1 regulatory system exists widely in human epithelial cell lines. We also found that the transcription activator NF1 was more highly expressed in C33a cell than in HT3 cell, indicating an unevenly distribution of functional proteins in various cell lines.

Cells, Cultured↗
PubMed1994

Overlapping Egr-1 and Sp1 sites function in the regulation of transcription of the mouse thrombospondin 1 gene.

We have evaluated the basis for the constitutive and serum-regulated expression of the mouse thrombospondin (TSP) gene in both transiently and stably transfected NIH-3T3 cells. Experiments with deleted and mutated mouse promoter/CAT constructs and gel mobility assays demonstrated that an Egr-1 binding site in the proximal promoter, flanked by overlapping GC boxes and an adjacent GC-rich region, functioned to positively regulate the constitutive activity of the gene. These motifs, and their cognate transcription factors, appear to act in concert, with partial redundancy, so that discrete mutations were only partially effective in reducing transcriptional activity. The Egr-1 site corresponds in position to an NF-Y binding site which functions synergistically with a distal serum-response element to mediate the serum response of the human TSP1 gene. However, neither the Egr-1 motif nor the surrounding proximal promoter region upstream from the TATA box participates in the serum response of mouse TSP1. These experiments add support to the growing realization that similar physiologic responses of homologous genes in mouse and man need not utilize similarly placed cis-acting elements.

3T3 Cells↗
PubMed1996

PIC 1, a novel ubiquitin-like protein which interacts with the PML component of a multiprotein complex that is disrupted in acute promyelocytic leukaemia.

Acute promyelocytic leukaemia (APL) arises following a reciprocal translocation t(15;17) that fuses PML with retinoic acid receptor alpha (RARA). The PML-RARA fusion protein targets and disrupts nuclear multiprotein complexes called PODs, ND10 or NBs, a process which is associated with a block in myeloid differentiation leading to APL. A human B-cell cDNA library was screened for PML-interacting clones and a single positive clone (PIC1) was isolated. The sequence of PIC1 shows 52% identity to a S. cerevisiae ubiquitin-like protein that was cloned as a suppressor of mutations in MIF2, a protein required for mitotic spindle integrity during anaphase. Transient transfection of NIH3T3 cells with PIC1 results in a nuclear staining pattern coincident with that of endogenous mouse PML. Further, cotransfection of PIC1 with human PML produces a completely overlapping staining pattern between the two proteins. An antibody raised against PIC1 detects a punctate staining pattern in HeLa cells that is coincident with endogenous human PML. There is no significant colocalisation observed between the staining of PML/ PML-RARA and PIC1 in an APL-derived cell line NB4, as compared to cells expressing only wild type PML. However, following all trans retinoic acid treatment of NB4 cells a significant relocalisation of PIC1 and PML is observed. PIC1 is the first identified NB-associated protein that interacts with PML, the function of which may lead to a fuller understanding of the molecular events leading to APL.

3T3 Cells↗
PubMed1998

Synergistic activation of transcription by nuclear factor Y and sterol regulatory element binding protein.

The current studies define the role of three distinct cis-elements in the proximal promoter of the rat farnesyl diphosphate (FPP) synthase gene. The three cis-elements, a sterol regulatory element 3 (SRE-3) flanked by an ATTGG motif (inverted CCAAT box), and a CCAAT box, form a sterol regulatory unit that is necessary and sufficient for sterol-regulated expression of FPP synthase promoter-reporter genes. FPP synthase promoter-reporter genes, that contain promoters with either wild-type nucleotide sequences or mutations in one or more of the three cis-elements, were transiently transfected into CV-1 cells. The activity of the wild-type promoter-reporter gene increased when the cells were incubated in sterol-depleted media or when the cells were co-transfected with a plasmid encoding the mature form of SRE binding protein (SREBP-1a). The results with the mutant promoter-reporter genes demonstrated that all three cis-elements were necessary for normal expression/regulation of the reporter gene by either sterols or by co-expressed SREBP-1a. Gel mobility shift assays demonstrated that the synergistic binding of SREBP-1a to SRE-3 was dependent on the binding of recombinant nuclear factor Y (NF-Y) to the DNA, consistent with the in vivo regulation studies.

Alkyl and Aryl Transferases↗
PubMed1994

Transcriptional activation of human leukosialin (CD43) gene by Sp1 through binding to a GGGTGG motif.

Human leukosialin (CD43) is expressed on the surface of hematopoietic cells in cell-type specific and differentiation-stage-specific manners. Previously we found that the sequence from -53 to -40 was critically involved in the promoter function [Kudo, S. & Fukuda, M. (1991) J. Biol. Chem. 266, 8483-8489]. A transient-expression assay using a chloramphenicol acetyltransferase reporter gene revealed that the promoter could confer a high basal transcriptional activity in both leukosialin-producing and non-producing cells. The transcription factor interacting with the promoter sequence was determined by DNase I footprinting and gel-mobility-shift assays. The nuclear extracts from both leukosialin-producing Jurkat cells and non-producing Hela cells showed a footprint on the 5' flanking region from -58 to -34. Gel-mobility-shift assays revealed that DNA-protein complexes were formed with both nuclear extracts, and these complex formations were inhibited by an oligonucleotide containing the Sp1-binding consensus sequence. Prior incubation of anti-Sp1 antibody with nuclear extracts in this assay resulted in the supershift of the band for the DNA-protein complex. In addition, the footprint produced by the purified Sp1 transcription factor was identical to those produced by nuclear extracts of Jurkat and Hela cells. The mutational analyses revealed that the binding affinities of Sp1 to mutated promoter sequences were parallel to the transcriptional activity of these promoter sequences. Transient expression analyses in Drosophila Schneider cells demonstrated that cotransfection with Sp1 expression plasmid increased the transcriptional activity. These results establish that Sp1 can bind to the promoter and positively regulates the expression of the leukosialin gene. Even the stable expression of CAT constructs in non-producing Hela cells showed high transcriptional activity. The leukosialin expression thus appears to be regulated by the unique mechanism, that is the repression of high basal transcriptional activity rather than the activation of the basal transcriptional level. Tissue-specific expression is probably achieved by suppression of the basal transcriptional activity in non-producing cells.

Animals↗
PubMed1998

Active domains of human papillomavirus type 11 E1 protein for origin replication.

Viral proteins E1 and E2 are essential for transient human papillomavirus (HPV) DNA replication. E1 is a multifunctional protein which can bind DNA and complex with E2, has ATPase and helicase activities, and interacts with DNA polymerase alpha-primase. E2 is a transactivator-repressor protein, playing an important role in replication and transcriptional regulation. A series of deletion mutants of HPV-11 E1 were constructed and tested in functional assays to define those domains of HPV-11 E1 which are important for binding to the origin DNA and E2. The domain of HPV-11 E1 involved in binding to the origin was located between aa 186 and 649, and that for binding to E2 was between aa 346 and 649. Since E1 binds to the origin more efficiently in the presence of E2, we also mapped the DNA binding domain of E1 in the presence of E2, and found that when binding was enhanced, the region of E1 involved in binding was similar to that observed with E1 alone. The same deletion mutation constructs of E1 were subcloned into an expression vector for use in transient replication assays to study the effect of the deletions on the replication of the origin DNA in vivo and the data suggest that the C-terminal domain contains important functions for replication.

Binding Sites↗
PubMed1987

A new mutation in IVS-1 of the human beta globin gene causing beta thalassemia due to abnormal splicing.

A G to T transversion at the fifth nucleotide of the first intervening sequence (IVS-1) of the beta-globin gene has been identified in cloned beta-thalassemia genes of two unrelated individuals, one of Mediterranean and the other of Anglo Saxon ancestry. In each patient the mutation was present in a different beta globin gene framework, defined by intragenic restriction site polymorphisms, thereby suggesting the occurrence of independent mutations. The study of the RNA products of one of these cloned genes, after transfer and transient expression in HeLa cells, showed partial inactivation of the normal donor splice site of IVS-1 and activation of two major and one minor cryptic splice sites. Only one of the two major cryptic sites was utilized in a cell-free splicing extract. The effects of this mutation on messenger RNA (mRNA) splicing are similar to that of another beta thalassemia gene with a G to C transition at the same position.

Base Sequence↗
PubMed1994

A Pit-1 phosphorylation mutant can mediate both basal and induced prolactin and growth hormone promoter activity.

The transcription factor Pit-1 has been shown to be important for both the developmental and homeostatic regulation of expression of the PRL and GH genes in pituitary cells. However, little is known about possible covalent modifications in Pit-1 that might mediate its transactivational properties. Previous studies showing that Pit-1 is a phosphorylation substrate for either protein kinase A or C, or their cellular inducers, led us to investigate whether phosphorylation of Pit-1 is required for its function in either basal or induced cellular activity of either the PRL or GH promoters. The transactivational properties of wild type Pit-1 were compared with those of Pit-1(A3), mutated in the three known phosphorylation sites. At saturating levels of Pit-1 expression vectors, activation of transient basal expression in HeLa cells of constructs (-1957)PRL-CAT or (-244)GH-CAT by RSV-Pit-1(A3) was, respectively, about 50% and 65% as strong as by RSV-Pit-1. Hence, phosphorylation at the sites mutated in Pit-1(A3) is not critically required for basal transactivation of either promoter but may modulate this activity. RSV-Pit-1 and RSV-Pit-1(A3) were equally effective in mediating estrogen receptor stimulation of (-1957)PRL-CAT expression in HeLa cells, thus revealing no phosphorylation requirement for the prerequisite for Pit-1 in estrogen receptor action on the PRL estrogen response element.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

A mutation changes ligand selectivity and transmembrane signaling preference of the neurokinin-1 receptor.

We studied the biochemical properties of a genetically engineered neurokinin-1 receptor (NK1R) in which two residues lying on the extracellular edge of the fourth transmembrane domain were replaced by equivalently located elements of the neurokinin-2 receptor (G166C, Y167F NK1R mutant). The mutation produced two effects. The first is enhancement of the apparent binding affinity for heterologous tachykinins (substance K and neurokinin B) and for N- or C-terminal modified analogues of substance P, but not for substance P itself, its full-length analogues, and several peptide and nonpeptide antagonists. Only two antagonists, as exceptions, were found to exhibit a diminished affinity for the mutant receptor. The second effect is a shift in NK1R preference for distinct G protein-mediated signaling pathways. NK1R-mediated phosphoinositide hydrolysis was enhanced both in transiently and permanently transfected cells, while stimulation of cAMP accumulation did not change in transient expression experiments and was reduced in permanently expressing cells. The effect of the mutation on ligand affinity was not related to any obvious structural commonality, nor to the selectivity for different neurokinin receptors or the agonistic/antagonistic nature of the ligand. However, all ligands responding to the mutation appear to share the ability to induce phosphoinositide signaling more efficiently than cAMP responses when binding to NK1R. We suggest that the mutation shifts the internal equilibria of different functional forms of NK1R. A theoretical analysis according to a multistate allosteric model suggests that the link between binding and biological changes can result from altered stability constants of substates in the conformational space of the receptor.

Allosteric Site↗
PubMed2001

Structure and expression of the Ah receptor repressor gene.

The aryl hydrocarbon receptor (AhR) repressor (AhRR) gene has been isolated and characterized from a mouse genomic library. The gene is distributed as 11 exons in a total length of about 60 kilobase pairs. Fluorescence in situ hybridization analysis has shown that the AhRR gene is located at mouse chromosome 13C2, at rat chromosome 1p11.2, and at human chromosome 5p15.3. The AhRR gene has a TATA-less promoter and several transcription start sites. In addition, putative regulatory DNA sequences such as xenobiotic responsive element (XRE), GC box, and NF-kappaB-binding sites have been identified in the 5'-upstream region of the AhRR gene. Transient transfection analyses of HeLa cells with reporter genes that contain deletions and point mutations in the AhRR promoter revealed that all three XREs mediated the inducible expression of the AhRR gene by 3-methylcholanthrene treatment, and furthermore, GC box sequences were indispensable for a high level of inducible expression and for constitutive expression. Moreover, by using gel mobility shift assays we were able to show that the AhR/Arnt heterodimer binds to the XREs with very low affinity, which is due to three varied nucleotides outside the XRE core sequence. We have also shown that Sp1 and Sp3 can bind to the GC boxes. Finally, both transient transfection analysis and gel mobility shift assay revealed that the AhRR gene is up-regulated by a p65/p50 heterodimer that binds to the NF-kappaB site when the cells has been exposed to 12-O-tetradecanoylphorbol-13-acetate, and this inducible expression was further enhanced by cotreatment of 12-O-tetradecanoylphorbol-13-acetate and 3-methylcholanthrene.

5' Untranslated Regions↗
PubMed2005

Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity.

We have previously identified in the pol gene of human immunodeficiency virus type 1 (HIV-1) a new positive transcriptional regulatory element (nt 4481-4982) containing recognition sites for nuclear proteins (sites B, C, D and a GC-box) [C. Van Lint, J. Ghysdael, P. Paras, Jr, A. Burny and E. Verdin (1994) J. Virol. 68, 2632-2648]. In this study, we have further physically characterized each binding site and have shown that the transcription factors Oct-1, Oct-2, PU.1, Sp1 and Sp3 interact in vitro with the pol region. Chromatin immunoprecipitation assays using HIV-infected cell lines demonstrated in the context of chromatin that Sp1, Sp3, Oct-1 and PU.1 are recruited to the HS7 region in vivo. For each site, we have identified mutations abolishing factor binding to their cognate DNA sequences without altering the underlying amino acid sequence of the integrase. By transient transfection assays, we have demonstrated the involvement of the pol binding sites in the transcriptional enhancing activity of the intragenic region. Our functional results with multimerized wild-type and mutated pol binding sites separately (i.e. in the absence of the other sites) have demonstrated that the PU.1, Sp1, Sp3 and Oct-1 transcription factors regulate the transcriptional activity of a heterologous promoter through their respective HS7 binding sites. Finally, we have investigated the physiological role of the HS7 binding sites in HIV-1 replication and have shown that these sites are important for viral infectivity.

Animals↗
PubMed2006

Characterization of novel missense mutations in CYP21 causing congenital adrenal hyperplasia.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is the most common inherited disorder of steroid metabolism, with an incidence of 1/10,000 in the general Caucasian population. Although most patients carry a deletion of the CYP21 gene or any of nine pseudogene-derived point mutations, the number of reported rare mutations continues to increase, and consist today of more than 80 different point mutations. In this study, we report the characterization of four additional missense mutations in CYP21. Two of these, L166P and A391T, are novel missense mutations, whereas the R479L and R483Q mutations have been detected previously. Functional assays of mutagenized CYP21 were performed in transiently transfected mammalian cells in vitro, and enzymatic ability of substrate conversion of the two natural substrates of CYP21-17-hydroxyprogesterone and progesterone-was determined. All mutants displayed reduced in vitro enzyme activities compared with wild type, but to different extents, corresponding to clinical phenotypes that span the whole spectrum of disease severity. Functional studies are important to further establish the relationships between genotype and clinical phenotype as well as in vitro CYP21 activity in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. This has relevance for diagnosis, prognosis, and genetic counseling for affected families.

Adrenal Hyperplasia, Congenital↗
PubMed2005

[The effect of envelope protein mutation on hepatitis B virus assemble].

OBJECTIVE: To investigate the effect of envelope protein mutation on HBV assembly. METHODS: The envelope protein mutated vectors were constructed by the molecular clone in vitro, and then transfected transiently in the cell HepG2. The expression and secretion of S protein was assay by ELISA. HBV DNA was quantitatively evaluated by PCR. After co-transfection with pHBV-mS1S and adwR9 the DNA was quantitatively evaluated by PCR. RESULTS: There was no significant difference in expression and secretion of S protein assayed by ELISA in the cytoplasm and supernatant among pHBV-mS1, pHBV-mS, pHBV-mS1S and the wild HBV adwR9 plasmid. The DNA detected by real-time fluorescence quantitative PCR from those the cytoplasm of mutants was higher than that from the wild HBV adwR9 cytoplasm, especially from the cytoplasm of pHBV-mS1S plasmid. However, the DNA detected by real-time fluorescence quantitative PCR from the supernatant of those mutants was lower than that from the wild HBV adwR9 supernatant, especially from pHBV-mS1S. The DNA detected by real-time fluorescence quantitative PCR from the supernatant co-transfected with pHBV-mS1S and adwR9 was lower than that from the supernatant co-transfected with pcDNA3 and adwR9. CONCLUSION: There was no effect of envelope protein mutation on the expression and secretion of S protein. Envelope protein mutation could interfere the assembly of HBV particle and cause reduction of secretion of HBV.

Cell Line, Tumor↗