Retarded Band Research Articles

Transcriptional activation of insulin-like growth factor-binding protein-4 by 17beta-estradiol in MCF-7 cells: role of estrogen receptor-Sp1 complexes.

Insulin-like growth factor-binding protein-4 (IGFBP-4) is expressed in MCF-7 human breast cancer cells, and treatment of these cells with 17beta-estradiol (E2) resulted in induction of IGFBP-4 gene expression (>3-fold) and protein secretion (>6-fold). To identify genomic sequences associated with E2 responsiveness, the 5'-promoter region (-1214 to +18) of the IGFBP-4 gene was cloned into a vector upstream from the firefly luciferase reporter gene, and E2 induced a 10-fold increase in luciferase activity in MCF-7 cells transiently transfected with this construct. Deletion analysis of this region of the IGFBP-4 gene promoter identified two GC-rich sequences at -559 to -553 and -72 to -64 that were important for E2-induced trans-activation. Gel mobility shift assays using 32P-labeled -569 to -540 and -83 to -54 oligonucleotides from the IGFBP-4 gene promoter showed that Sp1 protein bound these oligonucleotides to form a retarded band, and the intensity of the band was competitively decreased after coincubation with unlabeled IGFBP-4-derived and consensus Sp1 oligonucleotides. Mutation of the GC-rich sites within these sequences resulted in loss of the retarded band formation. Wild-type human estrogen receptor did not bind directly to the IGFBP-4 oligonucleotides; however, human estrogen receptor enhanced Sp1-DNA binding in a concentration-dependent manner. The results of this study demonstrate that at least two GC-rich sequences at -559 to -553 and -72 to -64 are required for induction of IGFBP-4 gene expression by E2 in MCF-7 cells.

The two phyto-oestrogens genistein and quercetin exert different effects on oestrogen receptor function.

We compared the oestrogenic and anti-oestrogenic properties of the two well-known phyto-oestrogens, genistein and quercetin, on the oestrogen-sensitive breast cancer cell line MCF-7. Genistein exerted a biphasic effect on growth of MCF-7 cells, stimulating at low and inhibiting at high concentrations, whereas quercetin was only growth inhibitory. At doses which did not inhibit cell growth, respectively 5 and 1 μM, genistein and quercetin counteracted oestrogen- and transforming growth factor-α-promoted cell growth stimulation. Furthermore, genistein promoted transcription of the oestrogen-regulated genes pS2 and cathepsin-D, whereas quercetin interfered with the oestrogen-induced expression of the proteins. In in vitro binding experiments, genistein competed with oestradiol for binding to the oestrogen receptor (ER), but quercetin did not. Quercetin and genistein down-regulated cytoplasmic ER levels and promoted a tighter nuclear association of the ER, but only genistein was able to up-regulate progesterone receptor protein levels. In gel mobility assays, ER preincubation with oestradiol or with the two phyto-oestrogens led to the appearance of the same retarded band, excluding differences between the various complexes in binding to the consensus sequence. The data allowed us to conclude that quercetin acts like a pure anti-oestrogen, whereas genistein displays mixed agonist/antagonist properties, and to formulate a hypothesis on the possible mechanism of action of such phyto-oestrogens. © 1999 Cancer Research Campaign

Structure and regulatory expression of a single copy alternative oxidase gene from the yeast Pichia anomala.

To investigate the regulatory mechanism of alternative oxidase gene expression, genomic DNA was cloned from the yeast Pichia anomala. Genomic Southern blot analysis suggested that a single copy nuclear gene encoded an alternative oxidase in the yeast. The nucleotide sequence showed an uninterrupted coding region for the alternative oxidase protein. In the upstream region from the transcription initiation site found by primer extension analysis, CCAAT, TATAA, and UAS2-like elements were detected. The UAS2 is the element involved in transcriptional regulation by carbon source and the target site for the factor, HAP2/3/4/5 protein complex, in Saccharomyces cerevisiae. By a gel mobility shift assay, a specific retardation band was detected when a protein extract from cells grown on an inducing carbon source was incubated with a UAS2-containing probe. These results suggest that carbon source regulation of alternative oxidase gene expression is mediated by the UAS2-like element and a HAP-like factor in P. anomala.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense.

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma 54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

A genome-derived (gaa.ttc)24 trinucleotide block binds nuclear protein(s) specifically and forms triple helices.

The properties of simple trinucleotide repeats generate increased interest as expansions of certain trinucleotide blocks cause human diseases. Here, we studied protein binding and structural features of a perfect (gaa.ttc)24 tract in its original genomic environment. Electrophoretic mobility shift assays revealed that HeLa nuclear proteins bind to the DNA fragment containing the (gaa.ttc)24 block. Competition experiments using simple (gt.ac)n repeats differing in length and flanking regions showed no cross-reactivity with the major retarded band. For the specific (gaa. ttc)n/protein complex, a binding constant of 9.3x10-9 mol/l was determined. DNase I footprinting revealed protein binding sites located exclusively within the repeat with a preference for the (gaa)24 strand. OsO4 and DEPC modifications followed by electrophoretic and electron microscopical analyses showed that the (gaa.ttc)24 block forms different types of intramolecular triple helices: Under superhelical stress, different H-DNA isomers are evident, whereas exclusively H-Y forms were detected in the relaxed state. Together, these data have functional implications for genomic (gaa.ttc)n tracts.

Identification of the Rhodobacter sphaeroides SOS box.

Gel-mobility shift assays with crude cell extracts of Rhodobacter sphaeroides, which belongs to the alpha group of the proteobacteria, have shown that a protein binds to the promoter of its recA gene, resulting in two retardation bands. Analysis of the minimal region of the R. sphaeroides recA gene required for the formation of the DNA-protein complexes, revealed the presence of the motifs GTTCN7GATC and GAACN7GAAC, which are centred at positions -21 and +8 from the transcriptional starting point respectively. Using PCR mutagenesis, we have demonstrated that these two motifs are required for the formation of both DNA-protein complexes in vitro as well as for the DNA damage-mediated inducibility of the recA gene in vivo. Furthermore, the level of the recA gene expression in the constitutive mutants is the same as that achieved by the wild-type cells after DNA damage, indicating that the binding protein must be a repressor. The motif GTTCN7GTTC is also present upstream of the R. sphaeroides uvrA promoter, which in vitro specifically binds to a protein and whose expression is DNA damage inducible. Mutagenesis of this motif abolishes both the binding of this protein to the uvrA promoter and the DNA damage-mediated expression of this gene. The fact that the recA and uvrA wild-type promoters compete with each other for the retardation band formation, but not with their mutant derivatives in any of these motifs, indicates that the same repressor binds to the operator of both genes. All these results lead us to propose the sequence GTTCN7GTTC as the SOS box of R. sphaeroides. This is the first SOS box known whose sequence is a direct repeat and not a palindrome.

The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperativity.

The products of the mammalian Pbx and Drosophila exd genes are able to interact with Hox proteins specifically and to increase their DNA binding affinity and selectivity. In the accompanying paper we show that Pbx proteins exist as stable heterodimers with a novel homeodomain protein, Prep1. Here we show that Prep1-Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N-terminal portions of both proteins. The Prep1-Pbx protein-protein interaction is essential for DNA-binding activity. Prep1-Pbx complexes are present in early mouse embryos at a time when Pbx is also interacting with Hox proteins. The use of different interaction surfaces could allow Pbx to interact with Prep1 and Hox proteins simultaneously. Indeed, we observe the formation of a ternary Prep1-Pbx1-HOXB1 complex on a HOXB1-responsive target in vitro. Interaction with Prep1 enhances the ability of the HOXB1-Pbx1 complex to activate transcription in a cooperative fashion from the same target. Our data suggest that Prep1 is an additional component in the transcriptional regulation by Hox proteins.

Estrogen Does Not Inhibit 2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated Effects in MCF-7 and Hepa 1c1c7 Cells

The estrogen receptor and aryl hydrocarbon receptor (AhR) are coexpressed in several Ah and estrogen-responsive human breast cancer cell lines. However, a recent study reported that 17beta-estradiol (E2) inhibited Ah responsiveness in mouse Hepa 1c1c7 hepatoma cells (Kharat, I., and Saatcioglu, F. (1996) J. Biol. Chem. 271, 10533-10537), and therefore, estrogen receptor-AhR cross-talk was reinvestigated in MCF-7 and mouse Hepa 1c1c7 cells. Treatment of MCF-7 or Hepa 1c1c7 cells with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in induction of CYP1A1-dependent activity and mRNA levels. Treatment of both cell lines with E2 had no effect on basal or TCDD-inducible CYP1A1-dependent activity or mRNA levels. In MCF-7 and Hepa 1c1c7 cells transiently transfected with an Ah-responsive plasmid containing the 5'-regulatory region of the human CYP1A1 gene fused to the chloramphenicol acetyltransferase reporter gene 10 nM TCDD significantly induced chloramphenicol acetyltransferase activity; in cells cotreated with TCDD plus E2 the induced response was not affected by the hormone. Nuclear extracts from cells treated with dimethyl sulfoxide, E2, TCDD, and TCDD plus E2 were incubated with the [32P]dioxin-responsive element and analyzed by gel electrophoretic mobility shift assays. A retarded band associated with formation of a [32P]dioxin-responsive element-AhR complex was observed in nuclear extracts from cells treated with TCDD or TCDD plus E2 (cotreated). Collectively these studies suggest that E2 does not modulate AhR-mediated CYP1A1 gene expression in MCF-7 or Hepa 1c1c7 cells.

DNA Bending Is Essential for the Site-specific Recognition of DNA Response Elements by the DNA Binding Domain of the Tumor Suppressor Protein p53

We have used circular permutation assays to determine the extent and location of the DNA bend induced by the DNA binding domain of human wild type p53 (p53DBD) upon binding to several naturally occurring DNA response elements. We have found that p53DBD binding induces axial bending in all of the response elements investigated. In particular, response elements having a d(CATG) sequence at the junction of two consensus pentamers in each half-site favor highly bent complexes (bending angle is approximately 50 degrees ), whereas response elements having d(CTTG) bases at this position are less bent (bending angles from approximately 37 to approximately 25 degrees ). Quantitative electrophoretic mobility shift assays of different complexes show a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with the response elements, i.e. the greater the stability of the complex, the more the DNA is bent by p53DBD binding. The study provides evidence that the energetics of DNA bending, as determined by the presence or absence of flexible sites in the response elements, may contribute significantly to the overall binding affinity of the p53DBD for different sequences. The results therefore suggest that both the structure and the stability of the p53-DNA complex may vary with different response elements. This variability may be correlated with variability in p53 function.

Identification of a functional imperfect estrogen-responsive element in the 5'-promoter region of the human cathepsin D gene.

17beta-Estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast cancer cells. Previous studies have identified an Sp1-imperfect estrogen-responsive element (ERE) half-site [GGGCGG(N)23ACGGG] (-199 to -165) in the promoter region which forms an Sp1-estrogen receptor (ER) complex and confers E2 responsiveness on the corresponding Sp1-ERE-chloramphenicol acetyl transferase (CAT) construct. Further analysis of downstream regions of the promoter identified a CGCCC(N)3TGACC sequence (-119 to -107) which is homologous to the adenovirus major late promoter element (MLPE) and binds the ER to form a retarded band in a gel electrophoretic mobility shift assay. The corresponding promoter-CAT construct is also E2-inducible. The MLPE resembles an imperfect palindromic ERE containing imperfect (5') and perfect (3') ERE half-sites; analysis of oligonucleotides with mutations in these half-sites shows that only the perfect ERE half-site is required for binding the ER, whereas both sites are required for transactivation. In vivo exonuclease III footprinting showed that treatment with E2 also enhanced binding at the MLPE site. Identification of this second functional enhancer sequence in the 5'-promoter region of cathepsin D is consistent with the increasingly complex cell-specific regulation of hormone-responsive genes.

Growth hormone modulates insulin regulation of hepatic insulin-like growth factor binding protein-1 transcription.

Hepatic transcription of insulin-like growth factor-binding protein-1 (IGFBP-1) is enhanced in hypophysectomized (hypox) rats and can be rapidly down-regulated by GH administration. Here we examined the effect of insulin on IGFBP-1 messenger RNA abundance in hypox rats and the effects of insulin and GH on IGFBP-1/chloramphenicol acetyltransferase (CAT) reporter plasmids transiently transfected into isolated hepatocytes from pituitary-intact and hypox rats. Unlike GH, administration of insulin to hypox rats in doses of 10 or 50 micrograms/100 g BW had no effect on hepatic IGFBP-1 messenger RNA abundance. Insulin at 10(-7) M resulted in a 42.1 +/- 9.8% suppression of CAT activity in hepatocytes from pituitary-intact animals transfected with a CAT reporter plasmid containing 1671 bp of the 5'-flanking region of the rat IGFBP-1 gene. In the same assay, GH at a concentration of 2.3 x 10(-8) M significantly reduced CAT activity. In contrast, insulin had no effect on CAT activity in hepatocytes from hypox rats, whereas GH resulted in comparable suppression of CAT activity in hepatocytes from hypox rats and pituitary-intact rats, 13.6 +/- 2.3% vs. 18.2 +/- 3.2%. Deletional analysis and mobility shift assays were used to identify the GH-responsive regions in the IGFBP-1 gene. GH suppression of CAT activity was lost when the IGFBP-1 5'-flanking region was deleted down to -277 bp, whereas insulin suppression was retained for all but the smallest fragment of the IGFBP-1 gene. Mobility shift assays were used to compare nuclear extracts from sham-operated, hypox, and GH-treated hypox rats. When hepatic nuclear extracts from hypox rats were incubated with the -277 to -82 and the -556 to -368 bp fragments, retarded bands were apparent that were not present in the extracts from sham-operated rats. GH treatment of hypox rats 15 or 30 min before death completely normalized the retardation pattern seen with the -277 to -82 bp fragment, but did not affect the pattern seen with the -556 to -368 bp fragment. A 20-bp fragment corresponding to the previously identified insulin response element, -108 to -89 bp, was also analyzed. An additional retarded band, not seen with nuclear extracts from sham-operated rats, was apparent when nuclear extracts of hypox rats or GH-treated hypox rats were used. These data provide the first in vitro evidence that GH directly regulates transcription of IGFBP-1 expression. In addition, our findings suggest that GH modulates insulin regulation of IGFBP-1 transcription, possibly by altering the milieu of trans-acting factors that interact with both the insulin response element and distinct upstream sites.

Activation of Epidermal Growth Factor Receptor Gene Transcription by Phorbol 12-Myristate 13-Acetate Is Mediated by Activator Protein 2

The response of the epidermal growth factor (EGF) receptor gene to phorbol 12-myristate 13-acetate (PMA) was analyzed using nuclei and nuclear extracts prepared from PMA-treated KB cells. Transient transfection assays and nuclear run-off experiments showed that PMA increased EGF receptor gene transcription. Cell-free transcription with promoter mutants revealed that the region of the promoter containing nucleotides -150 to -16 was sufficient for PMA inducibility. A promoter fragment containing nucleotides -167 to -105 showed increased binding of a factor present in extracts prepared from PMA-treated cells. When this factor was partially purified by column chromatography, it showed specific PMA-dependent binding to an EGF receptor promoter fragment. This binding was competed by an SV40 fragment containing binding sites for Sp1, AP1, and AP2. Purified AP2 was used in DNase I footprinting experiments to show that this factor can bind to the EGF receptor promoter. Oligonucleotides corresponding to the AP2 binding sites found in the EGF receptor promoter showed the ability to bind AP2 and compete for the binding of a factor induced by PMA treatment. The addition of AP2 to nuclear extract resulted in increased transcription from the EGF receptor promoter. These results demonstrate that AP2 can activate EGF receptor gene expression and may mediate the PMA response of this gene.

Inhibition of vitamin D receptor-retinoid X receptor-vitamin D response element complex formation by nuclear extracts of vitamin D-resistant New World primate cells.

Most New World primate (NWP) genera evolved to require high circulating levels of steroid hormones and vitamin D. We hypothesized that an intracellular vitamin D binding protein (IDBP), present in both nuclear and cytoplasmic fractions of NWP cells, or another protein(s) may cause or contribute to the steroid hormone-resistant state in NWP by disruption of the receptor dimerization process and/or by interference of receptor complex binding to the consensus response elements present in the enhancer regions of steroid-responsive genes. We employed electromobility shift assay (EMSA) to screen for the presence of proteins capable of binding to the vitamin D response element (VDRE). Nuclear and post-nuclear extracts were prepared from two B-lymphoblastoid cell lines known to be representative of the vitamin D-resistant and wild type phenotypes, respectively. The extracts were compared for their ability to retard the migration of radiolabeled double stranded oligomers representative of the VDREs of the human osteocalcin and the mouse osteopontin gene promoters. A specific, retarded band containing VDR-RXR was identified when wild type cell but not when vitamin D-resistant cell nuclear extract was used in the binding reaction with either probe. In addition, vitamin D-resistant cell nuclear extract contained a protein(s) which was bound specifically to the VDRE and was capable of completely inhibiting VDR-RXR-VDRE complex formation; these effects were not demonstrated with nuclear extract from the wild type cell line or with the post-nuclear extract of the vitamin D-resistant cell line. We conclude that a VDRE-binding protein(s), distinct from IDBP and present in nuclear extract of cells from a prototypical vitamin D-resistant NWP, is capable of inhibiting normal VDR-RXR heterodimer binding to the VDRE.

Naringenin: A weakly estrogenic bioflavonoid that exhibits antiestrogenic activity

Treatment of immature 21-day-old female Sprague-Dawley rats with 17β-estradiol (E2) (0.5 μg/rat) caused a significant increase in uterine wet weight, DNA synthesis, progesterone receptor (PR) binding, and peroxidase activity. At doses as high as 40 mg/rat, the bioflavonoid naringenin did not cause a significant increase in any of these E2-induced responses. However, in rats cotreated with E2 (0.5 μg/rat) plus naringenin (30 mg/rat), there was a significant decrease in E2-induced uterine wet weight, DNA synthesis, PR binding, and peroxidase activity, indicating that naringenin exhibits antiestrogenic activity in the immature rodent uterus. The binding of uterine nuclear extracts to a 32P-labeled estrogen responsive element (ERE) or progesterone responsive element (PRE) was determined using gel electrophoretic band shift assays. Incubation of [ 32P]ERE with uterine nuclear extracts from rats treated with naringenin or E2 resulted in the formation of estrogen receptor (ER):ERE complexes; a higher mobility complex was prominent in the extracts from E2-treated rats, whereas a lower mobility complex was observed using nuclear extracts from naringenin-treated animals. There was a significant decrease in the intensity of the E2-induced complex using nuclear extracts from rats treated with E2 plus naringenin. In contrast, transformed cytosol from control rats gave an intense ER:ERE complex, whereas the intensity of the band was decreased markedly using transformed uterine cytosol from treated rats. Formation of a PR:PRE complex was also determined using transformed uterine cytosol. Cytosol from E2-treated rats gave an intense retarded band, whereas only weak bands were observed using cytosols from DMSO- (solvent), naringenin-, or naringenin plus E2-treated cells. The results of in vitro studies showed that 1 nM E2 increased (3- to 4-fold) the growth of MCF-7 human breast cancer cells, whereas 1–1000 nM naringenin had no effect on cell proliferation. In cells cotreated with 1 nM E2 plus 1000 nM naringenin, there was a significant decrease in E2-induced cell growth. In MCF-7 cells transiently transfected with a pS2 promoter-regulated luciferase reporter gene, naringenin exhibited weak estrogenic activity. In cells cotreated with 0.1 or 1.0 μM naringenin plus 1 nM E2, naringenin inhibited E2-induced luciferase activity. The results of these studies confirmed that naringenin is a weak estrogen that also exhibits partial antiestrogenic activity in the female rat uterus and MCF-7 human breast cancer cells.

Mutational Analysis of SpvR Binding to DNA in the Regulation of the Salmonella Plasmid Virulence Operon

The Salmonella plasmid-borne spvR gene encodes a 33-kDa regulatory protein that activates transcription of the spvABCD operon during the stationary phase of bacterial growth. We used gel mobility shift assays to demonstrate that SpvR recognizes a specific target DNA sequence within a 318-bp EcoRI- Apa1 fragment upstream of spvA. The addition of unlabeled target DNA to the radioactive labeled DNA-SpvR complex resulted in competitive inhibition of band retardation confirming the specificity of SpvR binding. Introduction of target DNA on a high copy number plasmid into wild-type Salmonella dublin Lane resulted in a substantial decrease of SpvB synthesis, confirming the binding properties of this DNA segment in vivo. Three SpvR mutants were constructed and were shown to abolish the positive regulatory function of SpvR: By site-specific mutagenesis of spvR, three single amino acids within the putative SpvR N-terminal α-helix domains were substituted by prolines. This resulted in loss of binding to the spvA promotor sequence and in loss of activation of the spvABCD genes. This study demonstrates that the regulatory function of SpvR is mediated by specific binding to the promotor region of the spvABCD operon.

Identification of a Strong Transcriptional Activator for the Copia Retrotransposon Responsible for Its Differential Expression in Drosophila hydei and Melanogaster Cell Lines

We have characterized the regulatory properties of a 72bp sequence located in the 5′ untranslated domain of the Drosophila copia retrotransposon, 3′ to the left LTR, by transient transfection assays with cell lines derived from either Drosophila hydei (DH33 cells) or Drosophila melanogaster (Schneider II and Kc cells). Reporter plasmids were constructed which contained the lacZ gene under the control of either the entire copia LTR with 5′ untranslated domain, or a minimal heterologous promoter flanked with the identified copia regulatory sequences. Upon transfection into the copia-free DH33 cells, the presence of the 72bp sequence resulted for all reporter plasmids in a 100-700 fold increase in expression level -as well as in reporter gene RNA levels- whereas this sequence had no enhancing effect upon transfection of the same plasmids into the copia-containing Schneider II or Kc cells. Moreover, mobility shift assays with the 72bp enhancer sequence disclosed two specific bands of retarded mobility with whole-cell extracts from DH33 cells, whereas no retarded band could be detected, under identical conditions, with extracts from Schneider II cells. UV crosslinking experiments between the enhancer sequence and DH33 extracts revealed a single protein species -of app. mel. wt. 50kD- for both retarded bands, thus strongly suggesting that they simply correspond to the sequential binding of two identical factor molecules to the enhancer sequence. These data demonstrate that the copia-free D. hydei cells express a strong transcriptional activator for the copia element and possible interpretations for the absence of this factor in the copia-containing D. melanogaster cells are discussed in terms of a possible "adaptation" of the "host" (D. melanogaster) to an otherwise highly mutagenic "parasite" (copia with its transcription factor).

Inhibition of estrogen-induced progesterone receptor in MCF-7 human breast cancer cells by aryl hydrocarbon (Ah) receptor agonists

17β-Estradiol (E 2) induces progesterone receptor (PR) binding, immunoreactive protein, nuclear PR formation and PR mRNA levels in MCF-7 human breast cancer cells. Gel mobility shift analysis of nuclear extracts from E 2-treated cells also exhibited a higher intensity retarded band associated with formation of a PR complex with a consensus [ 32P]progesterone/glucocorticoid responsive element. In contrast, 1nM2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) alone did not alter or decrease these same responses in MCF-7 cells; however, in cells co-treated with 1 nM TCDD plus 1 nM E 2, TCDD significantly inhibited all the E 2-induced responses. Scatchard analysis of PR binding demonstrated that TCDD decreased the number of E 2-induced PR cellular binding sites but not the binding affinity of the PR for a radiolabeled promegestrone. In parallel studies, 3-methylcholanthrene, a prototypical polynuclear aromatic hydrocarbon, also inhibited E 2-induced PR binding and immunoreactive protein. For a series of halogenated aromatics including 2,3,7,8- and 1,2,7,8-tetrachlorodibenzofuran, 1,3,7,8-TCDD and 6-methyl-l,3,8-trichlorodibenzofuran, their rank order potency for inhibiting E 2-induced PR binding paralleled their rank order binding to the aryl hydrocarbon (Ah) receptor. These results support a role for the Ah receptor in mediating the antiestrogenic activity of polynuclear and halogenated aromatic hydrocarbons and illustrate cross-talk between the Ah and estrogen receptor signal transduction pathways.

Cloning and expression of an equine herpesvirus 1 origin-binding protein

Equine herpesvirus 1 (EHV-1) is an important pathogen of horses and is closely related to several important human pathogens, herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and varicella-zoster virus. The EHV-1 genome contains open reading frames similar in sequence to the HSV-1 replication genes. PCR was used to clone EHV-1 gene 53, which is similar in sequence to the HSV-1 UL9 gene. The gene 53 product has regions of striking similarity to the HSV-1 UL9 and VZV gene 51 products. In vitro transcription and translation of this gene generated a protein of 87 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further characterization of this protein was accomplished through the use of gel shift analysis. The in vitro-synthesized protein bound sequence specifically to EHV-1 OriS as well as HSV-1 OriS. A site was used in gel shift analysis to show that the EHV-1 origin-binding protein bound to the same consensus site as the HSV-1 origin-binding protein, 5'-CGTTCGCACTT-3'. Using a nuclear extract of EHV-1-infected RK13 cells, we have identified an activity that interacts similarly with this consensus site. In gel shift assays, the retarded band arising from the nuclear extract migrated similarly to the retarded band arising from in vitro-translated EHV-1 gene 53. An N-terminal deletion of EHV-1 gene 53 was also created, expressed in vitro, and used in gel shift assays to localize the DNA-binding domain. Results of these experiments indicated that amino acids 1 to 499 were dispensable for binding and that the C-terminal fragment (amino acids 500 to 888) recognized the same consensus site as did the wild-type protein. Thus, the product of EHV-1 gene 53 is an origin-binding protein with a high degree of similarity to the HSV-1 and varicella-zoster virus origin-binding proteins and possibly serves as the initiator of DNA replication in EHV-1.

Transcriptional regulation of the MUC1 mucin gene in colon carcinoma cells by a soluble factor. Identification of a regulatory element.

We have investigated a mechanism of the regulation of mucin core polypeptide (MUC1) gene expression, which is induced by a soluble stimulatory factor, in KM12C human colon carcinoma cells. Conditioned media from normal human colon tissues elevated the level of expression of MUC1 mRNA. Transcriptional activation of the MUC1 gene was analyzed by transient expression of MUC1-CAT reporter plasmids containing the 5'-flanking sequence of the MUC1 gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. A region between base pairs -531 and -520 of the 5'-flanking sequence of the MUC1 gene was sufficient for the induction of CAT activity by normal colon conditioned medium (NCCM). Mutagenesis of 3 base pairs within the region corresponding to sequence -531 to -517 from ACAGGGAGCGGTTAG to ACAGGGAGATTTTAG substantially decreased the induction of CAT activity by NCCM. Nuclear extracts from untreated or NCCM-treated KM12C cells were tested for their interaction with 32P-labeled oligonucleotides corresponding to this sequence. A specifically retarded band was identified after electrophoretic analysis. The quantity or mobility of this band was not changed by NCCM treatment. When an oligonucleotide with three point mutations was used as a competitor, the retarded band remained at the same position. This element (positions -531 to -520), which we call the responsive mucin element, does not contain any sequence that corresponds to previously described cis-acting elements. A protein component complexed with this sequence was identified with a molecular mass of approximately 70 kDa by SDS-polyacrylamide gel electrophoresis.

Two thyroid hormone response elements are present in the promoter of human thyroid hormone receptor beta 1.

Autoregulation of the human thyroid hormone receptor beta 1 (hTR beta 1) promoter was assessed by chloramphenicol acetyltransferase and luciferase reporter assays of transient transfections into COS1 and GH3 cells, DNase I footprinting, and gel shift assays. A 5'-deletional analysis of the promoter showed that the region between -906 and -839 and the sequence from -438 to -130 were positively regulated by T3 in COS1 cells cotransfected with an hTR beta 1 expression vector. We also transfected deletion constructs into GH3 cells and showed similar effects of T3 on the trans-activation of the reporters. DNase I footprinting showed a protected inverted palindromic thyroid response element (TRE) at position -890 to -866 in the distal fragment and a direct repeat at position -190 to -166 in the proximal fragment, which were protected by TRs. Mutation of each TRE significantly decreased the trans-activation of the promoter by T3. Gel mobility shift assays showed both proximal and distal TREs formed a retarded band with hTR alpha 1 or hTR beta 1 expressed in COS1 cells and reticulocyte lysates. The bands formed on the distal TRE and the proximal TRE appear to be preferentially formed by a TR homodimer and a heterodimer, respectively. Furthermore, the band formed on the distal TRE disappeared after adding T3 but that on the proximal TRE did not. These results indicate that hTR beta 1 expression is directly regulated by hTR alpha 1, beta 1, and their ligand through two TREs. The different structure of the TREs in this promoter suggests their physiological role in transcriptional regulation may be different.