Transient Cotransfection Assays Research Articles

The cellular stress response enhances human T-cell lymphotropic virus type 1 basal gene expression through the core promoter region of the long terminal repeat.

Viral protein expression is postulated to play a critical role in the pathogenesis of human T-cell lymphotropic virus type 1 (HTLV-1)-associated diseases. Therefore, knowledge of the cellular events which initiate or enhance viral gene expression is important in understanding the mechanism of HTLV-1-induced disease. In this report, we examined the modulation of transcription of the HTLV-1 long terminal repeat (LTR) following induction of the cellular stress response. We demonstrate by both in vitro transcription assays and transient transfections that induction of the stress response increases basal transcription from the LTR. Transient cotransfection assays indicate that stress induction of viral transcription is Tax independent. In addition, we provide evidence that the sequences responsible for the enhanced transcription are -52 through +157 of the U3/R region of the HTLV-1 LTR. Finally, our data suggest that the increase in transcription is mediated through an intermediate polymerase II/polymerase III transcriptional complex, demonstrated by the inability to abolish the effect with low concentrations of alpha-amanitin.

Evidence that the UL84 gene product of human cytomegalovirus is essential for promoting oriLyt-dependent DNA replication and formation of replication compartments in cotransfection assays.

The protein products of 11 viral genomic loci cooperate in a transient cotransfection assay to mediate lytic-phase DNA replication of oriLyt, the human cytomegalovirus (HCMV) origin of replication. Six of these genes have homology with the well-characterized herpes simplex virus replication genes and encode core replication machinery proteins that are typically essential for DNA synthesis. The remaining five HCMV gene loci, initially referred to as auxiliary components, include several known immediate-early (IE) transcriptional regulatory proteins as well as genes encoding functionally uncharacterized polypeptides. Some or all of the auxiliary components may be necessary in trans to replicate the HCMV oriLyt only because they are required for efficient expression or transactivation of the native early promoters and 3' processing elements included in the genomic clones. Therefore, we reassessed the requirements for the auxiliary components by adding constitutive heterologous promoters and control signals to the coding regions and carrying out transient DpnI replication assays in cotransfected Vero cells. The results revealed that in the presence of the UL69 posttranscriptional activator and the remaining auxiliary polypeptides, UL84 was the only auxiliary component that could not be omitted to obtain oriLyt-dependent DNA replication. Nevertheless, in human diploid fibroblasts, some additional auxiliary loci as well as UL84 were critical. There was also an obligatory requirement for UL84, in cooperation with two other auxiliary factors, UL112-113 and IE2, and the core machinery, to constitute the minimal HCMV proteins necessary to direct oriLyt-dependent DNA amplification. However, the Epstein-Barr virus core replication genes could substitute for the HCMV core genes, and in these circumstances, UL84 alone directed amplification of HCMV oriLyt. Moreover, there was also an absolute requirement for UL84 along with the core and other auxiliary factors for the formation of intranuclear replication compartments as assayed by immunofluorescence in transient DNA cotransfection assays. These compartments were typical of those associated with active viral DNA replication in HCMV-infected cells, they incorporated pulse-labeled bromodeoxyuridine, and their formation was both phosphonoacetic acid sensitive and oriLyt dependent. These results demonstrate that UL84 is obligatory for both intranuclear replication compartment formation and origin-dependent DNA amplification and suggest that it is a key viral component in promoting the initiation of HCMV oriLyt-directed DNA replication.

Sp1 binding plays a critical role in Erb-B2- and v-ras-mediated downregulation of alpha2-integrin expression in human mammary epithelial cells.

The human alpha2-integrin gene is transcriptionally downregulated in a nontumorigenic human mammary epithelial cell line, MTSV1-7, and its clonal variant HB2, overexpressing the Erb-B2 oncogene. In this study, we have used deletion mutations within the alpha2-integrin promoter inserted 5' of the chloramphenicol acetyltransferase or luciferase reporter genes to identify the element that is responsible for the Erb-B2-mediated downregulation. The results of the transient-transfection assay showed that the Sp1 binding element located in the core region (positions --64 to +1) of the alpha2-integrin promoter plays an essential role in the alpha2-integrin promoter activity and its downregulation by Erb-B2. By gel shift assay, we have demonstrated that this element binds with a high degree of affinity not only to Sp1, but also to Sp3. The downregulation of the alpha2-integrin promoter activity could also be achieved by overexpression of v-Hras (v-ras), suggesting that the signals generated by Erb-B2, which lead to downregulation of the alpha2-integrin gene expression, may proceed through the ras pathway. Both the Erb-B2- and the v-ras-overexpressing cells exhibited a Sp1 DNA binding activity lower than that of the parental line, while the relative levels of Sp1 protein in these cells were not altered. The Erb-B2- and v-ras-mediated downregulation could be reversed by the overexpression of Sp1 and by a dominant negative variant of ras (rasN17), confirming the importance of Sp1 and the ras pathway. The inhibitory effects of Erb-B2 on transcriptional activity of the alpha2-integrin promoter were observed in transient-cotransfection assays using alpha2-integrin reporter plasmids and plasmids expressing the Erb-B2 or v-ras oncogene. The same effects were seen when an alpha2-integrin reporter gene construct was transfected into MTSV1-7 or HB2 cells permanently overexpressing Erb-B2 or v-ras. The effects of Erb-B2 or v-ras on the transcriptional activity of the alpha2-integrin promoter were observed in nontumorigenic luminal epithelial cell lines (MTSV1-7 and HB2) as well as in the breast cancer cell line T47D. These data suggest that in luminal epithelial cells and the breast cancers which develop from them, the Erb-B2 proto-oncogene signaling leads to inhibition of (alpha)2(beta)1-integrin gene expression and could contribute to the disruption of tissue architecture seen in breast cancers.

A significant decrease of the transcriptional activity of p53 mutants deriving from human functional adrenal tumors.

Recently, our laboratory has found a high incidence (77%) of p53 gene mutations in human functional adrenal tumors. Furthermore, the majority of mutant sites were assembled at codons 100, 102, and 249. These mutation sites are not common, and there have been no studies addressing whether or not these mutants points or mutant styles cause the p53 protein to lose function. It has been well known that p53 is a transcription factor. To examine the transcriptional activities of these mutant p53 genes from patients with functional adrenal tumors, we constructed p53 expression plasmids from tumors and paired adjacent normal adrenal gland tissues, using a transient co-transfection assay with a reporter gene in H358 cells. Wild-type p53 from normal adrenal gland tissues specifically trans-activates the expression of a chloramphenicol acetyltransferase (CAT) reporter gene in H358 cells. Three mutant p53 proteins (at codons 100, 102, and 249, respectively) from tumors showed a >90% loss of transcriptional activity. One mutant at codon 68, other than at hot spots, remained at approximately 65% transcriptional activity. An immunoprecipitation assay showed that the mutant proteins of codon 68 and codon 102 could respond to the three monoclonal antibodies (PAbDO-1, PAb1620, and PAb421), indicating that there were no obvious changes in the antigenicity of the proteins. However, the mutant protein of codon 249 could not respond to the carboxy-terminus-specific antibody PAb421 and conformation-specific antibody PAb1620, indicating that there were some obvious changes in the conformation of the mutant proteins. The mutant protein of codon 100 could not be detected by immunoprecipitation assay but could be analyzed by Western blot. In a further study using a DNA-binding assay, it was shown that the loss of transcriptional activity was caused by the loss of DNA-binding ability. These results show that the p53 mutants, derived from functional adrenal tumors, actually lost DNA-binding ability and decreased the transcriptional activity. However, the role of the mutant protein in the tumorigenesis of functional adrenal tumors requires further investigation.

Regulation of Mouse Mast Cell Protease 6 Gene Expression by Transcription Factor Encoded by the mi Locus

The mi locus of mice encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF). Because the expression of the mouse mast cell protease 6 (MMCP-6) gene is remarkably reduced in mast cells of mi/mi mutant mice, we investigated the effect of MITF on the transcription of the MMCP-6 gene. First, we introduced the normal (+) MITF cDNA into mi/mi cultured mast cells using the retroviral vector. Overexpression of +-MITF but not mi-MITF normalized the expression of the MMCP-6 gene, indicating the involvement of +-MITF in the MMCP-6 gene transactivation. Second, we analyzed the promoter of the MMCP-6 gene by the transient cotransfection assay. The luciferase construct under the control of the MMCP-6 promoter and the cDNA encoding +-MITF or mi-MITF were cotransfected into NIH/ 3T3 fibroblasts. The coexpression of +-MITF but not mi-MITF increased the luciferase activity 10-fold. We found a CACATG and a CATCTG motif in the MMCP-6 promoter, both of which are generally recognized by bHLH-Zip-type transcription factors. We also found a GACCTG motif that was strongly bound by +-MITF. These three motifs were necessary for the 10-fold transactivation ability of the MMCP-6 promoter by +-MITF. Mutations of each motif significantly reduced the transactivation, suggesting that +-MITF directly transactivated the MMCP-6 gene through these three motifs.

Cooperation of the tumour suppressors IRF-1 and p53 in response to DNA damage.

Normally growing cells promptly cease DNA synthesis when exposed to genotoxic stresses, such as radiation, and this cell-cycle arrest prevents the accumulation of mutations. The transcription factor interferon regulatory factor (IRF)-1 is essential for the regulation of the interferon system, inhibits cell growth, and manifests tumour-suppressor activities. Here we show that mouse embryonic fibroblasts (EFs) lacking IRF-1 are deficient in their ability to undergo DNA-damage-induced cell-cycle arrest. A similar phenotype has been observed in EFs lacking the tumour suppressor p53 (refs 8, 9), although the expression of IRF-1 and p53 are independent of one another. Furthermore, we show that transcriptional induction of the gene encoding p21 (WAF1, CIP1), a cell-cycle inhibitor, by gamma-irradiation is dependent on both p53 and IRF-1, and that the p21 promoter is activated, either directly or indirectly, by both in a transient cotransfection assay. These two tumour-suppressor transcription factors therefore converge functionally to regulate the cell cycle through the activation of a common target gene.

Identification of a binding site in c-Ab1 tyrosine kinase for the C-terminal repeated domain of RNA polymerase II.

The c-abl proto-oncogene encodes a nuclear tyrosine kinase that can phosphorylate the mammalian RNA polymerase II (RNAP II) on its C-terminal repeated domain (CTD) in vitro. Phosphorylation of the CTD has previously been shown to require the tyrosine kinase and the SH2 domain of Abl. We show here that a CTD-interacting domain (CTD-ID) at the C-terminal region of c-Abl is also required. Deletion of the CTD-ID causes the Km 0.4 microM to increase by 2 orders of magnitude. Direct binding of the CTD-ID to CTD and to RNAP II could be demonstrated in vitro. Phosphorylation of a recombinant glutathione S-transferase-CTD by c-Abl was observed in cotransfected COS cells. Mutant Abl proteins lacking the CTD-ID, while capable of autophosphorylation, neither phosphorylated nor associated with the glutathione S-transferase-CTD in vivo. Transient overexpression of c-Abl also led to a four- to fivefold increase in the phosphotyrosine content of the RNAP II large subunit. Moreover, the large subunit of RNAP II could be coprecipitated with c-Abl. Tyrosine phosphorylation of the coprecipitated RNAP II was again dependent on the presence of the CTD-ID in Abl. Finally, the ability of c-Abl to phosphorylate and associate with RNAP II could be correlated with the enhancement of transcription by c-Abl in transient cotransfection assays. Taken together, these observations demonstrate that c-Abl can function as a CTD kinase in vitro as well as in vivo.

Silencing of the Gene for the β Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

The transcription factor Oct-3/4 may be important in maintaining embryonic cells in an undifferentiated state. It is probably down-regulated at about the time that human chorionic gonadotropin (hCG) is first expressed in embryonic trophectoderm. Here we report that Oct-3/4 strongly inhibits the hCGbeta subunit (hCGbeta) promoter in JAr choriocarcinoma cells. Oct-3/4 reduced chloramphenicol acetyltransferase (CAT) reporter expression from the -305hCGbeta promoter by about 90% in transient co-transfection assays, but had no effect on expression from the -249hCGbeta promoter. The -305/-249 hCGbeta fragment specifically bound synthetic Oct-3/4 protein as measured in electrophoretic mobility shift assays, and the Oct-3/4-binding site was localized around -270 by methylation interference footprinting. Site-directed mutagenesis of this binding site abolished Oct-3/4 repression. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGbeta messenger RNA and hCG protein to less than 10% of controls. We suggest that silencing of Oct-3/4 in trophectoderm is a prerequisite for hCG up-regulation in early human embryos at the time of maternal recognition of pregnancy.

Molecular Cloning, Expression and Characterization of Human Peroxisome Proliferator Activated Receptors γ1 and γ2

We describe the molecular cloning and expression of cDNAs encoding human PPARγ1 and PPARγ2. Our sequences are distinct from the published sequence at 3 positions, resulting in nonconservative amino acid substitutions. In humans, PPARγ mRNA is expressed in spleen, bone marrow, liver, testis, skeletal muscle and brain, in addition to fat. Three thiazolidinediones were found to 1) displace a radiolabeled thiazolidinedione from both receptors with essentially the same IC50s and 2) to transactivate both PPARγ isoforms with similar EC50s in transient cotransfection assays utilizing the adipocyte-specific aP2 promoter. Saturating concentrations of these 3 thiazolidinediones altered the conformation ofin vitrosynthesized PPARγ protein producing a 27 kDa protease-resistant fragment. These results indicate that the antidiabetic effects of thiazolidinediones in humans are likely to be mediated via binding to and transactivation of PPARγ1 and γ2.

Expression and Regulation of the Lipoprotein Lipase Gene in Human Adrenal Cortex

Lipoprotein lipase (LPL), an enzyme which hydrolyzes triglycerides and participates in the catabolism of remnant lipoproteins, plays a crucial role in energy and lipid metabolism. The goal of this study was to analyze the expression and regulation of the LPL gene in human adrenals. Reverse transcriptase-polymerase chain reaction amplification and sequence analysis demonstrated the presence of LPL mRNA in fetal and adult human adrenal cortex. Furthermore, the human adrenocortical carcinoma cell line, NCI-H295, expresses LPL mRNA and protein, which is localized to the outer cellular membrane as demonstrated by immunofluorescence confocal microscopy and can be released in the medium by heparin addition. To asses whether the LPL gene is regulated by agents regulating adrenal steroidogenesis, NCI-H295 cells were treated with activators of second messenger systems. Whereas the calcium-ionophore A23187 did not affect LPL gene expression, treatment with phorbol 12-myristate 13-acetate decreased LPL mRNA levels in a time- and dose-dependent manner. This decrease after phorbol 12-myristate 13-acetate was associated with diminished heparin-releasable LPL mass and activity in the culture medium. Addition of the cAMP analog 8-Br-cAMP to NCI-H295 cells resulted in a rapid, but transient dose-dependent induction of LPL mRNA. Treatment with the protein synthesis inhibitor cycloheximide gradually induced, whereas simultaneous addition of cAMP and cycloheximide superinduced LPL mRNA levels. Nuclear run-on analysis indicated that the effects of cAMP and cycloheximide occurred at the transcriptional and post-transcriptional level, respectively. Transient co-transfection assays demonstrated that the first 230 base pairs of the proximal LPL promoter contain a cAMP-responsive element activated by protein kinase A and transcription factors belonging to the CREB/CREM family. These data indicate that LPL is expressed in human adrenal cortex and regulated in NCI-H295 adrenocortical carcinoma cells by activators of the protein kinase A and protein kinase C second messenger pathways in a manner comparable to P450scc, which catalyzes the first step in adrenal steroidogenesis. These observations suggest a role for LPL in adrenal energy and/or lipid metabolism and possibly in steroidogenesis.

Mechanism for transcriptional gain of function resulting from chromosomal translocation in alveolar rhabdomyosarcoma.

The t(2;13) translocation of alveolar rhabdomyosarcoma results in tumor-specific expression of a chimeric transcription factor containing the N-terminal DNA-binding domain of PAX3 and the C-terminal transactivation domain of FKHR. Here we have tested the hypothesis that PAX3-FKHR gains function relative to PAX3 as a consequence of switching PAX3 and FKHR transactivation domains, which were previously shown to have similar potency but distinct structural motifs. In transient cotransfection assays with human expression constructs, we have demonstrated the increased ability of PAX3-FKHR to activate transcription of a reporter gene located downstream of multimerized e5, PRS-9, or CD19 DNA-binding sites in three cell lines. For example, PAX3-FKHR was 100-fold more potent than PAX3 as an activator binding to e5 sites in NIH 3T3 cells. To compare transactivation potency independent of PAX3-specific DNA binding, we tested GAL4 fusions of full-length PAX3 and PAX3-FKHR or their respective C-terminal transactivation domains on a reporter with GAL4 DNA-binding sites. In this context, full-length PAX3-FKHR was also much more potent than PAX3. Additionally, the activity of each full-length protein was decreased relative to its C-terminal domain, demonstrating that N-terminal sequences are inhibitory. By deletion analysis, we mapped a bipartite cis-acting inhibitory domain to the same subregions within the DNA-binding domains of both PAX3 and PAX3-FKHR. We have shown, however, that the structurally distinct transactivation domains of PAX3 and PAX3-FKHR differ 10- to 100-fold in their susceptibility to inhibition, thus elucidating a mechanism by which PAX3 gains enhanced function during oncogenesis.

The Myeloid-Cell-Specific c-fes Promoter Is Regulated by Sp1, PU.1, and a Novel Transcription Factor

The protein product of the c-fps/fes (c-fes) proto-oncogene has been implicated in the normal development of myeloid cells (macrophages and neutrophils). mRNA for c-fes has been detected exclusively in myeloid cells and vascular endothelial cells in adult mammals. Although a 13-kilobase-pair (kb) human c-fes transgene exhibits high levels of expression in mice, the sequences that confer myeloid-cell-specific expression of the human c-fes gene have not been defined. Transient-transfection experiments demonstrated that plasmids containing 446 bp of c-fes 5'-flanking sequences linked to a luciferase reporter gene were active exclusively in myeloid cells. No other DNA element within the 13-kb human c-fes locus contained positive cis-acting elements, with the exception of a weakly active region within the 3'-flanking sequences. DNase I footprinting assays revealed four distinct sites that bind myeloid nuclear proteins (-408 to -386, -293 to -254, -76 to -65, and -34 to +3). However, the first two footprints resided in sequences that were largely dispensable for transient activity. Plasmids containing 151 bp of 5'-flanking sequences confer myeloid-cell-specific gene expression. Electrophoretic mobility shift analyses demonstrated that the 151-bp region contains nuclear protein binding sites for Sp1, PU.1, and/or Elf-1, and a novel factor. This unidentified factor binds immediately 3' of the PU.1/Elf-1 sites and appears to be myeloid cell specific. Mutation of the PU.1/Elf-1 site or the 3' site (FP4-3') within the context of the c-fes promoter resulted in substantially reduced activity in transient transfections. Furthermore, transient-cotransfection assay demonstrated that PU.1 (and not Elf-1) can transactivate the c-fes promoter in nonmyeloid cell lines. We conclude that the human c-fes gene contains a strong myeloid-cell-specific promoter that is regulated by Sp1, PU.1, and a novel transcription factor.

The efficacy of 9-cis retinoic acid in experimental models of cancer.

9-cis retinoic acid (9-cis RA) is a retinoid receptor pan-agonist that binds with high affinity to both retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Using a variety of in vivo and in vitro cancer models, we present experimental data that 9-cis RA has activity as a potential chemotherapeutic agent. Treatment of the human promyelocytic leukemia cell line HL-60 with 9-cis RA decreases cell proliferation, increases cell differentiation, and increases apoptosis. Induction of apoptosis correlates with an increase in tissue transglutaminase (type II) activity. In vivo, 9-cis RA induces complete tumor regression of an early passage human lip squamous cell carcinoma xenograft. Finally, 9-cis RA inhibits the anchorage-independent growth of the human breast cancer cell lines MCF-7 and LY2 (an antiestrogen-resistant MCF-7 variant). Transient co-transfection assays indicate that 9-cis RA inhibits estrogen receptor transcription of an ERE-tk-LUC reporter through RAR or RXR receptors. These data suggest that retinoid receptors can antagonize estrogen-dependent transcription and provides one possible mechanism for the inhibition of cell growth by 9-cis RA in breast cancer cell lines. In summary, these findings present evidence that 9-cis RA has a wide range of activities in human cancer models.

Human Cytomegalovirus Immediate-Early Protein IE2 Tethers a Transcriptional Repression Domain to p53

The IE2 gene of human cytomegalovirus has been implicated in the development of coronary restenosis, and the gene product appears to inhibit p53-dependent transactivation. Here we describe an analysis of the IE2-p53 interaction. Repression of p53 function by IE2 requires two separable domains of IE2. The N terminus of IE2 interacts with p53. IE2 has little effect on the ability of p53 to bind specific DNA sequences. Reduction of the transactivation activity of p53 is caused by a transcriptional repression function contributed by the C-terminal domain of IE2. These findings suggest that IE2 may function as a transcriptional repressor, which is recruited to p53's target genes by interacting with p53.

Interaction of bovine herpesvirus 4 (BHV-4) immediate early 2 gene product with BHV-4 thymidine kinase promoter-regulatory region.

To understand the mechanism of regulation of bovine herpesvirus 4 (BHV-4) early gene expression by immediate-early (IE) gene products, we studied activation of expression of the BHV-4 early gene encoding thymidine kinase (TK). BHV-4 encodes two IE RNAs. IE RNA 2 encodes a protein with predicted amino acid sequence similarity to the Epstein-Barr virus (EBV) R transactivator. The BHV-4 TK promoter-regulatory region was specifically transactivated approximately 9-fold by the IE2 gene product in transient expression cotransfection assays. Deletion analysis localized the IE2 responsive element(s) within a 72 bp fragment 5' to the start of transcription. Multiple copies of this 72 bp fragment conferred IE2-responsiveness on an enhancerless simian virus 40 (SV40) promoter and mediated IE2 transactivation in either orientation. Gel retardation assays demonstrated a sequence-specific complex between the IE2 gene product and the 72 bp DNA fragment, and indicated that the IE2 gene product binds DNA as a dimer. Sequences contained in a 25 bp subfragment were sufficient for IE2-DNA complex formation. No nucleotide sequence similarity was noted between the TK IE2-binding fragment and the consensus binding site for the EBV R transactivator. A single amino acid change in the putative DNA-binding domain of the IE2 gene product abolished both complex formation in vitro and transactivation activity in vivo.

Transcriptional effects in GH3 cells of Gs alpha mutants associated with human pituitary tumors: stimulation of adenosine 3',5'- monophosphate response element-binding protein-mediated transcription and of prolactin and growth hormone promoter activity via protein kinase A

Somatic mutations of the alpha-subunit of Gs (Gs alpha) have been detected previously at high frequency in human PRL- and/or GH-producing pituitary tumors. To test whether these mutants are responsible for the increased production of these hormones, we used transient cotransfection assays to analyze their genomic effects in GH3 rat pituitary cells. We first show that guanosine triphosphatase (GTPase)-deficient Gs alpha subunits (mutated at amino acid 201 or 227) stimulate transcription from a reporter construct bearing the consensus cAMP response element (CRE; TGACGTCA). Using GAL4-CRE-binding protein fusion constructs, we further show that this stimulatory effects of Gs alpha on the CRE is probably mediated by the transacting factor CRE-binding protein. Then, in experiments using a reporter gene driven by the human promoters for either the PRL (position -250 to 18) or GH (position -500 to 13) genes, we show that these mutant Gs alpha subunits stimulate expression driven by either the PRL or GH promoter. Finally, we show that a dominant inhibitory mutant of cAMP-dependent kinase (protein kinase A) completely blocks the ability of these Gs alpha mutants to stimulate the activity of either the PRL or GH promoter, implying that GTPase-deficient Gs alpha subunits stimulation of the activities of these promoters is mediated entirely via the cAMP/protein kinase A pathway. Taken together, these results imply that activation of this pathway by the GTPase-deficient mutants found in human pituitary tumors stimulates the expression of PRL and GH genes. The transcriptional effects exerted via this pathway may thus provide a basis for the secretory phenotype and endocrine disorders associated with these tumors.

Transcriptional effects in GH3 cells of Gs alpha mutants associated with human pituitary tumors: stimulation of adenosine 3',5'-monophosphate response element-binding protein-mediated transcription and of prolactin and growth hormone promoter activity via protein kinase A.

Somatic mutations of the alpha-subunit of Gs (Gs alpha) have been detected previously at high frequency in human PRL- and/or GH-producing pituitary tumors. To test whether these mutants are responsible for the increased production of these hormones, we used transient cotransfection assays to analyze their genomic effects in GH3 rat pituitary cells. We first show that guanosine triphosphatase (GTPase)-deficient Gs alpha subunits (mutated at amino acid 201 or 227) stimulate transcription from a reporter construct bearing the consensus cAMP response element (CRE; TGACGTCA). Using GAL4-CRE-binding protein fusion constructs, we further show that this stimulatory effects of Gs alpha on the CRE is probably mediated by the transacting factor CRE-binding protein. Then, in experiments using a reporter gene driven by the human promoters for either the PRL (position -250 to 18) or GH (position -500 to 13) genes, we show that these mutant Gs alpha subunits stimulate expression driven by either the PRL or GH promoter. Finally, we show that a dominant inhibitory mutant of cAMP-dependent kinase (protein kinase A) completely blocks the ability of these Gs alpha mutants to stimulate the activity of either the PRL or GH promoter, implying that GTPase-deficient Gs alpha subunits stimulation of the activities of these promoters is mediated entirely via the cAMP/protein kinase A pathway. Taken together, these results imply that activation of this pathway by the GTPase-deficient mutants found in human pituitary tumors stimulates the expression of PRL and GH genes. The transcriptional effects exerted via this pathway may thus provide a basis for the secretory phenotype and endocrine disorders associated with these tumors.

Myc inhibits CCAAT/enhancer-binding protein alpha-gene expression in HIB-1B hibernoma cells through interactions with the core promoter region.

The product of the c-myc proto-oncogene, Myc, has been implicated in the transcriptional regulation of several genes, acting either as an activator or repressor of gene expression. To determine whether Myc is involved in the modulation of the expression of the CCAAT/enhancer-binding protein alpha (C/EBP alpha) gene, we used both stable cell lines overexpressing Myc and transient co-transfection assays. We show that the endogenous C/EBP alpha protein level is repressed in stable cell lines overexpressing Myc. We also show that enforced expression of Myc in mouse hibernoma HIB-1B cells dramatically repressed the expression of C/EBP alpha--promoter-reporter fusion genes. This effect of Myc was mediated through the core promoter region. Mutation of the initiator site could not abolish this affect, indicating that Myc may interact with some component(s) of the basal transcription machinery.

Suppression of simian immunodeficiency virus replication by human immunodeficiency virus type 1 trans-dominant negative rev mutants.

We demonstrate that trans-dominant negative rev mutants are able to suppress simian immunodeficiency virus provirus replication in both transient cotransfection assays and stably transduced HUT 78 cells. These studies suggest that the efficacy of trans-dominant rev strategies in reducing viral burden may be evaluated in a simian immunodeficiency virus-rhesus macaque animal model.

Estrogen receptor-induced DNA bending: orientation of the bend and replacement of an estrogen response element with an intrinsic DNA bending sequence.

The estrogen receptor (ER) binds to DNA fragments containing estrogen response elements (EREs) and causes them to bend. To characterize this ER-induced DNA bend and determine if it is involved in transcription activation, three different lines of investigation were used. Using MCF-7 human breast cancer cell extracts and circular permutation analysis, it was determined that molybdate-stabilized, unoccupied cytosolic ER was unable to bind to ERE-containing DNA fragments when maintained at 4 C, but that thermal activation enabled the cytosolic receptor to bind and bend ERE-containing DNA fragments to the same extent as ER present in whole cell extracts. DNA phasing analysis was utilized to determine that ER binding induced DNA fragments containing EREs to bend toward the major groove of the DNA helix. The orientation of this bend was the same with thermally activated, unoccupied cytosolic ER and with unoccupied ER, 17 beta-estradiol-occupied ER, and 4-hydroxytamoxifen-occupied ER present in whole cell extracts. Using transient cotransfection assays, the ability of an intrinsically bent DNA sequence to replace an ERE was tested. When a single consensus ERE, which is induced to bend 56 degrees on ER binding, was replaced with a 54 degrees intrinsic DNA bending sequence, transcription was effectively activated. Similar levels of transcription were also observed when promoters contained either a 108 degrees intrinsic DNA bending sequence or two consensus EREs. However, the 54 degrees DNA bending sequence and a single ERE were unable to cooperatively activate transcription. Because the magnitude and orientation of ER-induced DNA bends are the same with the unoccupied and occupied receptor, DNA bending alone probably does not function as a transcriptional switch to turn on gene transcription. However, DNA bending may be required to provide the architecture needed for modulation of target genes.