Virus Long Terminal Repeat Promoter Research Articles

Trim28 acts as restriction factor of prototype foamy virus replication by modulating H3K9me3 marks and destabilizing the viral transactivator Tas

BackgroundPrototype foamy virus (PFV) is nonpathogenic complex retroviruses that express a transcriptional transactivator Tas, which is essential for the activity of viral long terminal repeat (LTR) promoter and internal promoter (IP). Tripartite motif-containing protein 28 (Trim28) is well known as a scaffold protein normally enriched in gene promoter region to repress transcription. We sought to determine if whether Trim28 could be enriched in PFV promoter region to participate the establishment of PFV latency infection.ResultsIn this study, we show that Trim28 restricts Tas-dependent transactivation activity of PFV promoter and negatively regulates PFV replication. Trim28 was found to be enriched in LTR instead of IP promoter regions of PFV genome and contribute to the maintenance of histone H3K9me3 marks on the LTR promoter. Furthermore, Trim28 interacts with Tas and colocalizes with Tas in the nucleus. Besides, we found that Trim28, an E3 ubiquitin ligase, binds directly to and promotes Tas for ubiquitination and degradation. And the RBCC domain of Trim28 is required for the ubiquitination and degradation of Tas.ConclusionsCollectively, our findings not only identify a host factor Trim28 negatively inhibits PFV replication by acting as transcriptional restriction factor enriched in viral LTR promoter through modulating H3K9me3 mark here, but also reveal that Trim28 mediated ubiquitin proteasome degradation of Tas as a mechanism underlying Trim28 restricts Tas-dependent transcription activity of PFV promoter and PFV replication. These findings provide new insights into the process of PFV latency establishment.Graphical

Response to fish specific reproductive hormones and endocrine disrupting chemicals of a Sertoli cell line expressing endogenous receptors from an endemic cyprinid Gnathopogon caerulescens

Fish Sertoli cells play a critical role in spermatogenesis by mediating androgen and progestogen signaling. Their hormonal response, however, considerably differ among species. Therefore it would be ideal to use Sertoli cells originated from the fish of interest to investigate the effects of hormones as well as endocrine disrupting chemicals (EDCs). The aim of this study was to investigate the responses to reproductive hormones and EDCs of a Sertoli cell line that we established from an endemic cyprinid Gnathopogon caerulescens. As the Sertoli cell line expressed endogenous androgen and progestogen receptors, we were able to detect hormone responses by transfecting only a reporter vector (pGL4.36) expressing luciferase under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter into the cell line. Unlike previous reporter gene assays using fish steroid hormone receptors expressed in mammalian cell lines, luciferase activities were induced by the fish specific androgen (11-ketotestosterone) and progestogen (17α,20β-dihydroxy-4-pregnen-3-one), but not by testosterone and progesterone, at physiologically relevant concentrations. Furthermore, we found 4-nonylphenol (NP) but not bisphenol A showed strong anti-androgenic effects, implying that NP may have direct anti-androgenic effects on fish Sertoli cells in vivo. This is the first evidence, to the best of our knowledge, of anti-androgenic effects of NP in a fish Sertoli cell line. In addition, neither NP nor BPA showed anti-progestogenic effects. These results suggest that the Sertoli cell line established from the fish of interest can be a useful in vitro tool for investigating the mechanisms of reproductive hormones and EDCs in the specific fish.

Identification and functional characterization of BTas transactivator as a DNA-binding protein

The genome of bovine foamy virus (BFV) encodes a transcriptional transactivator, namely BTas, that remarkably enhances gene expression by binding to the viral long-terminal repeat promoter (LTR) and internal promoter (IP). In this report, we characterized the functional domains of BFV BTas. BTas contains two major functional domains: the N-terminal DNA-binding domain (residues 1–133) and the C-terminal activation domain (residues 198–249). The complete BTas responsive regions were mapped to the positions −380/−140 of LTR and 9205/9276 of IP. Four BTas responsive elements were identified at the positions −368/−346, −327/−307, −306/−285 and −186/−165 of the BFV LTR, and one element was identified at the position 9243/9264 of the BFV IP. Unlike other foamy viruses, the five BTas responsive elements in BFV shared obvious sequence homology. These data suggest that among the complex retroviruses, BFV appears to have a unique transactivation mechanism.

Does Plasminogen Activator Inhibitor-1 Drive Lymphangiogenesis?

The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and by regulating endothelial cell survival and migration. Protease system's role in lymphangiogenesis is unknown yet. Thus, based on its important pro-angiogenic effect, we hypothesized that PAI-1 may regulate lymphangiogenesis associated at least with metastatic dissemination of cancer cells. To address this issue, we studied the impact of PAI-1 deficiency in various murine models of tumoral lymphangiogenesis. Wild-type PAI-1 proficient mice were used as controls. We provide for the first time evidence that PAI-1 is dispensable for tumoral lymphangiogenesis associated with breast cancers either induced by mammary carcinoma cell injection or spontaneously appearing in transgenic mice expressing the polyomavirus middle T antigen (PymT) under the control of a mouse mammary tumor virus long-terminal repeat promoter (MMTV-LTR). We also investigated inflammation-related lymphatic vessel recruitment by using two inflammatory models. PAI-1 deficiency did neither affect the development of lymphangioma nor burn-induced corneal lymphangiogenesis. These novel data suggest that vascular remodelling associated with lymphangiogenesis and angiogenesis involve different molecular determinants. PAI-1 does not appear as a potential therapeutic target to counteract pathological lymphangiogenesis.

Effectiveness of HSV-tk Suicide Gene Therapy Driven by the Grp78 Stress-Inducible Promoter in Esophagogastric Junction and Gastric Adenocarcinomas

BackgroundThe thymidine kinase gene of the herpes simplex virus (HSV-tk) is a suicide gene when administrated with the prodrug ganciclovir (GCV). This study investigated the effectiveness of HSV-tk activation as gene therapy for gastroesophageal junction and gastric adenocarcinomas using either the stress-inducible Grp78 promoter or the murine leukemia virus long-terminal repeat (LTR) promoter. MethodsThe HSV-tk gene, controlled by either the Grp78 promoter or the LTR promoter, was transduced into the gastroesophageal junction adenocarcinoma cell line SK-GT-5 and the gastric adenocarcinoma cell line MKN-74. Cell viability after exposure to varying concentrations of GCV was compared. The same cell lines were used to develop a nude mouse model for studies of the HSV-tk/GCV effect in vivo. The effect of intraperitoneal GCV injection on growth of the subcutaneous tumors was measured. HSV-TK expression was measured by Western blot and reverse transcription polymerase chain reaction. ResultsCell viability in vitro was significantly lower in the HSV-tk expressing (HSV-tk+) cells compared to control (no HSV-tk) cells after exposure to GCV. MKN-74tk+ cells were more sensitive to GCV killing than SK-GT-5tk+ cells. After culture with 1 μg/ml GCV for 10 days, MKN-74/tk cells were totally killed, whereas most SK-GT-5/tk cells survived. Cell viability was significantly lower under glucose starvation conditions when HSV-tk expression was regulated by the Grp78 promoter compared with the LTR promoter. MKN-74 tumors formed with HSV-tk+ cells in nude mice were eliminated after administration of GCV for 3 weeks, but GCV had no effect on tumors formed from HSV-tk− cells. Eradication of tumor formed with Grp78-tk cells was faster than that with LTR-tk cells. HSV-TK protein and mRNA were expressed in the transduced, but not the non-transduced tumors. ConclusionHSV-tk xwith ganciclovir suicide gene therapy results in significant cell killing in gastroesophageal junction and gastric adenocarcinoma cells both in vitro and in vivo, but complete tumor elimination only occurred with the gastric adenocarcinoma cell tumors. The most effective approach in this study used the Grp78 promoter in glucose-starvation stress conditions.

Correction of Laminin-5 Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors

Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer-restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.Molecular Therapy (2008) 16 12, 1977-1985 doi:10.1038/mt.2008.204.

Calpain and Proteasomal Regulation of Antiretroviral Zinc Finger Protein OTK18 in Human Macrophages: Visualization in Live Cells by Intramolecular FRET

As part of the innate immune defense against HIV infection, OTK18, a zinc finger protein, is upregulated in human macrophages and reduces viral replication through suppression of viral long-terminal repeat promoter activity. Although we know that the processing products of OTK18 accumulate in the cytoplasm of brain perivascular macrophages in advanced HIV encephalitis cases, the molecular mechanisms behind its post-translational processing are still poorly understood. To characterize OTK18 processing, we assessed a panel of protease inhibitors to identify the candidates involved in the OTK18 processing using human monocyte-derived macrophages (MDM) overexpressing OTK18 by recombinant adenoviral gene transfer. Viral infection of MDM strongly increased the processing of OTK18 into its N-terminal fragment. Treatment of OTK18-expressing MDM with calpain and proteasome inhibitors significantly accumulated either full-length or processed OTK18 fragments in time- and dose-dependent manners. A series of OTK18 truncation mutants and synthetic peptides were tested to locate the calpain cleavage sites after arginine 359. Finally, we developed an enhanced cyan and yellow fluorescent protein (ECFP and EYFP)-based intramolecular fluorescent resonance energy transfer (intramolecular FRET) system to monitor the OTK18 endoproteolysis in human microglia cell line. Inhibition of proteasome activity significantly increased the intramolecular FRET signal in the nucleus. These data suggest that calpain and proteasome are involved in OTK18 endoproteolysis and degradation. Additionally, intramolecular FRET has proven to be a useful tool for monitoring the processing in live cells.

Kit and PDGFR-α activities are necessary for Notch4/Int3-induced tumorigenesis

Transgenic mice overexpressing Notch4 intracellular domain (Int3) under the control of the whey acidic protein (WAP) or mouse mammary tumor virus-long terminal repeat promoters, develop mammary tumors. Microarray analysis of these tumors revealed high levels of c-Kit expression. Gleevec is a tyrosine kinase inhibitor that targets c-Kit, platelet-derived growth factor receptors (PDGFRs) and c-Abl. This led us to speculate that tyrosine kinase receptor activity might be a driving force in the development of Int3 mammary tumors. WAP-Int3 tumor-bearing mice were treated with continuous release of Gleevec using subcutaneously implanted Alzet pumps. Phosphorylation of c-Kit, PDGFRs and c-Abl is inhibited in Int3 transgenic mammary tumors by Gleevec. Inhibition of these enzymes is associated with a decrease in cell proliferation and angiogenesis, and an induction of apoptosis. To examine the signaling mechanisms underlying Notch4/Int3 tumorigenesis, we employed small interfering RNA (siRNA) to knock down c-Kit, PDGFRs and c-Abl alone or in combination and observed the effects on soft agar growth of HC11 cells overexpressing Int3. Only siRNA constructs for c-Kit and/or PDGFR-alpha were able to inhibit HC11-Int3 colony formation in soft agar. Our data demonstrate an inhibitory effect of Gleevec on Int3-induced transformation of HC11 cells and mammary tumors and indicate an oncogenic role for c-Kit and PDGFR-alpha tyrosine kinases in the context of Int3 signaling.

Functional analysis of human T lymphotropic virus type 2 Tax proteins

BackgroundThe Tax proteins encoded by human T lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) are transcriptional activators of both the viral long terminal repeat (LTR) and cellular promoters via the CREB and NFkB pathways. In contrast to HTLV-1, HTLV-2 has been classified into four distinct genetic subtypes A, B, C and D defined by phylogenetic analysis of their nucleotide sequences and the size and amino acid sequence of their Tax proteins. In the present study we have analysed and compared the transactivating activities of three Tax 2A and one Tax 2B proteins using LTR and NFkB reporter assays.ResultsWe found that with the exception of the prototype Tax 2A Mo protein, the other two Tax 2A proteins failed to transactivate either the viral LTR or NFkB promoter in Jurkat and 293T cells. Loss of activity was not associated with either expression levels or an alteration in subcellular distribution as all Tax 2 proteins were predominantly located in the cytoplasm of transfected cells. Analysis of the sequence of the two inactive Tax 2A proteins relative to Mo indicated that one had six amino acid changes and the other had one change in the central region of the protein. Mutations present at the amino and the extreme carboxy termini of Mo resulted in the loss of LTR but not NFkB activation whereas those occurring in the central region of the protein appeared to abolish transactivation of both promoters. Analysis of the transactivation phenotypes of Tax 1, Tax 2A Mo and Tax 2B containing mutations identified in the present study or previously characterised Tax mutations showed that domains required for LTR and NFkB activation are very similar but not identical in all three Tax proteins.ConclusionOur results suggest that loss of activity of two Tax 2A proteins derived from different isolates is associated with multiple amino acid changes relative to Mo in domains required for the activation of the CREB or CREB and NFkB pathways and that these domains are very similar but not identical in Tax 2B and Tax 1. The loss of Tax function in 2A viruses may have implications for their biological and pathogenic properties.

Spontaneous and controllable activation of suicide gene expression driven by the stress-inducible grp78 promoter resulting in eradication of sizable human tumors.

GRP78 is a stress-inducible chaperone protein with antiapoptotic properties that is overexpressed in transformed cells and cells under glucose starvation, acidosis, and hypoxic conditions that persist in poorly vascularized tumors. Previously we demonstrated that the Grp78 promoter is able to eradicate tumors using murine cells in immunocompetent models by driving expression of the HSV-tk suicide gene. Here, through the use of positron emission tomography (PET) imaging, we provide direct evidence of spontaneous in vivo activation of the HSV-tk suicide gene driven by the Grp78 promoter in growing tumors and its activation by photodynamic therapy (PDT) in a controlled manner. In this report, we evaluated whether this promoter can be applied to human cancer therapy. We observed that the Grp78 promoter, in the context of a retroviral vector, was highly activated by stress and PDT in three different types of human breast carcinomas independent of estrogen receptor and p53. Complete regression of sizable human tumors was observed after prodrug ganciclovir treatment of the xenografts in immunodeficient mice. In addition, the Grp78 promoter-driven suicide gene is strongly expressed in a variety of human tumors, including human osteosarcoma. In contrast, the activity of the murine leukemia virus (MuLV) long-terminal repeat (LTR) promoter varied greatly in different human breast carcinoma cell lines, and in some cases, stress resulted in partial suppression of the LTR promoter activity. In transgenic mouse models, the Grp78 promoter-driven transgene is largely quiescent in major adult organs but highly active in cancer cells and cancer-associated macrophages, which can diffuse to tumor necrotic sites devoid of vascular supply and facilitate cell-based therapy. Thus, transcriptional control through the use of the Grp78 promoter offers multiple novel approaches for human cancer gene therapy.

Stimulation of Tat-independent transcriptional processivity from the HIV-1 LTR promoter by matrix attachment regions.

The chromatin environment and the sites of integration in the host genome are critical determinants of human immunodeficiency virus (HIV) transcription and replication. Depending on the chromosomal location of provirus integration within the genome, HIV-1 long terminal repeat (LTR)-mediated transcription may vary from 0- to 70-fold. Cis-elements such as topoisomerase II cleavage sites, Alu repeats and matrix attachment regions (MARs) are thought to be targets for retroviral integration. Here we show that a novel MAR sequence from the T-cell receptor beta locus (MARbeta) and the IgH MAR mediate transcriptional augmentation when placed upstream of the HIV-1 LTR promoter. The effect of transcriptional augmentation is seen in both transient and stable transfection, indicating its effect even upon integration in the genome. MAR-mediated transcriptional elevation is independent of Tat, and occurs synergistically in the presence of Tat. Further, we show that MAR-mediated transcriptional elevation is specific to the HIV-1 LTR and the Moloney murine leukemia virus LTR promoter. In a transient transfection assay using over-expressed IkappaB, the inhibitor of NF-kappaB, we show that MAR-induced processive transcription is NF-kappaB dependent, signifying the role of local enhancers within the LTR promoter. Furthermore, by RNase protection experiments using proximal and distal probes, we show that MAR-mediated transcriptional upregulation is more prominent at the distal rather than the proximal end, thus indicating the potential role of MARs in promoting elongation.

Persistence and expression of the adenosine deaminase gene for 12 years and immune reaction to gene transfer components: long-term results of the first clinical gene therapy trial

The first human gene therapy experiment begun in September 1990 used a retroviral vector containing the human adenosine deaminase (ADA) cDNA to transduce mature peripheral blood lymphocytes from patients with ADA deficiency, an inherited disorder of immunity. Two patients who had been treated with intramuscular injections of pegylated bovine ADA (PEG-ADA) for 2 to 4 years were enrolled in this trial and each received a total of approximately 10(11) cells in 11 or 12 infusions over a period of about 2 years. No adverse events were observed. During and after treatment, the patients continued to receive PEG-ADA, although at a reduced dose. Ten years after the last cell infusion, approximately 20% of the first patient's lymphocytes still carry and express the retroviral gene, indicating that the effects of gene transfer can be remarkably long lasting. On the contrary, the persistence of gene-marked cells is very low (< 0.1%), and no expression of the transgene is detectable in lymphocytes from the second patient who developed persisting antibodies to components of the gene transfer system. Data collected from these original patients have provided novel information about the longevity of T lymphocytes in humans and persistence of gene expression in vivo from vectors driven by the Moloney murine leukemia virus long-terminal repeat (LTR) promoter. This long-term follow-up has also provided unique evidence supporting the safety of retroviral-mediated gene transfer and illustrates clear examples of both the potential and the pitfalls of gene therapy in humans.

Quantitative characterization of specific genomic promoters using agarose gel electrophoresis

Over the past decade a large number of studies have focused attention on the role of nucleosomes as negative and positive regulators of specific nuclear functions. Due to the lack of an analytical method to determine the higher order conformation of the nucleosomal arrays that encompass specific genetic loci (e.g., promoters, enhancers), research emphasis has mostly been centered on chromatin remodeling and histone posttranslational modifications. We have recently developed an agarose gel electrophoresis method that permits us to analyze the higher order structure of specific in vivo assembled chromatin fragments. After calibration using a well-defined in vitro system, we have been able to experimentally determine the size, shape, and conformational flexibility of the Mouse Mammary Tumor Virus long-terminal repeat promoter region in its repressed and activated states. These studies pave the way for widespread analyses of the higher order structure of specific, functionally important chromosomal loci, and in so doing enhance our understanding of the roles that the higher order structure of chromatin play in genome regulations.

Direct interaction of the Krüppel-like family (KLF) member, BTEB1, and PR mediates progesterone-responsive gene expression in endometrial epithelial cells.

The present study was undertaken to evaluate the underlying mechanism(s) by which PR and a Krüppel-like family member, basic transcription element binding protein (BTEB1), mediate endometrial epithelial expression of pregnancy-associated genes. Human endometrial carcinoma cell lines (Hec-1-A) expressing high and low levels of BTEB1 were transiently transfected with a human PR isoform (PR-B) expression construct and a luciferase reporter gene driven by the uteroferrin gene promoter that is responsive to both BTEB1 and the PR ligand progesterone. Unliganded PR inhibited luciferase activity in low and high BTEB backgrounds, and this effect was reversed by the synthetic progestin R5020 in both lines. Transactivation by PR of uteroferrin promoter activity (approximately 4-fold) was maximal at lower R5020 concentrations (10 nM) in endometrial cells with higher BTEB1 expression, suggesting that nuclear BTEB1content influenced target gene promoter sensitivity to progesterone. BTEB1 and PR-B were found to physically interact in a progesterone-independent manner, using a coimmunoprecipitation assay that employed antibodies specific to either protein. Moreover, the formation of the BTEB1/PR complex, independent of progesterone, occurred within the context of uterine endometrial proteins and was diminished in late-pregnancy endometrium. Mammalian two-hybrid assays using the entire open reading frame of BTEB1 and/or PR-B fused to either the GAL4 DNA-binding domain or VP16 activation domain and a reporter gene (pG5CAT) under the control of GAL4-binding sites were used to evaluate the formation of functional PR-B/BTEB1 dimer in Cos-1 cells. GAL4/PR-B and VP16/PR-B induced ( approximately 3- to 4-fold) chloramphenicol acetyltransferase (CAT) activity in a progesterone-dependent manner, suggesting PR-dimer formation. By contrast, VP16/PR-B and GAL4/BTEB1 had no effect on basal CAT activity. The combination of VP16- and GAL4-PR-B fusion proteins with the BTEB1 expression construct, pCDNA3-BTEB1 enhanced ligand-bound PR-mediated CAT activity by approximately 3-fold. In transient cotransfection assays using the CAT reporter gene driven by the mouse mammary tumor virus-long terminal repeat promoter, which is responsive to ligand-bound PR but not BTEB1, BTEB1 increased PR-B-mediated CAT activity in a progesterone-dependent manner, consistent with a BTEB1/PR-dimer complex occurring independent of BTEB1 binding to DNA. Unliganded PR-B disrupted the DNA-binding activity of BTEB1 in gel retardation assays, and this effect was enhanced by the presence of PR ligand. Together, these findings support the conclusion that BTEB1 and PR-B are coregulatory proteins that mediate progesterone responsiveness of target genes by direct interactions, leading to the formation of a functional BTEB1/PR-dimer complex.

Physiological Control of Smooth Muscle-specific Gene Expression through Regulated Nuclear Translocation of Serum Response Factor

Prolonged serum deprivation induces a structurally and functionally contractile phenotype in about 1/6 of cultured airway myocytes, which exhibit morphological elongation and accumulate abundant contractile apparatus-associated proteins. We tested the hypothesis that transcriptional activation of genes encoding these proteins accounts for their accumulation during this phenotypic transition by measuring the transcriptional activities of the murine SM22 and human smooth muscle myosin heavy chain promoters during transient transfection in subconfluent, serum fed or 7 day serum-deprived cultured canine tracheal smooth muscle cells. Contrary to our expectation, SM22 and smooth muscle myosin heavy chain promoter activities (but not viral murine sarcoma virus-long terminal repeat promoter activity) were decreased in long term serum-deprived myocytes by at least 8-fold. Because serum response factor (SRF) is a required transcriptional activator of these and other smooth muscle-specific promoters, we evaluated the expression and function of SRF in subconfluent and long term serum-deprived cells. Whole cell SRF mRNA and protein were maintained at high levels in serum-deprived myocytes, but SRF transcription-promoting activity, nuclear SRF binding to consensus CArG sequences, and nuclear SRF protein were reduced. Furthermore, immunocytochemistry revealed extranuclear redistribution of SRF in serum-deprived myocytes; nuclear localization of SRF was restored after serum refeeding. These results uncover a novel mechanism for physiological control of smooth muscle-specific gene expression through extranuclear redistribution of SRF and consequent down-regulation of its transcription-promoting activity.

Functional Differences between Human and Bovine Immunodeficiency Virus Tat Transcription Factors

Transcriptional transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element by the essential viral Tat protein requires recruitment of positive transcription elongation factor b (P-TEFb) to the viral TAR RNA target. The recruitment of P-TEFb, which has been proposed to be necessary and sufficient for activation of viral gene expression, is mediated by the highly cooperative interaction of Tat and cyclin T1, an essential component of P-TEFb, with the HIV-1 TAR element. Species, such as rodents, that encode cyclin T1 variants that are unable to support TAR binding by the Tat-cyclin T1 heterodimer are also unable to support HIV-1 Tat function. In contrast, we here demonstrate that the bovine immunodeficiency virus (BIV) Tat protein is fully able to bind to BIV TAR both in vivo and in vitro in the absence of any cellular cofactor. Nevertheless, BIV Tat can specifically recruit cyclin T1 to the BIV TAR element, and this recruitment is as essential for BIV Tat function as it is for HIV-1 Tat activity. However, because the cyclin T1 protein does not contribute to TAR binding, BIV Tat is able to function effectively in cells from several species that do not support HIV-1 Tat function. Thus, BIV Tat, while apparently dependent on the same cellular cofactor as the Tat proteins encoded by other lentiviruses, is nevertheless unique in terms of the mechanism used to recruit the BIV Tat-cyclin T1 complex to the viral LTR promoter.

CBP/p300 interact with and function as transcriptional coactivators of BRCA1.

BRCA1 is a breast and ovarian cancer-specific tumor suppressor, with properties of a transcription factor involved in DNA repair. We previously have shown the transactivation of heterologous promoters by the carboxyl terminus of BRCA1. We now describe that BRCA1-mediated transactivation is enhanced by p300/CBP (CREB binding protein) and that this effect was suppressed by the adenovirus E1A oncoprotein. We show a physical association of BRCA1 with the transcriptional coactivators/acetyltransferases p300 and CBP. Endogenous as well as overexpressed BRCA1 and p300 were found to associate in a phosphorylation-independent manner. BRCA1 interacts with the cAMP response element binding protein (CREB) domain of p300/CBP via both its amino and carboxyl termini. Finally, full-length BRCA1 is shown to transcriptionally activate the Rous sarcoma virus-long terminal repeat promoter, which was further stimulated by p300. Immunocolocalization analyses suggest that BRCA1 and p300 associate in a cell cycle-dependent manner. Our results support a role for BRCA1 in transcription.

P300/CREB-Binding Protein Enhances the Prolactin-Mediated Transcriptional Induction through Direct Interaction with the Transactivation Domain of Stat5, but Does Not Participate in the Stat5-Mediated Suppression of the Glucocorticoid Response

Stat5 was discovered as a PRL-induced member of the Stat (signal transducer and activator of transcription) family. Its induction by many other cytokines and interleukins suggests that Stat5 plays a crucial role not only in mammary epithelial, but also in hematopoietic cells. Cell type- and promoter- specific functions of Stat5 are most likely modulated by the interaction with other transcription factors. We recently showed cross-talk between Stat5 and the glucocorticoid receptor. The activated glucocorticoid receptor forms a complex with Stat5 and enhances Stat5-mediated transcriptional induction. Conversely, Stat5 diminishes the induction of glucocorticoid-responsive genes. Here, we investigated the role of p300/CBP(CREB-binding protein), a transcriptional coactivator of several groups of transcription factors, in Stat5-mediated transactivation and in the cross-talk between Stat5 and the glucocorticoid receptor. p300/CBP acts as a coactivator of Stat5. Its ectopic expression enhances PRL-induced Stat5-mediated transcriptional activation. Consistent with this observation, we find that the adenovirus E1A protein, which binds to p300/CBP, suppresses Stat5-induced transcriptional activation. This inhibition requires the Stat5 transactivation domain and the p300/CBP binding site of E1A. Coimmunoprecipitation and mammalian two-hybrid assays demonstrate a direct interaction between the carboxyl-terminal transactivation domain of Stat5 and p300/CBP. p300/CBP also positively interacts with the glucocorticoid receptor and enhances glucocorticoid receptor-dependent transcriptional activation of the mouse mammary tumor virus-long terminal repeat promoter. Overexpression of p300/CBP does not counteract the Stat5-mediated inhibition of glucocorticoid receptor-dependent transactivation, i.e. the repression of the glucocorticoid response by Stat5 is not a consequence of competition for limiting amounts of p300/CBP.

P300/CREB-binding protein enhances the prolactin-mediated transcriptional induction through direct interaction with the transactivation domain of Stat5, but does not participate in the Stat5-mediated suppression of the glucocorticoid response.

Stat5 was discovered as a PRL-induced member of the Stat (signal transducer and activator of transcription) family. Its induction by many other cytokines and interleukins suggests that Stat5 plays a crucial role not only in mammary epithelial, but also in hematopoietic cells. Cell type- and promoter-specific functions of Stat5 are most likely modulated by the interaction with other transcription factors. We recently showed cross-talk between Stat5 and the glucocorticoid receptor. The activated glucocorticoid receptor forms a complex with Stat5 and enhances Stat5-mediated transcriptional induction. Conversely, Stat5 diminishes the induction of glucocorticoid-responsive genes. Here, we investigated the role of p300/CBP(CREB-binding protein), a transcriptional coactivator of several groups of transcription factors, in Stat5-mediated transactivation and in the cross-talk between Stat5 and the glucocorticoid receptor. p300/ CBP acts as a coactivator of Stat5. Its ectopic expression enhances PRL-induced Stat5-mediated transcriptional activation. Consistent with this observation, we find that the adenovirus E1A protein, which binds to p300/CBP, suppresses Stat5-induced transcriptional activation. This inhibition requires the Stat5 transactivation domain and the p300/CBP binding site of E1A. Coimmunoprecipitation and mammalian two-hybrid assays demonstrate a direct interaction between the carboxyl-terminal transactivation domain of Stat5 and p300/CBP. p300/CBP also positively interacts with the glucocorticoid receptor and enhances glucocorticoid receptor-dependent transcriptional activation of the mouse mammary tumor virus-long terminal repeat promoter. Overexpression of p300/CBP does not counteract the Stat5-mediated inhibition of glucocorticoid receptor-dependent transactivation, i.e. the repression of the glucocorticoid response by Stat5 is not a consequence of competition for limiting amounts of p300/CBP.

Expression of ovine insulin-like growth factor-1 (IGF-1) stimulates alveolar bud development in mammary glands of transgenic mice.

To determine whether murine mammary growth is modulated by local insulin-like growth factor-1 (IGF-1) production, expression of recombinant IGF-1 was directed to the mammary glands of transgenic mice using an ovine prepro IGF-1 cDNA under control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter. Bioactivity of recombinant IGF-1 in transgenic mouse milk extracts was demonstrated by a concentration-dependent increase in [3H]thymidine incorporation in clonal bovine mammary epithelial cells (MAC-T) compared with control mouse milk extracts; moreover, addition of excess recombinant human insulin-like growth factor binding protein-3 (rhlGFBP-3) abolished the increase in [3H]thymidine incorporation attributed to recombinant IGF-1 in transgenic mouse milk. Recombinant IGF-1 was produced in mammary tissue of virgin and pregnant transgenic mice, and secreted into milk of lactating mice. However, recombinant IGF-1 was not detected in serum from transgenic mice; and ligand blot analysis of serum insulin-like growth factor binding proteins (IGFBPs) indicated no differences owing to transgene presence. In peripubertal virgin mice at 49 d of age, the frequency of appearance of mammary alveolar buds was significantly higher in MMTV-IGF-1 than in CD-1 mice, and was unaffected by ovariectomy or estradiol treatment. In conclusion, mammary synthesis of recombinant IGF-1 enhances the rate of development of alveolar buds in mammary glands of virgin transgenic mice.