RESUMO
Half of estrogen receptor-positive breast cancers contain a subpopulation of cytokeratin 5 (CK5)-expressing cells that are therapy resistant and exhibit increased cancer stem cell (CSC) properties. We and others have demonstrated that progesterone (P4) increases CK5+ breast cancer cells. We previously discovered that retinoids block P4 induction of CK5+ cells. Here we investigated the mechanisms by which progesterone receptors (PR) and retinoic acid receptors (RAR) regulate CK5 expression and breast CSC activity. After P4 treatment, sorted CK5+ compared to CK5- cells were more tumorigenic in vivo. In vitro, P4-treated breast cancer cells formed larger mammospheres and silencing of CK5 using small hairpin RNA abolished this P4-dependent increase in mammosphere size. Retinoic acid (RA) treatment blocked the P4 increase in CK5+ cells and prevented the P4 increase in mammosphere size. Dual small interfering RNA (siRNA) silencing of RARα and RARγ reversed RA blockade of P4-induced CK5. Using promoter deletion analysis, we identified a region 1.1 kb upstream of the CK5 transcriptional start site that is necessary for P4 activation and contains a putative progesterone response element (PRE). We confirmed by chromatin immunoprecipitation that P4 recruits PR to the CK5 promoter near the -1.1 kb essential PRE, and also to a proximal region near -130 bp that contains PRE half-sites and a RA response element (RARE). RA induced loss of PR binding only at the proximal site. Interestingly, RARα was recruited to the -1.1 kb PRE and the -130 bp PRE/RARE regions with P4, but not RA alone or RA plus P4. Treatment of breast cancer xenografts in vivo with the retinoid fenretinide reduced the accumulation of CK5+ cells during estrogen depletion. This reduction, together with the inhibition of CK5+ cell expansion through RAR/PR cross talk, may explain the efficacy of retinoids in prevention of some breast cancer recurrences.
Assuntos
Neoplasias da Mama/genética , Queratina-5/genética , Receptores de Progesterona/genética , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Progesterona/genética , Progesterona/metabolismo , Regiões Promotoras Genéticas , Receptores de Estrogênio/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico/metabolismo , Transdução de Sinais , Tretinoína/metabolismo , Receptor gama de Ácido RetinoicoRESUMO
Glucocorticoids and progestins are two classes of steroid hormone with very distinct biological functions. However, the glucocorticoid receptor (GR) and the progesterone receptor (PR) share many structural and functional similarities. One way that glucocorticoids and progestins can exert different biological effects is through their different abilities to regulate the expression of certain target genes. A strategy employing a retroviral promoter-trap and Cre/loxP-mediated site-specific recombination has been developed to identify genes that are differentially regulated by glucocorticoids and progestins. A mouse fibroblast cell line (4F) stably expressing both GR and PR and containing a single copy of a multifunctional selection plasmid is generated. This line is transduced with a self-inactivating retroviral promoter-trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Integration of the provirus places Cre expression under the control of a genomic flanking sequence. Activation of Cre expression from integration into active genes results in a permanent switch between the selectable marker genes that converts the cells from neomycin-resistant to hygromycin-resistant. Selection for hygromycin resistance after hormone treatment yields recombinants in which Cre sequences in the U3 region are expressed from hormone-inducible upstream cellular promoters. Because Cre-mediated recombination is a permanent event, the expression of the selectable marker genes is independent of ongoing Cre expression. Thus this system permits the identification of genes that are transiently or weakly induced by hormone.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Congêneres da Progesterona/farmacologia , Animais , Sequência de Bases , Linhagem Celular , Dexametasona/farmacologia , Vetores Genéticos , Integrases/genética , Camundongos , Plasmídeos/genética , Promegestona/farmacologia , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/genética , Receptores de Progesterona/genética , Retroviridae/genética , Transfecção , Proteínas Virais/genéticaAssuntos
Proteínas Luminescentes , Receptores de Esteroides/metabolismo , Animais , Transporte Biológico , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas/genética , Proteínas/metabolismo , Receptores de Esteroides/genética , Espectrometria de Fluorescência/métodos , Frações SubcelularesAssuntos
Acetiltransferases/metabolismo , Cromatina , Histonas/genética , Hormônios/metabolismo , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Esteroides/metabolismo , Acetilação , Animais , Histona Acetiltransferases , Histona Desacetilases/metabolismo , Testes de Precipitina/métodosRESUMO
Glucocorticoid receptor (GR) and progesterone receptor (PR) are closely related members of the steroid receptor family of transcription factors. The two receptors share a similar domain structure, substantial sequence identity, DNA binding specificity, and the ability to induce many of the same genes. Despite these similarities, the unliganded GR is localized predominantly in the cytoplasm, while unliganded PR is found predominantly in the nucleus. By expressing green fluorescent protein (GFP)-tagged receptors and assessing subcellular localization in living cells by confocal microscopy, we have investigated the structural basis for the differential localization of GR and PR. By constructing a series of GFP-tagged receptor chimeras between GR and PR, we have shown that multiple features in the N-terminal half of the ligand-binding domain (LBD) are the critical determinants that mandate the differential localization of GR and PR. Replacement of residues encompassing helices 1-5 of GR with those of PR yields a receptor that is nuclear. However, this domain is unable to mediate nuclear import by itself when removed from the context of the receptor. The chimeric receptors also indicate that regions encompassing helices 6 and 7 are key determinants of the ligand binding potential and the transactivation potential of receptors. Thus, the determinants specifying localization of hormone-free receptors are separable from those governing ligand binding character.
Assuntos
Núcleo Celular/química , Citoplasma/química , Receptores de Glucocorticoides/análise , Receptores de Progesterona/análise , Animais , Sítios de Ligação , Células COS , Linhagem Celular , DNA/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde , Ligantes , Proteínas Luminescentes/genética , Camundongos , Microscopia Confocal , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/química , Receptores de Progesterona/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Ativação Transcricional , TransfecçãoRESUMO
Ligand binding to estrogen receptor (ER) is presumed to regulate the type and timing of ER interactions with different cofactors. Using fluorescence microscopy in living cells, we characterized the recruitment of five different green fluorescent protein (GFP)-labeled ER-interacting peptides to the distinct subnuclear compartment occupied by blue fluorescent protein (BFP)-labeled ER alpha. Different ligands promoted the recruitment of different peptides. One peptide was recruited in response to estradiol (E2), tamoxifen, raloxifene, or ICI 182,780 incubation whereas other peptides were recruited specifically by E2 or tamoxifen. Peptides containing different sequences surrounding the ER-interacting motif LXXLL were recruited with different time courses after E2 addition. Complex temporal kinetics also were observed for recruitment of the full-length, ER cofactor glucocorticoid receptor-interacting protein 1 (GRIP1); rapid, E2-dependent recruitment of GRIP1 was blocked by mutation of the GRIP1 LXXLL motifs to LXXAA whereas slower E2 recruitment persisted for the GRIP1 LXXAA mutant. This suggested the presence of multiple, temporally distinct GRIP 1 recruitment mechanisms. E2 recruitment of GRIP1 and LXXLL peptides was blocked by coincubation with excess ICI 182,780. In contrast, preformed E2/ER/GRIP1 and E2/ER/LXXLL complexes were resistant to subsequent ICI 182,780 addition whereas ICI 182,780 dispersed preformed complexes containing the GRIP1 LXXAA mutant. This suggested that E2-induced LXXLL binding altered subsequent ligand/ER interactions. Thus, alternative, ligand-selective recruitment and dissociation mechanisms with distinct temporal sequences are available for ER alpha action in vivo.
Assuntos
Núcleo Celular/metabolismo , Estradiol/análogos & derivados , Peptídeos/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Compartimento Celular , Linhagem Celular , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Fulvestranto , Proteínas de Fluorescência Verde , Indicadores e Reagentes/metabolismo , Cinética , Ligantes , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Substâncias Macromoleculares , Dados de Sequência Molecular , Coativador 2 de Receptor Nuclear , Estrutura Terciária de Proteína , Receptores de Estrogênio/agonistas , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/farmacologiaRESUMO
The estrogen receptor (ER) regulates the expression of genes involved in the growth, proliferation and differentiation of skeletal, cardiovascular, neural and reproductive tissues. A basic scheme for the mechanism for ER action has been developed, but precise details on the interactions between ER and the cellular signaling and transcription machinery required for receptor-mediated regulation of specific target genes are still lacking. We have developed a genetic approach to explore the functional interactions of ER. In this work, we describe the development of an estrogen responsive system in the fruit fly, Drosophila melanogaster. Transgenic flies carrying the human ER alpha and an estrogen responsive green fluorescent protein (GFP) reporter gene were constructed. In vivo expression of the GFP reporter gene was observed when larvae were grown on a food source containing steroidal or nonsteroidal estrogens. The induction of the reporter gene by estrogens was blocked upon treatment with tamoxifen, an estrogen antagonist. However, we failed to recapitulate ligand-independent activation of the receptor in vivo or in cultured Drosophila cells. An estrogen responsive Drosophila system could be used to identify and characterize the complex functional interactions between ER and the other components of the cellular transcriptional apparatus.
Assuntos
Drosophila melanogaster/metabolismo , Antagonistas de Estrogênios/farmacologia , Estrogênios/farmacologia , Receptores de Estrogênio/efeitos dos fármacos , Animais , Animais Geneticamente Modificados/genética , Receptor alfa de Estrogênio , Expressão Gênica , Genes Reporter/fisiologia , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes , Proteínas Luminescentes/genética , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/genética , Tamoxifeno/farmacologiaRESUMO
To investigate the role of chromatin structure in the regulation of transcription by RNA polymerase II, we developed a chromatin transcription system in which periodic nucleosome arrays are assembled with purified recombinant ATP-utilizing chromatin assembly and remodeling factor (ACF), purified recombinant nucleosome assembly protein 1 (dNAP1), purified native core histones, plasmid DNA, and ATP. With this chromatin, we observed robust activation of transcription with three different transcription factor sets (nuclear factor kappaB p65 + Sp1, estrogen receptor, and Gal4-VP16) added either before or after chromatin assembly. In fact, the efficiency of activated transcription from the ACF + dNAP1-assembled chromatin was observed to be comparable with that from naked DNA templates or chromatin assembled with a crude Drosophila extract (S190). With ACF + dNAP1-assembled chromatin, we found that transcriptional activation is dependent upon acetyl-CoA. This effect was not seen with naked DNA templates or with crude S190-assembled chromatin. We further determined that acetyl-CoA is required at the time of preinitiation complex assembly but not during assembly of the chromatin template. These findings suggest that there is at least one key acetylation event that is needed to assemble a functional transcription preinitiation complex with a chromatin template.
Assuntos
Acetilcoenzima A/metabolismo , Cromatina/metabolismo , Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Ciclo Celular , Proteínas de Drosophila , Células HeLa , Humanos , Proteínas Nucleares , Proteína 1 de Modelagem do Nucleossomo , Proteínas Recombinantes/metabolismoRESUMO
Coactivators of nuclear receptors are integral components of the signal transduction pathways of steroid hormones. Here, we show that one of the major coactivators of the glucocorticoid receptor (GR), CREB-binding protein (CBP), can also function conditionally as a negative regulator of its activities. Indeed, CBP suppressed the responsiveness of the mouse mammary tumor virus (MMTV) promoter to dexamethasone in a dose-dependent fashion in HeLa and A204 cells. Similarly, this protein suppressed the responsiveness of several glucocorticoid-responsive element (GRE)-containing synthetic promoters. Using deletion mutants of CBP, we localized the repressor effect of this protein to its N-terminal domain and showed that it was independent of the histone acetyltransferase and coactivator-binding domains but dependent upon its GR-binding domain. We also demonstrated functional differentiation between CBP and other coactivators, including SRC-1 and the CBP-related protein p300, both of which influenced GR signaling in a positive fashion. In fact, p300 completely antagonized the suppressive effects of CBP in a dose-dependent fashion, probably by competing with this protein at the level of the transcription complex. These findings suggest that CBP and p300 may function additively or antagonistically to each other depending on their relative concentrations and type of target tissue, to influence the sensitivity of tissues to glucocorticoids.
Assuntos
Proteínas Nucleares/fisiologia , Receptores de Glucocorticoides/fisiologia , Transativadores/fisiologia , Animais , Proteína de Ligação a CREB , Cloranfenicol O-Acetiltransferase/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteína p300 Associada a E1A , Células HeLa , Humanos , Luciferases/metabolismo , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Regiões Promotoras Genéticas , beta-Galactosidase/metabolismoRESUMO
Mouse mammary tumor virus (MMTV) expression is restricted primarily to mammary epithelial cells. Sequences responsible for both the mammary-specific expression of MMTV and the activation of cellular oncogenes are located within two enhancer elements at the 5'-end of the long terminal repeat. Whereas the Ban2 enhancer (-1075 to -978) has been well characterized, the mammary-specific enhancer of MMTV from -956 to -862 has only recently been recognized as a key determinant of mammary-specific oncogene activation by MMTV. The present study identifies and characterizes three binding sites located within this element. Transient transfection of deletion and mutation constructs shows that all three sites contribute to the basal expression of MMTV in mammary cells. One of the binding activities (footprint I) is restricted to mammary cells, whereas the other two sites bind factors found in both mammary and nonmammary cells. The multimerized mammary-specific enhancer of MMTV on its own can enhance a minimal promoter in a mammary-specific fashion. However, the FpI binding site alone cannot mediate this effect. Thus, it is the binding of multiple factors in a combinatorial fashion that mediates the mammary-restricted expression of MMTV.
Assuntos
Elementos Facilitadores Genéticos , Vírus do Tumor Mamário do Camundongo/fisiologia , Replicação Viral/genética , Animais , Sequência de Bases , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT , Pegada de DNA , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Dexametasona/farmacologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Proteínas Nucleares/metabolismo , Sequências Repetitivas de Ácido Nucleico/efeitos dos fármacos , TropismoAssuntos
Núcleo Celular/metabolismo , Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/análise , Animais , Proteínas de Fluorescência Verde , Humanos , Aumento da Imagem , Indicadores e Reagentes/análise , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The mouse mammary tumor virus (MMTV) promoter has target sequences recognized by several steroid receptors. We present evidence for a novel mechanism that confers hormone specificity to this promoter. We show that remodeling of MMTV chromatin and the concomitant activation of the MMTV promoter are induced equally by glucocorticoids and progestins in one chromosomal context but are selective for glucocorticoids in another. Furthermore, increased histone acetylation modulates MMTV promoter regulation disparately at the two chromosomal locations. Together, these data indicate that chromosomal architecture commands a crucial role in gene regulation, imposing locus-specific selectivity between regulators with similar sequence recognition.
Assuntos
Cromatina/metabolismo , Vírus do Tumor Mamário do Camundongo/genética , Regiões Promotoras Genéticas , Esteroides/metabolismo , Ativação Transcricional , Acetilação , Sequência de Bases , Linhagem Celular , Primers do DNA , Histonas/metabolismo , Hibridização in Situ Fluorescente , Luciferases/genética , Integração ViralRESUMO
Integration of mouse mammary tumor virus (MMTV) near the int genes results in the inappropriate expression of these proto-oncogenes and initiates events that lead to the formation of mammary adenocarcinomas. In most cases, the MMTV provirus integrates in a transcriptional orientation opposite that of the int genes. We have used a novel, vector-based system designed to recapitulate the integration of MMTV upstream of the int-2 promoter. Compared to a cellular promoter or another retroviral promoter, the MMTV long terminal repeat (LTR) in this configuration is particularly efficacious at activating the int-2 promoter. The sequences responsible for enhancing the activity of the int-2 promoter map to two domains in the 5' end of the MMTV LTR. One domain is a previously defined element; the second is an element delineated by these studies that acts synergistically with the first. Both of these elements display mammary cell-specific activity. Thus, even though the MMTV promoter itself is weak without hormonal stimulation, viral integration can position the 5' LTR elements to efficiently activate transcription from cellular proto-oncogenes. Other functional elements in the LTR have little effect on the activation of the int-2 promoter. Even stimulation of the MMTV promoter with steroid hormones only modestly activates transcription from the int-2 promoter, suggesting that the 5' elements of the LTR are the predominant determinants of the tissue- and orientation-specific activation of cellular promoters by MMTV.
Assuntos
Vírus do Tumor Mamário do Camundongo/genética , Vírus do Tumor Mamário do Camundongo/patogenicidade , Proto-Oncogenes , Animais , Sequência de Bases , Linhagem Celular , DNA/genética , Elementos Facilitadores Genéticos , Feminino , Fator 3 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Vetores Genéticos , Células HeLa , Humanos , Neoplasias Mamárias Experimentais/etiologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/virologia , Vírus do Tumor Mamário do Camundongo/efeitos dos fármacos , Camundongos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes/efeitos dos fármacos , Provírus/genética , Infecções por Retroviridae/etiologia , Infecções por Retroviridae/genética , Infecções por Retroviridae/virologia , Esteroides/farmacologia , Sequências Repetidas Terminais , Transfecção , Infecções Tumorais por Vírus/etiologia , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/virologia , Integração Viral/genéticaRESUMO
The question of how glucocorticoid, progesterone, androgen, and mineralocorticoid receptors can generate distinct patterns of gene expression despite similar, if not identical, DNA sequence recognition properties is a central question in steroid biology. This study addresses the hypothesis that glucocorticoid and progesterone receptors can differentially utilize the promoter context created by a series of receptor recognition sites. This hypothesis predicts that for different receptors an individual (often suboptimal) binding site contributes to the overall response element activity to a different degree. Therefore, mutations to an individual binding site of a multipartite hormone response element may differentially affect the response to different steroids. This study tests this hypothesis by introducing mutations into a receptor recognition site that is part of a hormone responsive promoter derived from the mouse mammary tumor virus. We find that weakening one site of a multipartite element differentially affects glucocorticoid and progestin signaling both in fibroblasts and in mammary carcinoma cells. A similar test was done in a simplified promoter context comprised only of a TATA box and a pair of receptor recognition sites. Mutations introduced into one of these sites impaired glucocorticoid response more than the progestin response. However, high receptor expression can partially overcome the effect of some target site mutations. Thus both receptor expression levels and the inherent ability to utilize the context created by a multipartite response element contribute to quantitative differences in the response to receptors that share DNA sequence recognition properties.
Assuntos
Regulação Neoplásica da Expressão Gênica , Vírus do Tumor Mamário do Camundongo/genética , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Animais , Sequência de Bases , Neoplasias da Mama , Cloranfenicol O-Acetiltransferase/genética , Dexametasona/farmacologia , Feminino , Fibroblastos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Luciferases/genética , Camundongos , Mutagênese Sítio-Dirigida , Receptores de Glucocorticoides/genética , TATA Box , Ativação Transcricional , Transfecção , Células Tumorais CultivadasRESUMO
Lipopolysaccharide (LPS) preconditioning induces cardiac resistance to subsequent LPS or ischemia. This study tested the hypothesis that resistance to LPS and resistance to ischemia are two manifestations of cardiac cross-resistance which may involve reprogramming of cardiac gene expression. Rats were preconditioned with a single dose of LPS (0.5 mg/kg ip). Cardiac resistance to LPS was examined with a subsequent LPS challenge. Cardiac resistance to ischemia was determined by subjecting hearts to ischemia-reperfusion. Total RNA was extracted from myocardium for Northern analysis of mRNAs encoding protooncoproteins, antioxidant enzymes, and contractile protein isoforms. Rats preconditioned with LPS 1-7 days earlier acquired cardiac resistance to endotoxemic depression. This resistance temporally correlated with resistance to ischemia. Pretreatment with cycloheximide (0.5 mg/kg ip) abolished resistance to both LPS and ischemia. LPS preconditioning induced the expression of c-jun and c-fos mRNAs. LPS also transiently increased mRNAs encoding catalase and Mn-containing superoxide dismutase. The expression of both alpha- and beta-myosin heavy chain mRNAs was upregulated, whereas the expression of cardiac alpha-actin mRNA was suppressed. We conclude that 1) LPS induces sustained cardiac resistance to both LPS and ischemia, 2) resistance to ischemia and resistance to LPS seem to be two mechanistically indistinct components of cardiac cross-resistance, and 3) the cardiac cross-resistance is associated with reprogramming of myocardial gene expression.
Assuntos
Regulação da Expressão Gênica , Coração/fisiologia , Precondicionamento Isquêmico Miocárdico , Lipopolissacarídeos/farmacologia , Miocárdio/metabolismo , Proto-Oncogenes , Análise de Variância , Animais , Cicloeximida/farmacologia , Endotoxemia/metabolismo , Endotoxemia/fisiopatologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos , Genes jun , Coração/efeitos dos fármacos , Coração/fisiopatologia , Humanos , Técnicas In Vitro , Cinética , Masculino , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Proto-Oncogênicas c-jun/genética , Proto-Oncogenes/efeitos dos fármacos , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Transcrição GênicaRESUMO
We previously reported that the chromatin high-mobility group protein 1 (HMG-1) enhances the sequence-specific DNA binding activity of progesterone receptor (PR) in vitro, thus providing the first evidence that HMG-1 may have a coregulatory role in steroid receptor-mediated gene transcription. Here we show that HMG-1 and the highly related HMG-2 stimulate DNA binding by other steroid receptors, including estrogen, androgen, and glucocorticoid receptors, but have no effect on DNA binding by several nonsteroid nuclear receptors, including retinoid acid receptor (RAR), retinoic X receptor (RXR), and vitamin D receptor (VDR). As highly purified recombinant full-length proteins, all steroid receptors tested exhibited weak binding affinity for their optimal palindromic hormone response elements (HREs), and the addition of purified HMG-1 or -2 substantially increased their affinity for HREs. Purified RAR, RXR, and VDR also exhibited little to no detectable binding to their cognate direct repeat HREs but, in contrast to results with steroid receptors, the addition of HMG-1 or HMG-2 had no stimulatory effect. Instead, the addition of purified RXR enhanced RAR and VDR DNA binding through a heterodimerization mechanism and HMG-1 or HMG-2 had no further effect on DNA binding by RXR-RAR or RXR-VDR heterodimers. HMG-1 and HMG-2 (HMG-1/-2) themselves do not bind to progesterone response elements, but in the presence of PR they were detected as part of an HMG-PR-DNA ternary complex. HMG-1/-2 can also interact transiently in vitro with PR in the absence of DNA; however, no direct protein interaction was detected with VDR. These results, taken together with the fact that PR can bend its target DNA and that HMG-1/-2 are non-sequence-specific DNA binding proteins that recognize DNA structure, suggest that HMG-1/-2 are recruited to the PR-DNA complex by the combined effect of transient protein interaction and DNA bending. In transient-transfection assays, coexpression of HMG-1 or HMG-2 increased PR-mediated transcription in mammalian cells by as much as 7- to 10-fold without altering the basal promoter activity of target reporter genes. This increase in PR-mediated gene activation by coexpression of HMG-1/-2 was observed in different cell types and with different target promoters, suggesting a generality to the functional interaction between HMG-1/-2 and PR in vivo. Cotransfection of HMG-1 also increased reporter gene activation mediated by other steroid receptors, including glucocorticoid and androgen receptors, but it had a minimal influence on VDR-dependent transcription in vivo. These results support the conclusion that HMG-1/-2 are coregulatory proteins that increase the DNA binding and transcriptional activity of the steroid hormone class of receptors but that do not functionally interact with certain nonsteroid classes of nuclear receptors.
Assuntos
DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Receptores de Esteroides/metabolismo , Transcrição Gênica , Animais , Células COS , Cloranfenicol O-Acetiltransferase/genética , Genes Reporter , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Mamíferos , Receptores Androgênicos/metabolismo , Receptores de Calcitriol/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Receptores de Esteroides/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Ácido Nucleico , Ativação TranscricionalRESUMO
Hormone response elements (HREs) are considered enhancers, activating transcription in a relatively position- and orientation-independent fashion. Upon binding to an HRE, steroid receptors presumably contact coactivators and/or proteins associated with the transcription initiation complex. As a receptor target site is moved further from a fixed position such as the TATA box, not only will the spatial separation of the receptor with respect to its interaction partners change, so will the orientation due to the rotation of the DNA helix. Additional constraints may be imposed by the assembly of DNA into chromatin. Therefore, we have endeavored to test rigorously the assertion that HRE action is position independent. We have constructed a series of 42 chloramphenicol acetyltransferase expression vectors that contain a single progesterone/glucocorticoid receptor-binding site separated from a TATA box by 4 to 286 bp. The enhancer activity of the HRE was assessed after transient transfection of progesterone receptor-expressing fibroblasts. We find that the position of the HRE has a dramatic influence on induction by progestins. When closely juxtaposed to the TATA box, the HRE was unable to support a hormone response, perhaps due to direct steric hindrance with the transcription initiation complex. Full activity was gained by moving the HRE 10 bp further from the TATA sequence. As the HRE was moved incrementally further, activity remained near maximal over the next 26 bp. HRE activity then declined over the subsequent 26 bp and remained low for another 2.5 helical turns. Surprisingly, a narrow window of HRE activity occurred at an HRE-TATA box separation of 90-100 bp. Little or no hormone-induced transcriptional activity was observed when the HRE was positioned further from the TATA box. The addition of a second HRE or a basal (nuclear factor-1) element failed to relieve this constraint. A similar series of experiments was carried out in a mammary carcinoma cell line that expressed high levels of both glucocorticoid and progestin receptors. Data in these cells indicate that glucocorticoids and progestins supported a similar HRE position-activity profile, but this pattern of HRE activity was quite distinct from that seen in fibroblasts. This may be indicative of cell type-specific interactions between steroid receptors and adapter/coactivator proteins or cell type-specific activities such as acetylases or deacetylases participating in the steroid response.
Assuntos
Dexametasona/farmacologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica/efeitos dos fármacos , Congêneres da Progesterona/farmacologia , Promegestona/farmacologia , Receptores de Glucocorticoides/genética , Receptores de Progesterona/genética , Sequência de Bases , Sítios de Ligação , Neoplasias da Mama/patologia , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/genética , Feminino , Fibroblastos , Genes Reporter , Vetores Genéticos/genética , Humanos , Dados de Sequência Molecular , Receptores de Glucocorticoides/biossíntese , Receptores de Progesterona/biossíntese , TATA Box , Transcrição GênicaRESUMO
Three types of transfection experiments were used to detect the abilities of different classes of antagonists to stimulate binding of progesterone receptor (PR) to progesterone response elements (PRE) in intact mammalian cells. These included a promoter interference assay, in which PR binding to PREs positioned between the TATA box and the start of transcription is detected as a reduction of expression of a constitutively active reporter gene, competition of PR antagonist and glucocorticoid receptor agonist for a common glucocorticoid response element/PRE-controlled reporter construct, and activation of a chimeric receptor (PR-VP16) containing the constitutive trans-activation domain derived from the VP16 protein of herpes simplex virus. By each approach, all antagonists tested were equally effective in stimulating PR binding to PREs in the cell. This included previously designated type I (ZK98299) and type II (RU486, ZK98734, and ZK112993) 11beta-aryl substituted steroid analogs. Stimulation of PR binding to PREs in the cell by ZK98299 was of interest because this antagonist has been reported to lack the ability to stimulate PR-DNA binding in vitro by electrophoretic gel mobility shift assay compared with RU486, which promotes efficient binding of PR to PREs. To clarify the apparent discrepancy between intact cell and in vitro results with ZK98299, we altered electrophoretic gel mobility shift assay conditions to allow detection of less stable DNA complexes. Under these conditions, ZK98299 induced the formation of specific PR-PRE complexes. Further analysis of the ZK98299-induced DNA complexes revealed that they exhibited an electrophoretic mobility different from that of the complexes induced by RU486, and the off-rate of PR from DNA was faster than that of the PR bound to agonist. This suggests that ZK98299 promotes a conformational change within PR distinct from that induced by RU486. The present results are consistent with the conclusions that ZK98299 stimulates PR binding to target DNA sequences and that ZK98299 and RU486 represent two mechanistic classes of antagonists based on inducing different conformational changes in PR.
Assuntos
DNA/metabolismo , Gonanos/farmacologia , Antagonistas de Hormônios/farmacologia , Mifepristona/farmacologia , Conformação Proteica/efeitos dos fármacos , Receptores de Progesterona/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Células COS , Humanos , Camundongos , Progesterona/antagonistas & inibidores , Progesterona/farmacologia , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/química , Receptores de Progesterona/genética , Proteínas Recombinantes de Fusão , Sequências Reguladoras de Ácido Nucleico , TATA Box , TransfecçãoRESUMO
We examined the mechanism(s) by which the progesterone receptor (PR) is able to inhibit glucocorticoid receptor (GR) activation from the mouse mammary tumor virus (MMTV) promoter in vivo. Using specific hormone antagonists, we demonstrate that the PR complexed with an type II antiprogestin blocks glucocorticoid-induced activation of the MMTV promoter. However, when complexed with a type I antiprogestin the PR is unable to block glucocorticoid-induced activation. PR repression of GR activity results from the inhibition of the ability of the GR to remodel chromatin such that the antiprogestin-occupied/PR prevents the glucocorticoid induced assembly of a preinitiation complex at MMTV promoter. These experiments suggest that the specific chromatin organization of the MMTV promoter provides a mechanism for regulating cross-talk between the GR and PR in vivo.