RESUMO
Over 25 years ago, eukaryotic cells were shown to contain a highly specific system for the selective degradation of short-lived proteins, this system is known as the ubiquitin-proteasome pathway. In this pathway, proteins are targeted for degradation by covalent modification by a small highly conserved protein named ubiquitin. Ubiquitin-mediated degradation of regulatory proteins plays an important role in numerous cell processes, including cell cycle progression, signal transduction and transcriptional regulation. Recent experiments have shown that the ubiquitin-proteasome pathway is also involved in nuclear hormone receptor (NR)-mediated transcriptional regulation. The idea that the ubiquitin-proteasome pathway is involved in NR-mediated transcription is strengthened by experiments showing that ubiquitin-proteasome components are recruited to NR target gene promoters. However, it is not clear how these components modulate NR-mediated chromatin remodeling and gene expression. In this review, we postulate the role of the ubiquitin-proteasome pathway on NR-mediated chromatin remodeling and gene regulation based on the current knowledge from studies implicating the pathway in chromatin structure modifications that are applicable to NR function. Since evidence from this laboratory, using the glucocorticoid receptor responsive mouse mammary tumor virus (MMTV) promoter organized as chromatin, suggest that the ubiquitin-proteasome system may be involved in the elongation phase of transcription, we particularly concentrate on chromatin modifications associated with the elongation phase.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo , Animais , Cromatina/química , Cromatina/metabolismo , Regulação da Expressão Gênica , Conformação de Ácido Nucleico , RNA Polimerase II/metabolismo , Transcrição GênicaRESUMO
The glucocorticoid receptor (GR) is a ligand-activated transcription factor that induces expression of many genes. The GR has been useful for understanding how chromatin structure regulates steroid-induced transcription in model systems. However, the effect of glucocorticoids on chromatin structure has been examined on few endogenous mammalian promoters. We investigated the effect of glucocorticoids on the in vivo chromatin structure of the glucocorticoid-responsive I kappa B alpha gene promoter, the inhibitor of the ubiquitous transcription factor, nuclear factor kappa B (NF kappa B). Glucocorticoids inhibit NF kappa B activity in some tissues by elevating the levels of I kappa B alpha. We found that glucocorticoids activated the I kappa B alpha promoter in human T47D/A1-2 cells containing the GR. We then investigated the chromatin structure of the I kappa B alpha promoter in the absence and presence of glucocorticoids with the use of micrococcal nuclease, restriction enzyme, and deoxyribonuclease (DNaseI) analyses. In untreated cells, the promoter assembles into regularly positioned nucleosomes, and glucocorticoid treatment did not alter nucleosomal position. Restriction enzyme accessiblity studies indicated that the I kappa B alpha promoter is assembled as phased nucleosomes that adopt an "open" chromatin architecture in the absence of hormone. However, glucocorticoids may be required for transcription factor binding, because DNaseI footprinting studies suggested that regulatory factors bind to the promoter upon glucocorticoid treatment.
Assuntos
Cromatina , Proteínas de Ligação a DNA/genética , Proteínas I-kappa B , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Ativação Transcricional , Pegada de DNA , Desoxirribonuclease I , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Humanos , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Nucleossomos , Células Tumorais CultivadasRESUMO
Understanding the role for DNA methylation in tumorigenesis has evolved from defining the location and extent of methylation in a variety of cancer-related genes to clarifying the functional and site-specific effects of aberrant methylation on gene expression. Our objectives were to characterize the functional effects of DNA methylation in the BRCA1 promoter and to clarify the functional status of the BRCA1 CRE (cAMP response element) motif. Luciferase reporter assays confirm that an intact CRE is important for BRCA1 expression in transient transfections. Luciferase activities were decreased in constructs where the CRE recognition sequence was altered and when constructs were methylated in vitro. Gel mobility shift and competition assays identified a DNA-protein complex recognizing the CRE motif that we were able to supershift using CREB-specific antibody. Furthermore this CRE is methylation sensitive, and we localized this methylation effect to a CpG dinucleotide within the BRCA1 CRE motif. The consequences of aberrant DNA methylation at specific transcription factor motifs, along with the multiple mutational events that can occur in a variety of essential genes such as BRCA1, paint a complex picture where both genetic and epigenetic changes contribute to tumour formation.
Assuntos
Ilhas de CpG , Metilação de DNA , Genes BRCA1/genética , Regiões Promotoras Genéticas , Sequência de Bases , Neoplasias da Mama/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Humanos , Luciferases/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transfecção , Células Tumorais CultivadasRESUMO
The authors have described three approaches for analyzing the chromatin architecture of a steroid-responsive promoter. Mnase allows one to map the positions of nucleosomes on the target gene. The more sensitive restriction enzyme hypersensitivity procedure permits detection of changes in chromatin architecture upon hormonal stimulation. Additional insight into transcriptional regulation of a gene can be obtained by using the related ExoIII footprinting protocol, which provides complementary data on transcription factor binding to chromatin templates. The use of these in vivo chromatin analysis techniques have provided evidence for a role of chromatin structure in regulation of transcription of steroid-responsive promoters including MMTV (2,7,10,14), tyrosine aminotransferase (15), TR beta A (16-19), and retinoic acid receptor beta (RAR beta) (20).
Assuntos
Cromatina/fisiologia , Vírus do Tumor Mamário do Camundongo/genética , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Receptores de Esteroides/metabolismo , Ativação Transcricional , Animais , HumanosRESUMO
We have previously shown a connection between histone H1 phosphorylation and the transcriptional competence of the hormone inducible mouse mammary tumor virus (MMTV) promoter. Prolonged exposure of mouse cells to dexamethasone concurrently dephosphorylated histone H1 and rendered the MMTV promoter refractory to hormonal stimulation and, therefore, transcriptionally unresponsive. Using electrospray mass spectrometry, we demonstrate here that prolonged dexamethasone treatment differentially effects a subset of the six somatic H1 isoforms in mouse cells. H1 isoforms H1.0, H1.1, and H1.2 are non-responsive to hormone whereas prolonged dexamethasone treatment effectively dephosphorylated the H1.3, H1.4, and H1.5 isoforms. The protein kinase inhibitor staurosporine, shown to dephosphorylate histone H1 and down-regulate MMTV in cultured cells, appears only to completely dephosphorylate the H1.3 isoform. These results suggest that dephosphorylation of specific histone H1 isoforms may contribute to the previously observed decrease in transcriptional competence of the MMTV promoter through the modulation of chromatin structure. In a broader sense, this work advances the hypothesis that post-translational modifications of individual histone H1 isoforms directly influence the transcriptional activation/repression of specific genes.
Assuntos
Histonas/química , Histonas/metabolismo , Hormônios/farmacologia , Animais , Antineoplásicos Hormonais/farmacologia , Western Blotting , Linhagem Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Dexametasona/farmacologia , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Camundongos , Fosforilação , Regiões Promotoras Genéticas , Isoformas de Proteínas , Espectrometria de Massas por Ionização por Electrospray , Estaurosporina/farmacologia , Transcrição GênicaRESUMO
Transcriptional activation of the mouse mammary tumor virus (MMTV) promoter by ligand-bound glucocorticoid receptor (GR) is transient. Previously, we demonstrated that prolonged hormone exposure results in displacement of the transcription factor nuclear factor 1 (NF1) and the basal transcription complex from the promoter, the dephosphorylation of histone H1, and the establishment of a repressive chromatin structure. We have explored the mechanistic link between histone H1 dephosphorylation and silencing of the MMTV promoter by describing the putative kinase responsible for H1 phosphorylation. Both in vitro kinase assays and in vivo protein expression studies suggest that in hormone-treated cells the ability of cdk2 to phosphorylate histone H1 is decreased and the cdk2 inhibitory p21 protein level is increased. To address the role of cdk2 and histone H1 dephosphorylation in the silencing of the MMTV promoter, we used potent cdk2 inhibitors, Roscovitine and CVT-313, to generate an MMTV promoter which is associated predominantly with the dephosphorylated form of histone H1. Both Roscovitine and CVT-313 block phosphorylation of histone H1 and, under these conditions, the GR is unable to remodel chromatin, recruit transcription factors to the promoter, or stimulate MMTV mRNA accumulation. These results suggest a model where cdk2-directed histone H1 phosphorylation is a necessary condition to permit GR-mediated chromatin remodeling and activation of the MMTV promoter in vivo.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/fisiologia , Histonas/fisiologia , Vírus do Tumor Mamário do Camundongo/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Cromatina/fisiologia , Quinase 2 Dependente de Ciclina , Camundongos , Fosforilação , Regiões Promotoras Genéticas/fisiologia , Transcrição Gênica , Replicação ViralRESUMO
The compaction of DNA into chromatin provides an additional level of gene regulation in eukaryotes that may not be available to prokaryotes. When packaged as chromatin, most promoters are transcriptionally repressed, and transcription factors have reduced access to their binding sites. The glucocorticoid receptor (GR) is a ligand-activated transcription factor that regulates the activity of genes involved in many physiological processes. To regulate eukaryotic genes, the GR binds to target sites within promoter regions of genes assembled as chromatin. This interaction alters the nucleosomal architecture to allow binding of other transcription factors, and formation of the preinitiation complex. The mouse mammary tumor virus (MMTV) promoter has been used extensively as a model to explore the processes by which the GR remodels chromatin and activates transcription. Significant progress has been made in our understanding of the mechanisms used by the GR to modify chromatin structure, and the limits placed on the GR by post-translational modifications of histones. We will describe recent developments in the processes used by the GR to activate transcription in vivo via chromatin remodeling complexes, histone H1 phosphorylation, and recruitment of diverse coactivators.
Assuntos
Cromatina/genética , Receptores de Glucocorticoides/fisiologia , Animais , Cromatina/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Modelos Biológicos , Fosforilação , Regiões Promotoras Genéticas/genéticaRESUMO
The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin. This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc from Saccharomyces cerevisiae. However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc. Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions. Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved domains that could be important for its function. First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the beta-globin locus. Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted. This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro. Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Proteínas Nucleares , Ativação Transcricional , Sequência de Aminoácidos , Clonagem Molecular , DNA Helicases , Proteínas de Ligação a DNA/genética , Globinas/genética , Humanos , Dados de Sequência Molecular , Família Multigênica , Estrutura Terciária de Proteína , Receptores de Glucocorticoides/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Fatores de TranscriçãoRESUMO
Steroid receptors represent a class of transcription regulators that act in part by overcoming the often repressive nature of chromatin to modulate gene activity. The mouse mammary tumor virus (MMTV) promoter is a useful model for studying transcriptional regulation by steroid hormone receptors in the context of chromatin. The chromatin architecture of the promoter prevents the assembly of basal transcription machinery and binding of ubiquitous transcription factors. However, in human breast carcinoma T47D cells lacking the glucocorticoid receptor (GR), but expressing the progesterone receptor (PR), nucleosome B (nuc B) assumes a constitutively hypersensitive chromatin structure. This correlation led us to test the hypothesis that the chromatin structure of nuc B was dependent on GR expression in T47D cells. To examine this possibility, we stably co-transfected the MMTV promoter and the GR into T47D cells that lacked both the GR and the PR. We found that in T47D cells that lack both the GR and the PR or express only the GR, nuc B assumes a constitutively "open" chromatin structure, which allows hormone independent access by restriction endonucleases and transcription factors. These results suggest that in GR(+)/pr(-) T47D cells, the MMTV chromatin structure permits GR transcriptional activation, independent of chromatin remodeling.
Assuntos
Neoplasias da Mama/virologia , Proteínas Estimuladoras de Ligação a CCAAT , Cromatina/metabolismo , Vírus do Tumor Mamário do Camundongo/genética , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição , Western Blotting , Núcleo Celular/metabolismo , Cromatina/química , Proteínas de Ligação a DNA/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Humanos , Hipersensibilidade , Fatores de Transcrição NFI , Proteínas Nucleares , Testes de Precipitina , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Proteína 1 de Ligação a Y-BoxRESUMO
Steroid hormones regulate the transcription of numerous genes via high affinity receptors that act in concert with chromatin remodeling complexes, coactivators and corepressors. We have compared the activities of a variety of glucocorticoid receptor (GR) antagonists in breast cancer and osteosarcoma cell lines engineered to stably maintain the mouse mammary tumor virus promoter. In both cell types, GR activation by dexamethasone occurs via the disruption of mouse mammary tumor virus chromatin structure and the recruitment of receptor coactivator proteins. However, when challenged with a variety of antagonists the GR displays differential ability to activate transcription within the two cell types. For the breast cancer cells, the antagonists fail to activate the promoter and do not promote the association of the GR with either remodeling or coactivator proteins. In contrast, in osteosarcoma cells, the antiglucocorticoids, RU486 and RU43044, exhibit partial agonist activity. The capacity of these antagonists to stimulate transcription in the osteosarcoma cells is reflected in the ability of the RU486-bound receptor to remodel chromatin and associate with chromatin-remodeling proteins. Similarly, the observation that the RU486-bound receptor does not fully activate transcription is consistent with its inability to recruit receptor coactivator proteins.
Assuntos
Dexametasona/farmacologia , Regulação Neoplásica da Expressão Gênica , Glucocorticoides/farmacologia , Antagonistas de Hormônios/farmacologia , Hidroxicorticosteroides , Vírus do Tumor Mamário do Camundongo/genética , Mifepristona/farmacologia , Receptores de Glucocorticoides/metabolismo , Animais , Neoplasias da Mama , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Osteossarcoma , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Tumour suppressor genes and growth regulatory genes are frequent targets for methylation defects that can result in aberrant expression and mutagenesis. We have established a methylation map of the promoter region of the neurofibromatosis (NF1) gene and demonstrated functional sensitivity for methylation at specific sites for the SP1 and CRE binding (CREB) proteins in the NF1 regulatory region. We evaluated the methylation status of CpG dinucleotides within five promoter subregions in the human and mouse homologues of the neurofibromatosis (NF1) genes. Three 5' subregions were found to be consistently methylated in all the tissues analysed. In contrast, DNA methylation was absent in the vicinity of the transcription start site bounded by SP1 recognition sequences. Gelshift assays showed that methylation specifically inhibits the CREB transcription factor from binding to its recognition site at the NF1 transcription start site. Furthermore, SP1 elements within the NF1 promoter are methylation sensitive, particularly when methylation is present on the antisense strand. We propose that for NF1 as with several other tumour suppressor genes, CpG methylation occurs in a complex, site-specific manner with the maintenance of a methylation-free promoter region bounded by SP1 binding sites that allow an accessible promoter to be retained. When these SP1 boundaries are breached, methylation can sweep in, rendering the promoter inaccessible for specific methylation-sensitive transcription factors and leading to a loss of functional integrity of the methylation-free CpG island.
Assuntos
Ilhas de CpG , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Metilação de DNA , DNA/metabolismo , Genes da Neurofibromatose 1 , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Regulação da Expressão Gênica , Humanos , Camundongos , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Ligação ProteicaAssuntos
Cromatina/genética , Cromatina/ultraestrutura , Exodesoxirribonucleases , Nucleossomos/genética , Nucleossomos/ultraestrutura , Animais , Fracionamento Celular/métodos , Núcleo Celular/ultraestrutura , Células Cultivadas , Primers do DNA , DNA Viral/genética , Genes Reporter , Humanos , Indicadores e Reagentes , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Sequências Repetitivas de Ácido Nucleico , Taq Polimerase , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
The biological actions of glucocorticoids in target organs are determined at least in part by the local expression of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which is responsible for the inactivation of glucocorticoids. The human endometrium is a glucocorticoid target tissue, and is known to express 11beta-HSD2. However, little is known about the function and regulation of 11beta-HSD2 in the endometrium, probably owing to the lack of in vitro model systems (i.e., cell lines) that express 11beta-HSD2. Here, we describe the characterization of 11beta-HSD expression in Ishikawa cells, a well-differentiated human endometrial adenocarcinoma cell line. The 11beta-HSD activity in intact Ishikawa cells was characteristic of 11beta-HSD2 in that it only possessed dehydrogenase activity (cortisol to cortisone) and had a high affinity for cortisol (apparent Km of 34 nM). The exclusive expression of 11beta-HSD2 in Ishikawa cells was confirmed by RT-PCR which demonstrated the presence of the mRNA for 11beta-HSD2 but not that for 11beta-HSD1. To investigate the regulation of 11beta-HSD2 in Ishikawa cells, we treated these cells with sex steroid hormones, glucocorticoids and epidermal growth factor (EGF), and determined the effects of these treatments on 11beta-HSD2 activity by an established intact cell radiometric conversion assay. Treatment with estradiol-17beta (E2, 10 nM) and medroxyprogesterone acetate (MPA, 100 nM) produced a classic sex steroid effect; the greatest increase (330% of the control) in the level of 11beta-HSD2 activity was caused by the combined treatment, followed by MPA (240% of the control) with E2 being the least effective (156% of the control). The stimulatory effect of E2 was blocked by the pure antiestrogen ICI 182,780. The synthetic glucocorticoid dexamethasone (Dex) increased 11beta-HSD2 activity in a time- and dose-dependent manner (200% of the control; 100 nM for 48 h), and the endogenous glucocorticoid cortisol was equally effective in this regard. The antiprogesterone-antiglucocorticoid RU486 did not counteract with MPA or Dex but rather acted as an agonist; increased 11beta-HSD2 activity (160% of the control; 100 nM for 72 h). By contrast, treatment with EGF caused a dose- and time-dependent decrease in 11beta-HSD2 activity (60% of the control; 10 ng/ml for 72 h). In addition, semi-quantitative RT-PCR analysis revealed that there were corresponding changes in the level of 11beta-HSD2 mRNA following the treatment of Ishikawa cells with these steroid hormones and EGF, indicating that the effects of these hormones and EGF are mediated, at least in part, at the level of 11beta-HSD2 gene transcription. In conclusion, we have demonstrated for the first time that the human Ishikawa endometrial cell line expresses exclusively the 11beta-HSD2 isozyme. Moreover, we have presented the first direct evidence that sex steroid hormones and glucocorticoids stimulate while EGF inhibit the expression of 11beta-HSD2 in Ishikawa cells, suggesting that endometrial 11beta-HSD2 is under the control of steroid hormones and EGF. Thus, the Ishikawa cell line represents an excellent model in which the function and regulation of endometrial 11beta-HSD2 may be studied.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hidroxiesteroide Desidrogenases/genética , Hidroxiesteroide Desidrogenases/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2 , Adenocarcinoma , Dexametasona/farmacologia , Neoplasias do Endométrio , Endométrio , Estradiol/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Acetato de Medroxiprogesterona/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Overexpression of cathepsin D (CD), a ubiquitous lysosomal protease, is closely associated with a poor clinical outcome for patients with breast cancer. Estrogen greatly induces transcription of the CD gene in estrogen receptor (ER)-positive breast cancer cells. In this report, we transiently introduced a human CD promoter/chloramphenicol acetyltransferase reporter gene into human MCF-7 breast cancer cells to study the mechanisms by which the ER activates the promoter. Using an in vivo Exonuclease III footprinting assay, we found that estrogen stimulation of MCF-7 cells induced loading of a transcription factor(s) to a portion of the promoter (-124 to -104) that is homologous to the adenovirus major late promoter element. Subsequent gel mobility shift assays with a 21-bp CD -124/-104 probe and nuclear extracts prepared from naive and estrogen-stimulated cells detected a single sequence-specific protein-DNA complex. Southwestern and UV cross-linking experiments detected two proteins of 44 kDa and 43 kDa that were specifically bound to the 21-bp fragment of the promoter. Gel super-shift assays with upstream stimulatory factor 1 (USF-1) and USF-2 antibodies demonstrated that USF-1 and USF-2 bound to the E box probe. Sequence specific binding was abolished by a 2-bp change shown previously to prevent the binding of USF to the E box. Incorporation of a mutant E box into the wild-type CD promoter/chloramphenicol acetyltransferase gene abolished USF binding and reduced the levels of both basal and estrogen-stimulated transcription. These results suggest that the ER targeting of USF-1 and USF-2 is a critical step in hormone activation of CD gene transcription in human breast cancer cells.
Assuntos
Catepsina D/genética , Proteínas de Ligação a DNA , Sequências Hélice-Alça-Hélice , Zíper de Leucina , Regiões Promotoras Genéticas , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sequência de Bases , Sítios de Ligação/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Pegada de DNA , Feminino , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Análise de Sequência de DNA , Transfecção , Células Tumorais Cultivadas , Fatores Estimuladores UpstreamRESUMO
The assembly of transcriptional regulatory DNA sequences into chromatin plays a fundamental role in modulating gene expression. The promoter of the mouse mammary-tumour virus (MMTV) is packaged into a regular array of nucleosomes when it becomes stably integrated into mammalian chromosomes, and has been used to investigate the relationship between chromatin architecture and transcriptional activation by the hormone-bound glucocorticoid and progesterone receptors. In mammalian cells that express both of these receptors, the progesterone receptor activates transcription from transiently transfected MMTV DNA but not from organized chromatin templates. Moreover, the activated progesterone receptor inhibits the chromatin remodelling and consequent transcriptional stimulation that is mediated by the glucocorticoid receptor. Here we investigate the mechanism of this inhibition by characterizing the interaction of the glucocorticoid receptor with transcriptional co-activator and chromatin remodelling protein complexes. We show that when this receptor is prevented from interacting with the hBRG1/BAF chromatin remodelling complex, it can activate transcription from transiently transfected DNA but not from organized chromatin templates. Our results indicate that it may be possible to separate the transcriptional activation and chromatin remodelling activities of proteins that interact with hormone receptors.
Assuntos
Cromatina/fisiologia , Proteínas Nucleares/fisiologia , Receptores de Glucocorticoides/fisiologia , Transativadores , Fatores de Transcrição/fisiologia , Animais , DNA Helicases , Estrenos/farmacologia , Furanos/farmacologia , Regulação da Expressão Gênica , Antagonistas de Hormônios/farmacologia , Vírus do Tumor Mamário do Camundongo/genética , Vírus do Tumor Mamário do Camundongo/fisiologia , Proteínas Nucleares/metabolismo , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/fisiologia , Fatores de Transcrição/metabolismo , Ativação Transcricional , Células Tumorais CultivadasRESUMO
Breast cancer is a genetic disease arising from a series of germ-line and/or somatic DNA changes in a variety of genes, including BRCA1 and BRCA2. DNA modifications have been shown to occur by a number of mechanisms that include DNA methylation. In some cases, the aberrant methylation of CpGs within 5' regulatory regions has led to suppression of gene activity. In this report we describe a variation in the pattern of DNA methylation within the regulatory region of the BRCA1 gene. We found no evidence of methylation at CpGs within the BRCA1 promoter in a variety of normal human tissues. However, screening of a series of randomly sampled breast carcinomas revealed the presence of CpG methylation adjacent to the BRCA1 transcription start site. One such methylated CpG occurs at a putative CREB (cAMP-responsive element binding) transcription factor binding site in the BRCA1 promoter. Gelshift assays with methylated and unmethylated BRCA1/CREB binding site oligonucleotides demonstrate that this site is sensitive to site-specific CpG methylation. These data suggest that aberrant DNA methylation at regulatory sequences in the BRCA1 locus may play a role in the transcriptional inactivation of the BRCA1 gene within subclones of breast tumors. This study represents the first evidence suggesting a role for DNA methylation in the transcriptional inactivation of the BRCA1 in human breast cancer.
Assuntos
Neoplasias da Mama/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Metilação de DNA , Fosfatos de Dinucleosídeos/metabolismo , Genes BRCA1 , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Sequência de Bases , Sítios de Ligação , Éxons , Feminino , Humanos , Modelos Genéticos , Oligodesoxirribonucleotídeos , Neoplasias Ovarianas/genética , Valores de Referência , Transcrição GênicaRESUMO
Glucocorticoids rapidly induce transcription from the mouse mammary tumour virus (MMTV) promoter via a glucocorticoid receptor (GR)-mediated chromatin disruption event. This remodelling of chromatin is transient such that upon prolonged exposure to hormone the promoter becomes refractory to glucocorticoids. We demonstrate that this refractory state requires the continual presence of hormone and can be reversed by its removal. Our experiments show that the promoter is inactivated via a mechanism whereby histone H1 is dephosphorylated in response to glucocorticoids. Removal of glucocorticoids results in the rephosphorylation of histone H1 and the reacquisition of transcriptional competence by the promoter. This response is specific for the MMTV promoter assembled as chromatin and is not observed for another inducible gene or transiently transfected MMTV DNA. Finally, we demonstrate that H1 on the MMTV promoter is dephosphorylated when the promoter is unresponsive to glucocorticoids. These studies indicate that phosphorylated H1 is intimately linked with the GR-mediated disruption of MMTV chromatin in vivo.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Histonas/metabolismo , Vírus do Tumor Mamário do Camundongo/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica/fisiologia , Animais , Linhagem Celular , Cromatina/metabolismo , DNA Viral/metabolismo , Inibidores Enzimáticos/farmacologia , Metalotioneína/genética , Camundongos , Fosforilação , Proteína Quinase C/antagonistas & inibidores , RNA Mensageiro/biossíntese , Receptores de Glucocorticoides/fisiologia , Estaurosporina/farmacologia , Ativação Transcricional/fisiologiaRESUMO
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.
Assuntos
Cromatina/química , Antagonistas de Hormônios/farmacologia , Progesterona/antagonistas & inibidores , Receptores de Glucocorticoides/efeitos dos fármacos , Dexametasona/farmacologia , Humanos , Vírus do Tumor Mamário do Camundongo/genética , Modelos Moleculares , Conformação Proteica , Receptores de Glucocorticoides/fisiologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Células Tumorais CultivadasRESUMO
17beta-Estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast cancer cells. Previous studies have identified an Sp1-imperfect estrogen-responsive element (ERE) half-site [GGGCGG(N)23ACGGG] (-199 to -165) in the promoter region which forms an Sp1-estrogen receptor (ER) complex and confers E2 responsiveness on the corresponding Sp1-ERE-chloramphenicol acetyl transferase (CAT) construct. Further analysis of downstream regions of the promoter identified a CGCCC(N)3TGACC sequence (-119 to -107) which is homologous to the adenovirus major late promoter element (MLPE) and binds the ER to form a retarded band in a gel electrophoretic mobility shift assay. The corresponding promoter-CAT construct is also E2-inducible. The MLPE resembles an imperfect palindromic ERE containing imperfect (5') and perfect (3') ERE half-sites; analysis of oligonucleotides with mutations in these half-sites shows that only the perfect ERE half-site is required for binding the ER, whereas both sites are required for transactivation. In vivo exonuclease III footprinting showed that treatment with E2 also enhanced binding at the MLPE site. Identification of this second functional enhancer sequence in the 5'-promoter region of cathepsin D is consistent with the increasingly complex cell-specific regulation of hormone-responsive genes.
Assuntos
Catepsina D/genética , Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , DNA , Humanos , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
A number of important nuclear processes including replication, recombination, repair, and transcription involve the interaction of soluble nuclear proteins with DNA assembled as chromatin. Recent progress in a number of experimental systems has focused attention on the influence chromatin structure may exert on gene regulation in eukaryotes. With the advent of new technologies for the analysis of chromatin structure in vivo, studies evaluating the influence of chromatin structure on gene transcription have become feasible for a number of systems. This article serves as an introduction to the use of restriction endonucleases to define nucleosomal organization and characterize changes in this organization that accompany transcriptional activation in vivo. The procedure includes the isolation of intact transcriptionally competent nuclei, limited digestion with specific restriction endonucleases, and purification of the DNA. This DNA serves as the substrate for a linear amplification using single primers that generate enzyme-specific DNA fragments, which are then resolved by electrophoresis. Specific examples related to our studies of the influence of chromatin structure on steroid hormone regulation of transcription from the mouse mammary tumor virus promoter are provided to illustrate this technique and several novel variations. Alternative methods for analysis of chromatin architecture using DNase I, micrococcal nuclease, permanganate, and methidiumpropyl-EDTA-iron(II) are also described. Through the use of these methodologies one is able to determine both the translational and the rotational positions for a given nucleosome as well as quantify changes at a specific nucleosome in response to regulatory and developmental signals.