RESUMO
The PML protein and PML nuclear bodies (PML-NB) are implicated in multiple cellular functions relevant to tumor suppression, including DNA damage response. In most cases of acute promyelocytic leukemia, the PML and retinoic acid receptor alpha (RARA) genes are translocated, resulting in expression of oncogenic PML-RARα fusion proteins. PML-NB fail to form normally, and promyelocytes remain in an undifferentiated, abnormally proliferative state. We examined the involvement of PML protein and PML-NB in homologous recombinational repair (HRR) of chromosomal DNA double-strand breaks. Transient overexpression of wild-type PML protein isoforms produced hugely enlarged or aggregated PML-NB and reduced HRR by ~2-fold, suggesting that HRR depends to some extent upon normal PML-NB structure. Knockdown of PML by RNA interference sharply attenuated formation of PML-NB and reduced HRR by up to 20-fold. However, PML-knockdown cells showed apparently normal induction of H2AX phosphorylation and RAD51 foci after DNA damage by ionizing radiation. These findings indicate that early steps in HRR, including recognition of DNA double-strand breaks, initial processing of ends, and assembly of single-stranded DNA/RAD51 nucleoprotein filaments, do not depend upon PML-NB. The HRR deficit in PML-depleted cells thus reflects inhibition of later steps in the repair pathway. Expression of PML-RARα fusion proteins disrupted PML-NB structure and reduced HRR by up to 10-fold, raising the possibility that defective HRR and resulting genomic instability may figure in the pathogenesis, progression and relapse of acute promyelocytic leukemia.
Assuntos
Quebras de DNA de Cadeia Dupla , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Reparo de DNA por Recombinação , Trióxido de Arsênio , Arsenicais/farmacologia , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Reparo do DNA por Junção de Extremidades , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Instabilidade Genômica , Histonas/metabolismo , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Óxidos/farmacologia , Fosforilação , Proteína da Leucemia Promielocítica , Interferência de RNA , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The alternation/deficiency in activation-3 (ADA3) is an essential component of the human p300/CBP-associated factor (PCAF) and yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) histone acetyltransferase complexes. These complexes facilitate transactivation of target genes by association with transcription factors and modification of local chromatin structure. It is known that the yeast ADA3 is required for nuclear receptor (NR)-mediated transactivation in yeast cells; however, the role of mammalian ADA3 in NR signaling remains elusive. In this study, we have investigated how the human (h) ADA3 regulates retinoic acid receptor (RAR) α-mediated transactivation. We show that hADA3 interacts directly with RARα in a hormone-dependent manner and this interaction contributes to RARα transactivation. Intriguingly, this interaction involves classical LxxLL motifs in hADA3, as demonstrated by both 'loss' and 'gain' of function mutations, as well as a functional coactivator pocket of the receptor. Additionally, we show that hADA3 associates with RARα target gene promoter in a hormone-dependent manner and ADA3 knockdown impairs RARß2 expression. Furthermore, a structural model was established to illustrate an interaction network within the ADA3/RARα complex. These results suggest that hADA3 is a bona fide transcriptional coactivator for RARα, acting through a conserved mechanism involving direct contacts between NR boxes and the receptor's co-activator pocket.
Assuntos
Receptores do Ácido Retinoico/química , Fatores de Transcrição/química , Ativação Transcricional , Motivos de Aminoácidos , Sítios de Ligação , Linhagem Celular , Humanos , Modelos Moleculares , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Fatores de Transcrição/metabolismoRESUMO
Estrogen-related receptors (ERR) are orphan members of the nuclear receptor superfamily most closely related to estrogen receptors (ER). Although ERalpha is a successful target for treating breast cancer, there remains an unmet medical need especially for estrogen-independent breast cancer. Although estradiol is not an ERR ligand, ER and ERR share many commonalities and overlapping signaling pathways. An endogenous ERR ligand has not been identified; however, novel synthetic ERRalpha subtype-specific antagonists have started to emerge. In particular, we recently identified a novel compound, N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5H dibenzo[a,d][7]annulen-5-amine (termed compound A) that acts specifically as an ERRalpha antagonist. Here, we show that compound A inhibited cell proliferation in ERalpha-positive (MCF-7 and T47D) and ERalpha-negative (BT-20 and MDA-MD-231) breast cancer cell lines. Furthermore, we report the differential expression of 83 genes involved in ERRalpha signaling in MCF-7 and BT-20 breast cancer cell lines. We show that compound A slowed tumor growth in MCF-7 and BT-20 mouse xenograft models, and displayed antagonistic effects on the uterus. Furthermore, a subset of genes involved in ERRalpha signaling in vitro were evaluated and confirmed in vivo by studying uterine gene expression profiles from xenograft mice. These results suggest for the first time that inhibition of ERRalpha signaling via a subtype-specific antagonist may be an effective therapeutic strategy for ER-positive and ER-negative breast cancers.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Receptores de Estrogênio/antagonistas & inibidores , Tiazolidinas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Regulação para Baixo/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Camundongos , Camundongos Nus , Especificidade por Substrato/efeitos dos fármacos , Tiazolidinas/farmacologia , Células Tumorais Cultivadas , Útero/efeitos dos fármacos , Útero/metabolismo , Útero/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
The silencing mediator for retinoid and thyroid hormone receptors (SMRT) serves as a platform for transcriptional repression elicited by several steroid/nuclear receptors and transcription factors. SMRT exists in two major splicing isoforms, alpha and tau, with SMRTalpha containing only an extra 46-amino acid sequence inserted immediately downstream from the C-terminal corepressor motif. Little is known about potential functional differences between these two isoforms. Here we show that the pregnane X receptor (PXR) interacts more strongly with SMRTalpha than with SMRTtau both in vitro and in vivo. It is interesting that the PXR-SMRTalpha interaction is also resistant to PXR ligand-induced dissociation, in contrast to the PXR-SMRTtau interaction. SMRTalpha consistently inhibits PXR activity more efficiently than does SMRTtau in transfection assays, although they possess comparable intrinsic repression activity and association with histone deacetylase. We further show that the mechanism for the enhanced PXR-SMRTalpha interaction involves both the 46-amino acid insert and the C-terminal corepressor motif. In particular, the first five amino acids of the SMRTalpha insert are essential and sufficient for the enhanced binding of SMRTalpha to PXR. Furthermore, we demonstrate that Tyr2354 and Asp2355 residues of the SMRTalpha insert are most critical for the enhanced interaction. In addition, expression data show that SMRTalpha is more abundantly expressed in most human tissues and cancer cell lines, and together these data suggest that SMRTalpha may play a more important role than SMRTtau in the negative regulation of PXR.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Esteroides/metabolismo , Proteínas Repressoras/metabolismo , Animais , Inativação Gênica , Humanos , Camundongos , Correpressor 2 de Receptor Nuclear , Receptor de Pregnano X , Coelhos , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
The pregnane X receptor (PXR; PXR.1) can be activated by structurally diverse lipophilic ligands. PXR.2, an alternatively spliced form of PXR, lacks 111 nucleotides encoding 37 amino acids in the ligand binding domain. PXR.2 bound a classic CYP3A4 PXR response element (PXRE) in electrophoretic mobility shift assays, but transfected PXR.2 failed to transactivate a CYP3A4-promoter-luciferase reporter plasmid in HepG2 cells treated with various PXR ligands. Cotransfection experiments showed that PXR.2 behaved as a dominant negative, interfering with PXR.1/rifampin activation of CYP3A4-PXRE-LUC. In HepG2 and LS180 cells stably transduced with PXR.1, PXR target genes (CYP3A4, MDR1, CYP2B6, and UGT1A1) were higher than mock-transduced cells in the absence of ligand and were further induced in the presence of rifampin. In contrast, PXR.2 stably introduced into the same host cells failed to induce target genes over levels in mock-transfected cells after drug treatment. Our homology modeling suggests that ligands bind PXR.1 more favorably, probably because of the presence of a key disordered loop region, which is missing in PXR.2. Yeast two-hybrid assays revealed that, even in the presence of ligand, the corepressors remain tightly bound to PXR.2, and coactivators are unable to bind at helix 12. In summary, PXR.2 can bind to PXREs but fails to transactivate target genes because ligands do not bind the ligand binding domain of PXR.2 productively, corepressors remain tightly bound, and coactivators are not recruited to PXR.2.
Assuntos
Processamento Alternativo , Citocromo P-450 CYP3A/química , Isoformas de Proteínas/farmacologia , Receptores de Esteroides/química , Ativação Transcricional/efeitos dos fármacos , Domínio Catalítico , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Microssomos Hepáticos , Receptor de Pregnano X , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Conformação Proteica , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Rifampina/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
ANCO (ankyrin repeats-containing cofactor)-1 and ANCO-2 are a family of unique transcriptional co-regulators with dual properties: they interact with both the co-activators and the co-repressors [Zhang, Yeung, Li, Tsai, Dinh, Wu, Li and Chen (2004) J. Biol. Chem. 279, 33799-33805]. Specifically, ANCO-1 is thought to recruit HDACs (histone deacetylases) to the p160 co-activator to repress transcriptional activation by nuclear receptors. In the present study, we provide new evidence to suggest further that ANCO-1 and ANCO-2 also interact with the co-activator ADA3 (alteration/deficiency in activation 3). The interaction occurs between the conserved C-terminal domain of ANCO-1 and the N-terminal transactivation domain of ADA3. Several subunits of the P/CAF {p300/CBP [CREB (cAMP-response-element-binding protein)-binding protein]-associated factor} complex, including ADA3, ADA2alpha/beta and P/CAF, showed co-localization with ANCO-1 nuclear dots, indicating an in vivo association of ANCO-1 with the P/CAF complex. Furthermore, a transient reporter assay revealed that both ANCO-1 and ANCO-2 repress ADA3-mediated transcriptional co-activation on nuclear receptors, whereas ANCO-1 stimulated p53-mediated transactivation. These data suggest that ADA3 is a newly identified target of the ANCO proteins, which may modulate co-activator function in a transcription-factor-specific manner.
Assuntos
Repetição de Anquirina , Anquirinas/química , Regulação da Expressão Gênica , Fatores de Transcrição/química , Animais , Células COS , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Humanos , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Fatores de Transcrição/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Daxx plays a major role in several important signaling pathways including transcription and cell death. It has been postulated that Daxx regulates both events from the nucleus; however, the mechanism by which Daxx is localized in the nucleus remains obscure. Here we show that nuclear localization of Daxx is controlled by two independent signals and importin 3. Domain analysis reveals that Daxx contains two separate nuclear localizing domains. Site-directed mutagenesis reveals that the basic aa sequence RLKRK at residues 227-231 (NLS1) is responsible for nuclear localization of N-terminal domain, while aa sequence KKSRKEKK at residues 630-637 (NLS2) is responsible for nuclear localization of the C-terminal domain. Mutations of a NLS consensus sequence RKKRR at residues 391-395 and several other basic aa clusters have no effect on Daxx nuclear localization. In full-length Daxx, NLS1 contributes partially to nuclear localization, while NLS2 plays a major role. Markedly, it is essential to disrupt both NLS1 and NLS2 in order to completely block nuclear localization of the full-length protein and to prevent its association with PML nuclear bodies. Furthermore, Daxx interacts selectively with importin alpha3 through its NLS1 and NLS2 sequences. Conversely, importin alpha3 utilizes two NLS-binding sites for Daxx interaction, suggesting that the importin/mediates nuclear import of Daxx. Finally, we show that nuclear localization of Daxx is essential for its transcriptional effects on GR and p53. Together, these data unveil a molecular mechanism that controls nuclear localization of Daxx and support a nuclear role of Daxx in transcriptional regulation.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Sinais de Localização Nuclear/química , Proteínas Nucleares/química , Mapeamento de Interação de Proteínas , alfa Carioferinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS/metabolismo , Linhagem Celular/metabolismo , Chlorocebus aethiops , Proteínas Correpressoras , Células HeLa/metabolismo , Humanos , Chaperonas Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Transfecção , alfa Carioferinas/fisiologiaRESUMO
Daxx is a nuclear protein involved in apoptosis and transcriptional repression, and it interacts with the death receptor Fas, promyelocytic leukemia protein (PML), and several transcriptional repressors. The function of Daxx in apoptosis is controversial because opposite results were obtained in transient overexpression and genetic knockout studies. Furthermore, the roles of PML and transcriptional repression in Daxx-regulated apoptosis are currently unknown. In this study, we investigated the role of Daxx in Fas- and stress-induced apoptosis by small interfering RNA-mediated Daxx silencing in mammalian cells. Daxx silencing had no apparent cytotoxic effects on mammalian cells within 72 h. Intriguingly, Daxx silencing strongly sensitized cells to Fas- and stress-induced apoptosis, which was accompanied by caspase activation, cytochrome c release, and Jun N-terminal kinase activation. Consistently, endogenous Daxx was degraded rapidly upon induction of apoptosis by stress or anti-Fas antibody. Finally, PML silencing had no effect on Daxx silencing-mediated apoptotic events, while caspase gene expression was upregulated in the absence of Daxx. These data strongly suggest that Daxx may inhibit Fas and stress-mediated apoptosis by suppressing proapoptotic gene expression outside of PML domains.
Assuntos
Apoptose , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Western Blotting , Caspases/metabolismo , Divisão Celular , Núcleo Celular/metabolismo , Proteínas Correpressoras , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Inativação Gênica , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , MAP Quinase Quinase 4 , Microscopia de Fluorescência , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Chaperonas Moleculares , RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição Gênica , Transfecção , Raios Ultravioleta , Regulação para CimaRESUMO
Transcription factor NF-E2-related factor 2 (Nrf2) regulates the induction of Phase II detoxifying enzymes and antioxidant enzymes in response to many cancer chemopreventive compounds. In this study, we investigated the role of receptor associated coactivator (RAC3) or steroid receptor coactivator-3 (SRC3) and other nuclear co-regulators including CBP/p300 (CREB-binding protein), CARM1(Coactivator-associated arginine methyltransferase), PRMT1(Protein arginine methyl-transferase 1), and p/CAF (p300/CBP-associated factor) in the transcriptional activation of a chimeric Gal4-Nrf2-Luciferase system containing the transactivation domain (TAD) of Nrf2 in HepG2 cells. The results indicated that RAC3 up-regulated the transactivation activity of Gal4-Nrf2-(1-370) in a dose-dependent manner. The enhancement of transactivation domain activity of Gal4-Nrf2-(1-370) by RAC3 was dampened in the presence of dominant negative mutants of RAC3. Next we studied the effects of other nuclear co-regulators including CBP/ p300, CARM1, PRMT1 and p/CAF, and the results showed that they had different level of positive effects on this transactivation domain activity of Gal4-Nrf2-(1-370). But importantly, synergistic effects of these co-regulators in the presence of RAC3/SRC3 on the transactivation activity of Gal4-Nrf2-(1-370) were observed. In summary, our present study showed for the first time that the 160 RAC3/SRC3 is involved in the functional transactivation of TAD of Nrf2 and that the other nuclear co-regulators such as CBP/p300, CARM1, PRMT1 and p/CAF can also transcriptionally activate this TAD of Nrf2 and that they could further enhance the transactivation activity mediated by RAC3/SRC3.
Assuntos
Acetiltransferases/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Linhagem Celular Tumoral , Genes Reguladores , Histona Acetiltransferases , Humanos , Coativador 3 de Receptor Nuclear , Ligação Proteica , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Regulação para Cima , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
SRCAP (SNF2-related CBP activator protein) is a 350-kDa protein that shares homology with the SNF2 family of proteins whose members function in various aspects of transcriptional regulation. In various cell types, SRCAP is found in distinct multiprotein complexes that include proteins found in SWI/SNF chromatin remodeling complexes. SRCAP was identified by its ability to bind to CBP and was found to potentiate the ability of CBP to activate transcription. Studies in our laboratory have demonstrated that SRCAP functions as a coactivator for CREB-mediated transcription of a number of promoters, including that of the phosphoenolpyruvate carboxykinase gene. Our current studies demonstrate that SRCAP enhances phosphoenolpyruvate carboxykinase promoter transcription induced by glucocorticoids. SRCAP also enhances glucocorticoid receptor-mediated transcription of a simple promoter containing only two glucocorticoid response elements, indicating that SRCAP functions as a glucocorticoid receptor coactivator. In similar studies, SRCAP was also found to serve as a coactivator for the androgen receptor. SRCAP exhibits synergistic activation with nuclear receptor coactivators and functionally interacts in vivo with glucocorticoid receptor-interacting protein-1 and coactivator-associated arginine methyltransferase-1. We propose that SRCAP, by virtue of its ability to interact with CBP, functions as a coactivator to regulate transcription initiated by several signaling pathways.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Receptores de AMPA/metabolismo , Transcrição Gênica/genética , Animais , Células COS , Chlorocebus aethiops , Clonagem Molecular , Células HeLa , Humanos , Plasmídeos/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
BACKGROUND: The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) is a member of the nuclear receptor superfamily. It was identified through a search for genes encoding proteins related to estrogen receptor alpha (ERalpha). An endogenous ligand has not been found. Novel ERRalpha antagonists that are highly specific for binding to the ligand binding domain (LBD) of ERRalpha have been recently reported. Research suggests that ERRalpha may be a novel drug target to treat breast cancer and/or metabolic disorders and this has led to an effort to characterize the mechanisms of action of N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5H dibenzo[a,d][7]annulen-5-amine, a novel ERRalpha specific antagonist. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate this ERRalpha ligand inhibits ERRalpha transcriptional activity in MCF-7 cells by luciferase assay but does not affect mRNA levels measured by real-time RT-PCR. Also, ERalpha (ESR1) mRNA levels were not affected upon treatment with the ERRalpha antagonist, but other ERRalpha (ESRRA) target genes such as pS2 (TFF1), osteopontin (SPP1), and aromatase (CYP19A1) mRNA levels decreased. In vitro, the ERRalpha antagonist prevents the constitutive interaction between ERRalpha and nuclear receptor coactivators. Furthermore, we use Western blots to demonstrate ERRalpha protein degradation via the ubiquitin proteasome pathway is increased by the ERRalpha-subtype specific antagonist. We demonstrate by chromatin immunoprecipitation (ChIP) that the interaction between ACADM, ESRRA, and TFF1 endogenous gene promoters and ERRalpha protein is decreased when cells are treated with the ligand. Knocking-down ERRalpha (shRNA) led to similar genomic effects seen when MCF-7 cells were treated with our ERRalpha antagonist. CONCLUSIONS/SIGNIFICANCE: We report the mechanism of action of a novel ERRalpha specific antagonist that inhibits transcriptional activity of ERRalpha, disrupts the constitutive interaction between ERRalpha and nuclear coactivators, and induces proteasome-dependent ERRalpha protein degradation. Additionally, we confirmed that knocking-down ERRalpha lead to similar genomic effects demonstrated in vitro when treated with the ERRalpha specific antagonist.
Assuntos
Neoplasias da Mama/metabolismo , Receptores de Estrogênio/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Transcrição Gênica/efeitos dos fármacos , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
The ability of p53 to act as a transcription factor is critical for its function as a tumor suppressor. Ankyrin repeat domain 11, ANKRD11 (also known as ANR11 or ANCO1), was found to be a novel p53-interacting protein that enhanced the transcriptional activity of p53. ANKRD11 expression was shown to be downregulated in breast cancer cell lines. Restoration of ANKRD11 expression in MCF-7 (wild-type p53) and MDA-MB-468 (p53(R273H) mutant) cells suppressed their proliferative and clonogenic properties through enhancement of CDKN1A (p21(waf1)/CIP1) expression. ShRNA-mediated silencing of ANKRD11 expression reduced the ability of p53 to activate CDKN1A expression. ANKRD11 was shown to associate with the p53 acetyltransferases and cofactors, P/CAF and hADA3. Exogenous ANKRD11 expression enhanced the levels of acetylated p53 in both MCF-7 and MDA-MB-468 cells. ANKRD11 enhanced the DNA-binding properties of mutant p53(R273H) to the CDKN1A promoter, suggesting that ANKRD11 can mediate the restoration of normal p53 function in some cancer-related p53 mutations. In addition, ANKRD11 itself was found to be a novel p53 target gene. These findings demonstrate a role for ANKRD11 as a p53 coactivator and suggest the involvement of ANKRD11 in a regulatory feedback loop with p53.
Assuntos
Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , DNA/química , Inativação Gênica , Genes p53 , Células HeLa , Humanos , Modelos Biológicos , Mutação , Estrutura Terciária de Proteína , Transcrição GênicaRESUMO
The ankyrin repeats cofactor-1 (ANCO-1) was recently identified as a p160 coactivator-interacting protein that may inhibit transcriptional activity of nuclear receptors. Here, we have characterized the transcriptional regulatory domains of ANCO-1. Two intrinsic repression domains (RD) were identified: an N-terminal RD1 at residues 318-611 and a C-terminal RD2 at 2369-2663. ANCO-1 also contains an activation domain (AD) capable of stimulating transcription in both mammalian and yeast cells. The minimal AD was delimited to a 70-amino acid region at residues 2076-2145. Overall, full-length ANCO-1 exhibited transcriptional repressor activity, suggesting that RD domains may suppress the AD activity. We further demonstrated that ANCO-1 silencing by siRNA enhanced progesterone receptor-mediated transcription. Together, these results indicate that the transcriptional potential of ANCO-1 may be modulated by a combination of repression and activation signals.
Assuntos
Regiões Promotoras Genéticas/genética , Receptores Citoplasmáticos e Nucleares/genética , Elementos Reguladores de Transcrição/genética , Proteínas Repressoras/química , Fatores de Transcrição/genética , Transcrição Gênica/genética , Ativação Transcricional/genética , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
The ankyrin repeats cofactor-1 (ANCO-1) was recently identified as a novel nuclear receptor corepressor that regulates receptor-mediated transcription through interactions with p160 coactivators and histone deacetylases. Interestingly, exogenously expressed ANCO-1 is localized at distinct subnuclear domains. The relevance of these subnuclear domains and the mechanisms of nucleocytoplasmic translocation of ANCO-1 have not been determined. We report here the identification of an N-terminal signaling motif that is essential for both nuclear/subnuclear localization and transcription corepressor function of ANCO-1. This N-terminal motif at residues 80-86 of ANCO-1 constitutes a classical nuclear localization signal (NLS1). Disruption of NLS1 causes complete cytoplasmic accumulation of the full-length ANCO-1, and abolishes its corepressor function on receptor-mediated transcription. A second NLS (NLS2) is found at the C-terminal residues 2384-2390; however, its disruption abolishes only nuclear localization of isolated C-terminal fragments. We also identify a leucine-rich nuclear export signal (NES) at residues 2415-2424 of ANCO-1, and show that both the NLSs and NES sequences are capable of mediating nuclear import and export of heterologous protein, respectively. In addition, attachment of the NES sequence to a transcription factor impairs its activation function. These results suggest that ANCO-1 subnuclear localization is regulated by both nuclear import and export signals, and that proper subcellular localization of ANCO-1 is essential for its corepressor function.
Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Repetição de Anquirina , Células COS , Linhagem Celular , Chlorocebus aethiops , Análise Mutacional de DNA , Proteína Vmw65 do Vírus do Herpes Simples/genética , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares , Transcrição GênicaRESUMO
The nuclear receptor coactivator RAC3 (also known as SRC-3/ACTR/AIB1/p/CIP/TRAM-1) belongs to the p160 coactivator family, which are involved in several physiological processes and diseases. Here we have investigated how RAC3 is translocated into the nucleus and show that it is mediated through a bipartite NLS and importin alpha3. This bipartite NLS is located within the conserved bHLH domain, and its mutation abolished nuclear localization. The NLS is also sufficient to cause nuclear import of EGFP, and the activity requires basic amino acids within the NLS. RAC3 binds strongly to importin alpha3, which also depends on the basic amino acids. Functionally, RAC3 cytoplasmic mutant loses its ability to enhance transcription, suggesting that nuclear localization is essential for coactivator function. Together, these results reveal a previous unknown mechanism for nuclear translocation of p160 coactivators and a critical function of the conserved bHLH within the coactivator.
Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear/metabolismo , Fatores de Transcrição/metabolismo , alfa Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Humanos , Dados de Sequência Molecular , Coativador 3 de Receptor Nuclear , Ligação Proteica , Alinhamento de Sequência , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
The pregnane X receptor (PXR) is an orphan nuclear receptor predominantly expressed in liver and intestine. PXR coordinates hepatic responses to prevent liver injury induced by environmental toxins. PXR activates cytochrome P450 3A4 gene expression upon binding to rifampicin (Rif) and clotrimazole (CTZ) by recruiting transcriptional coactivators. It remains unclear whether and how PXR regulates gene expression in the absence of ligand. In this study, we analyzed interactions between PXR and the silencing mediator of retinoid and thyroid hormone receptors (SMRT) and determined the role of SMRT in regulating PXR activity. We show that SMRT interacts with PXR in glutathione S-transferase pull-down, yeast two-hybrid, and mammalian two-hybrid assays. The interaction is mediated through the ligand-binding domain of PXR and the SMRTs' nuclear receptor-interacting domain 2. The PXR-SMRT interaction is sensitive to species-specific ligands, and Rif causes an exchange of the corepressor SMRT with the p160 coactivator known as receptor-associated coactivator 3 (RAC3). Deletion of the PXR's activation function 2 helix enhances SMRT binding and abolishes ligand-dependent dissociation of SMRT. Coexpression of PXR with SMRT results in colocalization at discrete nuclear foci. Finally, transient transfection assays show that overexpression of SMRT inhibits PXR's transactivation of the Cyp3A4 promoter, whereas silencing of SMRT enhances the reporter expression. Taken together, our results suggest that the corepressor SMRT may bind to and regulate the transcriptional activity of PXR.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA , Imunofluorescência , Humanos , Correpressor 2 de Receptor Nuclear , Receptor de Pregnano X , Ligação Proteica , Técnicas do Sistema de Duplo-HíbridoRESUMO
The human pregnane X receptor (PXR), also known as steroid and xenobiotic receptor, is a member of the orphan nuclear receptors and mediates the mammalian xenobiotic response with broad specificity and implications for drug clearance. The mouse pregnane X receptor is highly similar to the human ortholog in structure but with subtle species differentiation in the ligand binding domain (LBD). The C-terminal helix named alphaAF or AF-2 helix in other nuclear receptors is responsible for transcription activation by recruiting coactivators through conformational change. In the absence of ligands, PXR can also repress gene expression by interacting with transcriptional corepressors, such as the silencing mediator for retinoid and thyroid hormone receptor (SMRT). We first constructed homology models of the complete LBD with two SMRT nuclear receptor (NR)-interacting domains (ID1 and ID2), respectively. We then performed energy minimization and molecular dynamics simulations on these systems to study the specific interactions between the interacting domains and LBD. Further experimental results supported and validated the observed preference of SMRT toward ID2 over ID1. Our modeling results revealed the key interactions that account for the binding preference. Here, we propose structural models of the PXR-LBD/SMRT-ID1 and PXR-LBD/SMRT-ID2 complexes to understand their molecular interactions and potential inhibitory mechanism.
Assuntos
Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/química , Receptores de Esteroides/fisiologia , Sequência de Aminoácidos , Animais , Simulação por Computador , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Receptor de Pregnano X , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
So far, two thioredoxin proteins, DHD and Trx-2, have been biochemically characterized in Drosophila melanogaster. Here, with the cloning and characterization of TrxT we describe an additional thioredoxin with testis-specific expression. TrxT and dhd are arranged as a gene pair, transcribed in opposite directions and sharing a 471 bp regulatory region. We show that this regulatory region is sufficient for correct expression of the two genes. This gene pair makes a good model for unraveling how closely spaced promoters are differentially regulated by a short common control region. Both TrxT and DHD proteins are localized within the nuclei in testes and ovaries, respectively. Use of a transgenic construct expressing TrxT fused to Enhanced Yellow Fluorescent Protein reveals a clear association of TrxT with the Y chromosome lampbrush loops ks-1 and kl-5 in primary spermatocytes. The association is lost in the absence of the Y chromosome. Our results suggest that nuclear thioredoxins may have regulatory functions in the germline.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Expressão Gênica , Proteínas de Membrana , Tiorredoxinas/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias , DNA Complementar/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Imunofluorescência , Componentes do Gene , Hibridização In Situ , Proteínas Luminescentes , Masculino , Dados de Sequência Molecular , Ovário/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Testículo/metabolismo , Tiorredoxinas/metabolismo , Transgenes/genética , Cromossomo Y/metabolismoRESUMO
RORgamma is a nuclear receptor that binds to DNA motifs as a monomer to constitutively activate target genes. RORgamma plays an important role in thymocyte development and lymph node organogenesis, while the regulation of RORgamma-mediated transcriptional activation is currently unclear. The purpose of this study was to identify other nuclear proteins that interact with RORgamma. A yeast two-hybrid screen with Y190 yeast cells under stringent conditions resulted in the identification of CHD4, also known as Mi-2beta, as a RORgamma-interacting protein. This interaction was confirmed by GST pull-down assays. This interaction occurred within the middle regulatory region (amino acids 719-1164) of Mi-2beta. Transfection of Gal4-RORgamma into HeLa cells resulted in constitutive transactivation of the MH100-tk-luc reporter. The addition of Mi-2beta resulted in a dramatic 50% decrease in Gal4-RORgamma-mediated transactivation. These data demonstrate that RORgamma forms a protein-protein interaction with the regulatory region of Mi-2beta, resulting in inhibition of RORgamma transcriptional activity. These results may provide evidence as to how RORgamma-mediated transactivation is regulated by other nuclear proteins.
Assuntos
Histona Desacetilases/metabolismo , Proteínas Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Ativação Transcricional/fisiologia , Animais , DNA Complementar/genética , Biblioteca Gênica , Genes Reporter/genética , Células HeLa , Histona Desacetilases/fisiologia , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/fisiologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Ligação Proteica , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Leveduras/genéticaRESUMO
Ajulemic acid (AJA) is a synthetic analog of the tetrahydrocannabinol (THC) metabolite THC-11-oic acid; THC is a major active ingredient of the drug marijuana derived from the plant cannabis. AJA has potent analgesic and anti-inflammatory activity without the psychotropic action of THC. Unlike the nonsteroidal anti-inflammatory drugs, AJA is not ulcerogenic at therapeutic doses, making it a promising anti-inflammatory drug. However, the mechanism of AJA action remains unknown. Here we report that AJA binds directly and specifically to the peroxisome proliferator-activated receptor gamma (PPARgamma), a pharmacologically important member of the nuclear receptor superfamily. Functional assay indicates that AJA activates the transcriptional activity of both human and mouse PPARgamma at pharmacological concentrations. Activation of PPARgamma by AJA requires the AF-2 helix of the receptor, suggesting that AJA activates PPARgamma through the ligand-dependent AF-2 function. AJA binding consistently enables PPARgamma to recruit nuclear receptor coactivators. In addition, we show that AJA inhibits interleukin-8 promoter activity in a PPARgamma-dependent manner, suggesting a link between the anti-inflammatory action of AJA and the activation of PPARgamma. Finally, we find that AJA treatment induces differentiation of 3T3 L1 fibroblasts into adipocytes, a process mediated by PPARgamma. Together, these data indicate that PPARgamma may be a molecular target for AJA, providing a potential mechanism for the anti-inflammatory action of AJA, and possibly other cannabinoids. These studies also implicate other potential therapeutic actions of AJA through PPARgamma activation in multiple signaling pathways.