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1.
Mol Cell ; 84(2): 202-220.e15, 2024 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-38103559

RESUMO

Compounds binding to the bromodomains of bromodomain and extra-terminal (BET) family proteins, particularly BRD4, are promising anticancer agents. Nevertheless, side effects and drug resistance pose significant obstacles in BET-based therapeutics development. Using high-throughput screening of a 200,000-compound library, we identified small molecules targeting a phosphorylated intrinsically disordered region (IDR) of BRD4 that inhibit phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication in HPV-containing keratinocytes. Proteomic profiling identified two DNA damage response factors-53BP1 and BARD1-crucial for differentiation-associated HPV genome amplification. pBRD4-mediated recruitment of 53BP1 and BARD1 to the HPV origin of replication occurs in a spatiotemporal and BRD4 long (BRD4-L) and short (BRD4-S) isoform-specific manner. This recruitment is disrupted by phospho-IDR-targeting compounds with little perturbation of the global transcriptome and BRD4 chromatin landscape. The discovery of these protein-protein interaction inhibitors (PPIi) not only demonstrates the feasibility of developing PPIi against phospho-IDRs but also uncovers antiviral agents targeting an epigenetic regulator essential for virus-host interaction and cancer development.


Assuntos
Infecções por Papillomavirus , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Papillomavirus Humano , Infecções por Papillomavirus/tratamento farmacológico , Infecções por Papillomavirus/genética , Proteômica , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Papillomaviridae/genética , Papillomaviridae/metabolismo , Proteínas Virais/genética , Replicação Viral/fisiologia , Reparo do DNA , Proteínas que Contêm Bromodomínio
2.
Mol Cell ; 78(6): 1133-1151.e14, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32402252

RESUMO

Precise control of the RNA polymerase II (RNA Pol II) cycle, including pausing and pause release, maintains transcriptional homeostasis and organismal functions. Despite previous work to understand individual transcription steps, we reveal a mechanism that integrates RNA Pol II cycle transitions. Surprisingly, KAP1/TRIM28 uses a previously uncharacterized chromatin reader cassette to bind hypo-acetylated histone 4 tails at promoters, guaranteeing continuous progression of RNA Pol II entry to and exit from the pause state. Upon chromatin docking, KAP1 first associates with RNA Pol II and then recruits a pathway-specific transcription factor (SMAD2) in response to cognate ligands, enabling gene-selective CDK9-dependent pause release. This coupling mechanism is exploited by tumor cells to aberrantly sustain transcriptional programs commonly dysregulated in cancer patients. The discovery of a factor integrating transcription steps expands the functional repertoire by which chromatin readers operate and provides mechanistic understanding of transcription regulation, offering alternative therapeutic opportunities to target transcriptional dysregulation.


Assuntos
RNA Polimerase II/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Acetilação , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica/genética , Histonas/metabolismo , Humanos , Oncogenes/genética , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/genética , RNA Polimerase II/genética , Proteína Smad2/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína 28 com Motivo Tripartido/genética
3.
Mol Cell ; 78(6): 1114-1132.e10, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32446320

RESUMO

Bromodomain-containing protein 4 (BRD4) is a cancer therapeutic target in ongoing clinical trials disrupting primarily BRD4-regulated transcription programs. The role of BRD4 in cancer has been attributed mainly to the abundant long isoform (BRD4-L). Here we show, by isoform-specific knockdown and endogenous protein detection, along with transgene expression, the less abundant BRD4 short isoform (BRD4-S) is oncogenic while BRD4-L is tumor-suppressive in breast cancer cell proliferation and migration, as well as mammary tumor formation and metastasis. Through integrated RNA-seq, genome-wide ChIP-seq, and CUT&RUN association profiling, we identify the Engrailed-1 (EN1) homeobox transcription factor as a key BRD4-S coregulator, particularly in triple-negative breast cancer. BRD4-S and EN1 comodulate the extracellular matrix (ECM)-associated matrisome network, including type II cystatin gene cluster, mucin 5, and cathepsin loci, via enhancer regulation of cancer-associated genes and pathways. Our work highlights the importance of targeted therapies for the oncogenic, but not tumor-suppressive, activity of BRD4.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genes Homeobox , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Invasividade Neoplásica , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Transcrição Gênica/genética , Neoplasias de Mama Triplo Negativas/genética
4.
J Virol ; 97(10): e0074723, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37712706

RESUMO

IMPORTANCE: Respiratory syncytial virus (RSV) matrix (M) protein is indispensable for virion assembly and release. It is localized to the nucleus during early infection to perturb host transcription. However, the function of RSV M protein in other cellular activities remains poorly understood. In this study, several interferon response-associated host factors, including RACK1, were identified by proteomic analysis as RSV M interactors. Knockdown of RACK1 attenuates RSV-restricted IFN signaling leading to enhanced host defense against RSV infection, unraveling a role of M protein in antagonizing IFN response via association with RACK1. Our study uncovers a previously unrecognized mechanism of immune evasion by RSV M protein and identifies RACK1 as a novel host factor recruited by RSV, highlighting RACK1 as a potential new target for RSV therapeutics development.


Assuntos
Receptores de Quinase C Ativada , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Proteínas da Matriz Viral , Humanos , Interferons , Proteínas de Neoplasias/genética , Proteínas , Proteômica , Receptores de Quinase C Ativada/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Proteínas da Matriz Viral/metabolismo
5.
J Virol ; 97(10): e0078223, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37712702

RESUMO

IMPORTANCE: Human papillomavirus 16 (HPV16) is a causative agent in around 3%-4% of all human cancers, and currently, there are no anti-viral therapeutics available for combating this disease burden. In order to identify new therapeutic targets, we must increase our understanding of the HPV16 life cycle. Previously, we demonstrated that an interaction between E2 and the cellular protein TopBP1 mediates the plasmid segregation function of E2, allowing distribution of viral genomes into daughter nuclei following cell division. Here, we demonstrate that E2 interaction with an additional host protein, BRD4, is also essential for E2 segregation function, and that BRD4 exists in a complex with TopBP1. Overall, these results enhance our understanding of a critical part of the HPV16 life cycle and presents several therapeutic targets for disruption of the viral life cycle.


Assuntos
Cromatina , Proteínas Oncogênicas Virais , Humanos , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Plasmídeos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Nature ; 529(7586): 413-417, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26735014

RESUMO

Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several models of cancer, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.


Assuntos
Azepinas/farmacologia , Azepinas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Nucleares/antagonistas & inibidores , Estrutura Terciária de Proteína/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Triazóis/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Ligação Competitiva/efeitos dos fármacos , Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cromatina/genética , Cromatina/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Humanos , Subunidade 1 do Complexo Mediador/metabolismo , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , Proteômica , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Cancer ; 127(6): 850-864, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33270909

RESUMO

BACKGROUND: Despite the significant societal burden of human papillomavirus (HPV)-associated cancers, clinical screening interventions for HPV-associated noncervical cancers are not available. Blood-based biomarkers may help close this gap in care. METHODS: Five databases were searched, 5687 articles were identified, and 3631 unique candidate titles and abstracts were independently reviewed by 2 authors; 702 articles underwent a full-text review. Eligibility criteria included the assessment of a blood-based biomarker within a cohort or case-control study. RESULTS: One hundred thirty-seven studies were included. Among all biomarkers assessed, HPV-16 E seropositivity and circulating HPV DNA were most significantly correlated with HPV-associated cancers in comparison with cancer-free controls. In most scenarios, HPV-16 E6 seropositivity varied nonsignificantly according to tumor type, specimen collection timing, and anatomic site (crude odds ratio [cOR] for p16+ or HPV+ oropharyngeal cancer [OPC], 133.10; 95% confidence interval [CI], 59.40-298.21; cOR for HPV-unspecified OPC, 25.41; 95% CI, 8.71-74.06; cOR for prediagnostic HPV-unspecified OPC, 59.00; 95% CI, 15.39-226.25; cOR for HPV-unspecified cervical cancer, 12.05; 95% CI, 3.23-44.97; cOR for HPV-unspecified anal cancer, 73.60; 95% CI, 19.68-275.33; cOR for HPV-unspecified penile cancer, 16.25; 95% CI, 2.83-93.48). Circulating HPV-16 DNA was a valid biomarker for cervical cancer (cOR, 15.72; 95% CI, 3.41-72.57). In 3 cervical cancer case-control studies, cases exhibited unique microRNA expression profiles in comparison with controls. Other assessed biomarker candidates were not valid. CONCLUSIONS: HPV-16 E6 antibodies and circulating HPV-16 DNA are the most robustly analyzed and most promising blood-based biomarkers for HPV-associated cancers to date. Comparative validity analyses are warranted. Variations in tumor type-specific, high-risk HPV DNA prevalence according to anatomic site and world region highlight the need for biomarkers targeting more high-risk HPV types. Further investigation of blood-based microRNA expression profiling appears indicated.


Assuntos
Anticorpos Antivirais/sangue , Neoplasias do Ânus/virologia , Biomarcadores/sangue , DNA Viral/sangue , Neoplasias Orofaríngeas/virologia , Infecções por Papillomavirus/complicações , Feminino , Papillomavirus Humano 16/isolamento & purificação , Humanos , Neoplasias do Colo do Útero/virologia
8.
Mol Cell ; 49(5): 843-57, 2013 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-23317504

RESUMO

Bromodomain-containing protein 4 (Brd4) is an epigenetic reader and transcriptional regulator recently identified as a cancer therapeutic target for acute myeloid leukemia, multiple myeloma, and Burkitt's lymphoma. Although chromatin targeting is a crucial function of Brd4, there is little understanding of how bromodomains that bind acetylated histones are regulated, nor how the gene-specific activity of Brd4 is determined. Via interaction screen and domain mapping, we identified p53 as a functional partner of Brd4. Interestingly, Brd4 association with p53 is modulated by casein kinase II (CK2)-mediated phosphorylation of a conserved acidic region in Brd4 that selectively contacts either a juxtaposed bromodomain or an adjacent basic region to dictate the ability of Brd4 binding to chromatin and also the recruitment of p53 to regulated promoters. The unmasking of bromodomains and activator recruitment, concurrently triggered by the CK2 phospho switch, provide an intriguing mechanism for gene-specific targeting by a universal epigenetic reader.


Assuntos
Caseína Quinase II/metabolismo , Cromatina/metabolismo , Marcação de Genes , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Caseína Quinase II/genética , Proteínas de Ciclo Celular , Cromatina/genética , Células HCT116 , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
9.
Nucleic Acids Res ; 46(13): 6576-6591, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29860315

RESUMO

Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator in response to hypoxia and its transcriptional activity is crucial for cancer cell mobility. Here we present evidence for a novel epigenetic mechanism that regulates HIF-1 transcriptional activity and HIF-1-dependent migration of glioblastoma cells. The lysine methyltransferases G9a and GLP directly bound to the α subunit of HIF-1 (HIF-1α) and catalyzed mono- and di-methylation of HIF-1α at lysine (K) 674 in vitro and in vivo. K674 methylation suppressed HIF-1 transcriptional activity and expression of its downstream target genes PTGS1, NDNF, SLC6A3, and Linc01132 in human glioblastoma U251MG cells. Inhibition of HIF-1 by K674 methylation is due to reduced HIF-1α transactivation domain function but not increased HIF-1α protein degradation or impaired binding of HIF-1 to hypoxia response elements. K674 methylation significantly decreased HIF-1-dependent migration of U251MG cells under hypoxia. Importantly, we found that G9a was downregulated by hypoxia in glioblastoma, which was inversely correlated with PTGS1 expression and survival of patients with glioblastoma. Therefore, our findings uncover a hypoxia-induced negative feedback mechanism that maintains high activity of HIF-1 and cell mobility in human glioblastoma.


Assuntos
Autoantígenos/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Proteínas da Matriz do Complexo de Golgi/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Transcrição Gênica , Hipóxia Celular , Linhagem Celular , Movimento Celular , Glioblastoma/metabolismo , Glioblastoma/fisiopatologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Lisina/metabolismo , Metilação , Elementos de Resposta
10.
EMBO J ; 34(2): 184-99, 2015 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-25425577

RESUMO

Acetylation of transcriptional regulators is normally dynamically regulated by nutrient status but is often persistently elevated in nutrient-excessive obesity conditions. We investigated the functional consequences of such aberrantly elevated acetylation of the nuclear receptor FXR as a model. Proteomic studies identified K217 as the FXR acetylation site in diet-induced obese mice. In vivo studies utilizing acetylation-mimic and acetylation-defective K217 mutants and gene expression profiling revealed that FXR acetylation increased proinflammatory gene expression, macrophage infiltration, and liver cytokine and triglyceride levels, impaired insulin signaling, and increased glucose intolerance. Mechanistically, acetylation of FXR blocked its interaction with the SUMO ligase PIASy and inhibited SUMO2 modification at K277, resulting in activation of inflammatory genes. SUMOylation of agonist-activated FXR increased its interaction with NF-κB but blocked that with RXRα, so that SUMO2-modified FXR was selectively recruited to and trans-repressed inflammatory genes without affecting FXR/RXRα target genes. A dysregulated acetyl/SUMO switch of FXR in obesity may serve as a general mechanism for diminished anti-inflammatory response of other transcriptional regulators and provide potential therapeutic and diagnostic targets for obesity-related metabolic disorders.


Assuntos
Regulação da Expressão Gênica , Inflamação/patologia , Hepatopatias/patologia , Obesidade/complicações , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Western Blotting , Citocinas/genética , Citocinas/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Perfilação da Expressão Gênica , Técnicas Imunoenzimáticas , Imunoprecipitação , Inflamação/etiologia , Inflamação/metabolismo , Hepatopatias/etiologia , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Dados de Sequência Molecular , NF-kappa B/genética , NF-kappa B/metabolismo , Obesidade/fisiopatologia , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteômica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Espectrometria de Massas em Tandem
11.
J Virol ; 91(2)2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27807239

RESUMO

Papillomaviruses are small, double-stranded DNA viruses that encode the E2 protein, which controls transcription, replication, and genome maintenance in infected cells. Posttranslational modifications (PTMs) affecting E2 function and stability have been demonstrated for multiple types of papillomaviruses. Here we describe the first phosphorylation event involving a conserved tyrosine (Y) in the bovine papillomavirus 1 (BPV-1) E2 protein at amino acid 102. While its phosphodeficient phenylalanine (F) mutant activated both transcription and replication in luciferase reporter assays, a mutant that may act as a phosphomimetic, with a Y102-to-glutamate (E) mutation, lost both activities. The E2 Y102F protein interacted with cellular E2-binding factors and the viral helicase E1; however, in contrast, the Y102E mutant associated with only a subset and was unable to bind to E1. While the Y102F mutant fully supported transient viral DNA replication, BPV genomes encoding this mutation as well as Y102E were not maintained as stable episomes in murine C127 cells. These data imply that phosphorylation at Y102 disrupts the helical fold of the N-terminal region of E2 and its interaction with key cellular and viral proteins. We hypothesize that the resulting inhibition of viral transcription and replication in basal epithelial cells prevents the development of a lytic infection. IMPORTANCE: Papillomaviruses (PVs) are small, double-stranded DNA viruses that are responsible for cervical, oropharyngeal, and various genitourinary cancers. Although vaccines against the major oncogenic human PVs are available, there is no effective treatment for existing infections. One approach to better understand the viral replicative cycle, and potential therapies to target it, is to examine the posttranslational modification of viral proteins and its effect on function. Here we have discovered that the bovine papillomavirus 1 (BPV-1) transcription and replication regulator E2 is phosphorylated at residue Y102. While a phosphodeficient mutant at this site was fully functional, a phosphomimetic mutant displayed impaired transcription and replication activity as well as a lack of an association with certain E2-binding proteins. This study highlights the influence of posttranslational modifications on viral protein function and provides additional insight into the complex interplay between papillomaviruses and their hosts.


Assuntos
Papillomavirus Bovino 1/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Transcrição Gênica , Tirosina/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Alphapapillomavirus/fisiologia , Animais , Bovinos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Genoma Viral , Humanos , Modelos Moleculares , Mutação , Fosforilação , Plasmídeos/genética , Conformação Proteica , Transporte Proteico , Proteínas Virais/química , Proteínas Virais/genética
12.
PLoS Pathog ; 12(1): e1005366, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26727473

RESUMO

We investigated the mechanism of how the papillomavirus E2 transcription factor can activate promoters through activator protein (AP)1 binding sites. Using an unbiased approach with an inducible cell line expressing the viral transcription factor E2 and transcriptome analysis, we found that E2 induces the expression of the two AP1 components c-Fos and FosB in a Brd4-dependent manner. In vitro RNA interference confirmed that c-Fos is one of the AP1 members driving the expression of viral oncogenes E6/E7. Mutation analysis and in vivo RNA interference identified an essential role for c-Fos/AP1 and also for the bromodomain protein Brd4 for papillomavirus-induced tumorigenesis. Lastly, chromatin immunoprecipitation analysis demonstrated that E2 binds together with Brd4 to a canonical E2 binding site (E2BS) in the promoter of c-Fos, thus activating c-Fos expression. Thus, we identified a novel way how E2 activates the viral oncogene promoter and show that E2 may act as a viral oncogene by direct activation of c-Fos involved in skin tumorigenesis.


Assuntos
Transformação Celular Viral/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/metabolismo , Proteínas Proto-Oncogênicas c-fos/biossíntese , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Ensaio de Desvio de Mobilidade Eletroforética , Genes Virais , Imunoprecipitação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/genética , Oncogenes , Papillomaviridae , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , Proteínas Proto-Oncogênicas c-fos/genética , Coelhos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Nucleic Acids Res ; 44(3): 1080-94, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26464433

RESUMO

The transcription start site (TSS) determines the length and composition of the 5' UTR and therefore can have a profound effect on translation. Yet, little is known about the mechanism underlying start site selection, particularly from promoters lacking conventional core elements such as TATA-box and Initiator. Here we report a novel mechanism of start site selection in the TATA- and Initiator-less promoter of miR-22, through a strictly localized downstream element termed DTIE and an upstream distal element. Changing the distance between them reduced promoter strength, altered TSS selection and diminished Pol II recruitment. Biochemical assays suggest that DTIE does not serve as a docking site for TFIID, the major core promoter-binding factor. TFIID is recruited to the promoter through DTIE but is dispensable for TSS selection. We determined DTIE consensus and found it to be remarkably prevalent, present at the same TSS downstream location in ≈20.8% of human promoters, the vast majority of which are TATA-less. Analysis of DTIE in the tumor suppressor p53 confirmed a similar function. Our findings reveal a novel mechanism of transcription initiation from TATA-less promoters.


Assuntos
Regiões Promotoras Genéticas , TATA Box/genética , Animais , Sequência de Bases , Células HEK293 , Humanos , MicroRNAs/genética , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica
14.
Nucleic Acids Res ; 42(8): 4996-5006, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24589584

RESUMO

Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Proteínas de Neoplasias/metabolismo , Terminação da Transcrição Genética , Animais , Antineoplásicos/toxicidade , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA , Endonucleases , Instabilidade Genômica , Humanos , Camundongos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
15.
PLoS Pathog ; 9(11): e1003777, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24278023

RESUMO

Replication foci are generated by many viruses to concentrate and localize viral DNA synthesis to specific regions of the cell. Expression of the HPV16 E1 and E2 replication proteins in keratinocytes results in nuclear foci that recruit proteins associated with the host DNA damage response. We show that the Brd4 protein localizes to these foci and is essential for their formation. However, when E1 and E2 begin amplifying viral DNA, Brd4 is displaced from the foci and cellular factors associated with DNA synthesis and homologous recombination are recruited. Differentiated HPV-infected keratinocytes form similar nuclear foci that contain amplifying viral DNA. We compare the different foci and show that, while they have many characteristics in common, there is a switch between early Brd4-dependent foci and mature Brd4-independent replication foci. However, HPV genomes encoding mutated E2 proteins that are unable to bind Brd4 can replicate and amplify the viral genome. We propose that, while E1, E2 and Brd4 might bind host chromatin at early stages of infection, there is a temporal and functional switch at later stages and increased E1 and E2 levels promote viral DNA amplification, displacement of Brd4 and growth of a replication factory. The concomitant DNA damage response recruits proteins required for DNA synthesis and repair, which could then be utilized for viral DNA replication. Hence, while Brd4 can enhance replication by concentrating viral processes in specific regions of the host nucleus, this interaction is not absolutely essential for HPV replication.


Assuntos
Replicação do DNA/fisiologia , DNA Viral/biossíntese , Genoma Viral/fisiologia , Papillomavirus Humano 16/fisiologia , Proteínas Nucleares/metabolismo , Infecções por Papillomavirus/metabolismo , Fatores de Transcrição/metabolismo , Replicação Viral/fisiologia , Proteínas de Ciclo Celular , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Cromatina/virologia , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/genética , Fatores de Transcrição/genética
16.
J Virol ; 87(3): 1497-507, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23152516

RESUMO

The p300, CBP, and pCAF lysine acetyltransferase (KAT) proteins have been reported to physically interact with bovine (BPV) and human (HPV) papillomavirus E2 proteins. While overexpression of these KAT proteins enhances E2-dependent transcription, the mechanism has not been determined. Using RNA interference (RNAi) to deplete these factors, we demonstrated that E2 transcriptional activity requires physiological levels of p300, CBP, and pCAF. Each protein appears to have a unique function in E2-dependent transcription, since overexpression of one KAT failed to compensate for RNAi knockdown of another KAT. Using an in vitro acetylation assay, we identified highly conserved lysines that are targeted by p300 for acetylation. The conservative changes of lysines at positions 111 and 112 to arginine were of particular interest. The K111R and the K111R/K112R mutants showed reduced transcriptional activity that was not responsive to p300 overexpression, while the K112R mutant retained activity. p300 and CBP were detected at the viral promoter; however, pCAF was not. We propose a model by which E2 transcriptional activity is controlled by p300-mediated acetylation of lysine 111. This model represents a novel mechanism regulating papillomavirus gene expression.


Assuntos
Papillomavirus Bovino 1/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Lisina/metabolismo , Transcrição Gênica , Proteínas Virais/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Bovinos , Linhagem Celular
17.
ACS Pharmacol Transl Sci ; 7(4): 1178-1190, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38633583

RESUMO

A promising alternative for cancer treatment involves targeted inhibition of the epigenetic regulator bromodomain-containing protein 4 (BRD4); however, available BRD4 inhibitors are constrained by their potency, oral bioavailability, and cytotoxicity. Herein, to overcome the drawback of the translational BRD4 inhibitors, we describe a novel BRD4-p53 inhibitor, SDU-071, which suppresses BRD4 interaction with the p53 tumor suppressor and its biological activity in MDA-MB-231 triple-negative breast cancer (TNBC) cells in vitro and in vivo. This novel small-molecule BRD4-p53 inhibitor suppresses cell proliferation, migration, and invasion by downregulating the expression of BRD4-targeted genes, such as c-Myc and Mucin 5AC, and inducing cell cycle arrest and apoptosis, as demonstrated in cultured MDA-MB-231 TNBC cells. Its antitumor activity is illustrated in an orthotopic mouse xenograft mammary tumor model. Overall, our results show that SDU-071 is a viable option for potentially treating TNBC as a new BRD4-p53 inhibitor.

18.
EMBO J ; 28(9): 1246-59, 2009 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-19339993

RESUMO

Covalent modification by small ubiquitin-related modifiers (SUMO) regulates p53 transcription activity through an undefined mechanism. Using reconstituted sumoylation components, we purified SUMO-1-conjugated p53 (Su-p53) to near homogeneity. Su-p53 exists in solution as a tetramer and interacts with p300 histone acetyltransferase as efficiently as the unmodified protein. Nevertheless, it fails to activate p53-dependent chromatin transcription because of its inability to bind DNA. With sequential modification assays, we found that sumoylation of p53 at K386 blocks subsequent acetylation by p300, whereas p300-acetylated p53 remains permissive for ensuing sumoylation at K386 and alleviates sumoylation-inhibited DNA binding. While preventing the free form of p53 from accessing its cognate sites, sumoylation fails to disengage prebound p53 from DNA. The sumoylation-deficient K386R protein, when expressed in p53-null cells, exhibits higher transcription activity and binds better to the endogenous p21 gene compared with the wild-type protein. These studies unravel a molecular mechanism underlying sumoylation-regulated p53 function and further uncover a new role of acetylation in antagonizing the inhibitory effect of sumoylation on p53 binding to DNA.


Assuntos
Cromatina/genética , DNA/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Transcrição Gênica/genética , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Cromatografia em Gel , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição de p300-CBP/metabolismo
19.
bioRxiv ; 2023 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-37292798

RESUMO

During the human papillomavirus 16 life cycle, the E2 protein binds simultaneously to the viral genome and host chromatin throughout mitosis, ensuring viral genomes reside in daughter cell nuclei following cell division. Previously, we demonstrated that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1, and that this interaction is required for optimum E2 mitotic chromatin association and plasmid segregation function. Others have implicated BRD4 in mediating the plasmid segregation function of E2 and we have demonstrated that there is a TopBP1-BRD4 complex in the cell. We therefore further investigated the role of the E2-BRD4 interaction in mediating E2 association with mitotic chromatin and plasmid segregation function. Using a combination of immunofluorescence and our novel plasmid segregation assay in U2OS and N/Tert-1 cells stably expressing a variety of E2 mutants, we report that direct interaction with the BRD4 carboxyl-terminal motif (CTM) and TopBP1 is required for E2 association with mitotic chromatin and plasmid segregation. We also identify a novel TopBP1 mediated interaction between E2 and the BRD4 extra-terminal (ET) domain in vivo . Overall, the results demonstrate that direct interaction with TopBP1 and the BRD4 CTM are required for E2 mitotic chromatin association and plasmid segregation function. Disruption of this complex offers therapeutic options for targeting segregation of viral genomes into daughter cells, potentially combatting HPV16 infections, and cancers that retain episomal genomes. Importance: HPV16 is a causative agent in around 3-4% of all human cancers and currently there are no anti-viral therapeutics available for combating this disease burden. In order to identify new therapeutic targets, we must increase our understanding of the HPV16 life cycle. Previously, we demonstrated that an interaction between E2 and the cellular protein TopBP1 mediates the plasmid segregation function of E2, allowing distribution of viral genomes into daughter nuclei following cell division. Here, we demonstrate that E2 interaction with an additional host protein, BRD4, is also essential for E2 segregation function, and that BRD4 exists in a complex with TopBP1. Overall, these results enhance our understanding of a critical part of the HPV16 life cycle and presents several therapeutic targets for disruption of the viral life cycle.

20.
J Biol Chem ; 286(47): 40974-86, 2011 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-21937452

RESUMO

Activator protein-1 (AP-1) regulates diverse gene responses triggered by environmental cues and virus-induced cellular stress. Although many signaling events leading to AP-1 activation have been described, the fundamental features underlying binding site selection and factor recruitment of dimeric AP-1 complexes to their target genes remain mostly uncharacterized. Using recombinant full-length human AP-1 dimers formed between c-Jun and Fos family members (c-Fos, FosB, Fra-1, Fra-2) for DNA binding and transcriptional analysis, we found that each of these AP-1 complex exhibits differential activity for distinct non-consensus AP-1 sites present in human papillomavirus (HPV), and each AP-1 complex is capable of activating transcription from in vitro-reconstituted HPV chromatin in a p300- and acetyl-CoA-dependent manner. Transcription from HPV chromatin requires AP-1-dependent and contact-driven recruitment of p300. Acetylation of dimeric AP-1 complexes by p300 enhances AP-1 binding to DNA. Using a human C-33A cervical cancer-derived cell line harboring the episomal HPV type 11 genome, we illustrate binding site selectivity recognized by c-Jun, JunB, JunD, and various Fos family members in a combinatorial and unique pattern, highlighting the diversity and importance of non-canonical binding site recognition by various AP-1 family proteins.


Assuntos
Cromatina/metabolismo , Papillomavirus Humano 11/genética , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Sobrevivência Celular , Cromatina/genética , Condiloma Acuminado/virologia , Sequência Conservada , DNA Viral/genética , DNA Viral/metabolismo , Proteína p300 Associada a E1A/metabolismo , Células HCT116 , Células HeLa , Histonas/metabolismo , Humanos , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Especificidade por Substrato
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