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 BromodomínioRESUMO
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/genéticaRESUMO
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/genéticaRESUMO
Bromodomain proteins (BRD) are key chromatin regulators of genome function and stability as well as therapeutic targets in cancer. Here, we systematically delineate the contribution of human BRD proteins for genome stability and DNA double-strand break (DSB) repair using several cell-based assays and proteomic interaction network analysis. Applying these approaches, we identify 24 of the 42 BRD proteins as promoters of DNA repair and/or genome integrity. We identified a BRD-reader function of PCAF that bound TIP60-mediated histone acetylations at DSBs to recruit a DUB complex to deubiquitylate histone H2BK120, to allowing direct acetylation by PCAF, and repair of DSBs by homologous recombination. We also discovered the bromo-and-extra-terminal (BET) BRD proteins, BRD2 and BRD4, as negative regulators of transcription-associated RNA-DNA hybrids (R-loops) as inhibition of BRD2 or BRD4 increased R-loop formation, which generated DSBs. These breaks were reliant on topoisomerase II, and BRD2 directly bound and activated topoisomerase I, a known restrainer of R-loops. Thus, comprehensive interactome and functional profiling of BRD proteins revealed new homologous recombination and genome stability pathways, providing a framework to understand genome maintenance by BRD proteins and the effects of their pharmacological inhibition.
Assuntos
Instabilidade Genômica , Estruturas R-Loop , Reparo de DNA por Recombinação/genética , Fatores de Transcrição/genética , Acetilação , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Células HEK293 , Células HeLa , Humanos , Transativadores/metabolismo , Fatores de Transcrição/análise , Ubiquitinação , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
BRD4, a bromodomain and extraterminal (BET) protein, is deregulated in multiple cancers and has emerged as a promising drug target. However, the function of the two main BRD4 isoforms (BRD4-L and BRD4-S) has not been analysed in parallel in most cancers. This complicates determining therapeutic efficacy of pan-BET inhibitors. In this study, using functional and transcriptomic analysis, we show that BRD-L and BRD4-S isoforms play distinct roles in fusion negative embryonal rhabdomyosarcoma. BRD4-L has an oncogenic role and inhibits myogenic differentiation, at least in part, by activating myostatin expression. Depletion of BRD4-L in vivo impairs tumour progression but does not impact metastasis. On the other hand, depletion of BRD4-S has no significant impact on tumour growth, but strikingly promotes metastasis in vivo. Interestingly, BRD4-S loss results in the enrichment of BRD4-L and RNA Polymerase II at integrin gene promoters resulting in their activation. In fusion positive alveolar rhabdomyosarcoma, BRD4-L is unrestricted in its oncogenic role, with no evident involvement of BRD4-S. Our work unveils isoform-specific functions of BRD4 in rhabdomyosarcoma.
Assuntos
Rabdomiossarcoma , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Isoformas de Proteínas/genética , Rabdomiossarcoma/genética , Proteínas que Contêm BromodomínioRESUMO
Bromodomain-containing protein 4 (BRD4) is overexpressed and functionally implicated in various myeloid malignancies. However, the role of BRD4 in normal hematopoiesis remains largely unknown. Here, utilizing an inducible Brd4 knockout mouse model, we find that deletion of Brd4 (Brd4Δ/Δ ) in the hematopoietic system impairs hematopoietic stem cell (HSC) self-renewal and differentiation, which associates with cell cycle arrest and senescence. ATAC-seq analysis shows increased chromatin accessibility in Brd4Δ/Δ hematopoietic stem/progenitor cells (HSC/HPCs). Genome-wide mapping with cleavage under target and release using nuclease (CUT&RUN) assays demonstrate that increased global enrichment of H3K122ac and H3K4me3 in Brd4Δ/Δ HSC/HPCs is associated with the upregulation of senescence-specific genes. Interestingly, Brd4 deletion increases clipped H3 (cH3) which correlates with the upregulation of senescence-specific genes and results in a higher frequency of senescent HSC/HPCs. Re-expression of BRD4 reduces cH3 levels and rescues the senescence rate in Brd4Δ/Δ HSC/HPCs. This study unveils an important role of BRD4 in HSC/HPC function by preventing H3 clipping and suppressing senescence gene expression.
Assuntos
Histonas , Fatores de Transcrição , Animais , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Histonas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Senescência Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular , HematopoeseRESUMO
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/metabolismoRESUMO
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/metabolismoRESUMO
Bromodomain-containing protein 4 (BRD4), as the most studied member of the bromodomain and extra-terminal (BET) family, is a chromatin reader protein interpreting epigenetic codes through binding to acetylated histones and non-histone proteins, thereby regulating diverse cellular processes including cell cycle, cell differentiation, and cell proliferation. As a promising drug target, BRD4 function is closely related to cancer, inflammation, cardiovascular disease, and liver fibrosis. Currently, clinical resistance to BET inhibitors has limited their applications but synergistic antitumor effects have been observed when used in combination with other tumor inhibitors targeting additional cellular components such as PLK1, HDAC, CDK, and PARP1. Therefore, designing dual-target inhibitors of BET bromodomains is a rational strategy in cancer treatment to increase potency and reduce drug resistance. This review summarizes the protein structures and biological functions of BRD4 and discusses recent advances of dual BET inhibitors from a medicinal chemistry perspective. We also discuss the current design and discovery strategies for dual BET inhibitors, providing insight into potential discovery of additional dual-target BET inhibitors.
Assuntos
Proteínas de Ciclo Celular , Neoplasias , Fatores de Transcrição , Proteínas de Ciclo Celular/antagonistas & inibidores , Química Farmacêutica , Histonas/química , Humanos , Neoplasias/patologia , Fatores de Transcrição/antagonistas & inibidoresRESUMO
Epigenetic regulation is important for cancer progression; however, the underlying mechanisms, particularly those involving protein acetylation, remain to be fully understood. Here, we show that p300/CBP-associated factor (PCAF)-dependent acetylation of the transcription factor intestine-specific homeobox (ISX) regulates epithelial-mesenchymal transition (EMT) and promotes cancer metastasis. Mechanistically, PCAF acetylation of ISX at lysine 69 promotes the interaction with acetylated bromodomain-containing protein 4 (BRD4) at lysine 332 in tumor cells, and the translocation of the resulting complex into the nucleus. There, it binds to promoters of EMT genes, where acetylation of histone 3 at lysines 9, 14, and 18 initiates chromatin remodeling and subsequent transcriptional activation. Ectopic ISX expression enhances EMT marker expression, including TWIST1, Snail1, and VEGF, induces cancer metastasis, but suppresses E-cadherin expression. In lung cancer, ectopic expression of PCAF-ISX-BRD4 axis components correlates with clinical metastatic features and poor prognosis. These results suggest that the PCAF-ISX-BRD4 axis mediates EMT signaling and regulates tumor initiation and metastasis.
Assuntos
Transição Epitelial-Mesenquimal , Neoplasias , Fatores de Transcrição , Acetilação , Epigênese Genética , Transição Epitelial-Mesenquimal/genética , Genes Homeobox , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
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 XenoenxertoRESUMO
Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have drawn significant attention in recent years. Here we describe the construction and characterization of a CRISPR-Cas13b-based tool for targeted demethylation of specific mRNA. A fusion protein, named dm6ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5-125 (dPspCas13b) to m6A demethylase AlkB homolog 5 (ALKBH5). dm6ACRISPR specifically demethylates m6A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate ß-catenin-encoding CTNNB1 mRNA that contains multiple m6A sites to trigger its translation. In addition, the dm6ACRISPR system incurs efficient demethylation of targeted epitranscriptome transcripts with limited off-target effects. Targeted demethylation of transcripts coding for oncoproteins such as epidermal growth factor receptor (EGFR) and MYC can suppress proliferation of cancer cells. Together, we provide a programmable and in vivo manipulation tool to study mRNA modification of specific genes and their related biological functions.
Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , RNA Mensageiro/metabolismo , Regiões 5' não Traduzidas , Adenosina/análogos & derivados , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Proliferação de Células , Desmetilação , Células HEK293 , Células HeLa , Humanos , Oncogenes , Prevotella/enzimologia , Engenharia de Proteínas , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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/virologiaRESUMO
Liver injury triggers adaptive remodeling of the hepatic transcriptome for repair/regeneration. We demonstrate that this involves particularly profound transcriptomic alterations where acute induction of genes involved in handling of endoplasmic reticulum stress (ERS) is accompanied by partial hepatic dedifferentiation. Importantly, widespread hepatic gene downregulation could not simply be ascribed to cofactor squelching secondary to ERS gene induction, but rather involves a combination of active repressive mechanisms. ERS acts through inhibition of the liver-identity (LIVER-ID) transcription factor (TF) network, initiated by rapid LIVER-ID TF protein loss. In addition, induction of the transcriptional repressor NFIL3 further contributes to LIVER-ID gene repression. Alteration to the liver TF repertoire translates into compromised activity of regulatory regions characterized by the densest co-recruitment of LIVER-ID TFs and decommissioning of BRD4 super-enhancers driving hepatic identity. While transient repression of the hepatic molecular identity is an intrinsic part of liver repair, sustained disequilibrium between the ERS and LIVER-ID transcriptional programs is linked to liver dysfunction as shown using mouse models of acute liver injury and livers from deceased human septic patients.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica/genética , Hepatopatias/metabolismo , Transcriptoma/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/genética , Sequenciamento de Cromatina por Imunoprecipitação , Regulação para Baixo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hepatopatias/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Tapsigargina/toxicidade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
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/metabolismoRESUMO
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 RespostaRESUMO
The epigenetic reader BRD4 plays a vital role in transcriptional regulation, cellular growth control, and cell-cycle progression. Dysregulation of BRD4 function has been implicated in the pathogenesis of a wide range of cancers. However, how BRD4 is regulated to maintain its normal function in healthy cells and how alteration of this process leads to cancer remain poorly understood. In this study, we discovered that BRD4 is hyperphosphorylated in NUT midline carcinoma and identified CDK9 as a potential kinase mediating BRD4 hyperphosphorylation. Disruption of BRD4 hyperphosphorylation using both chemical and molecular inhibitors led to the repression of BRD4 downstream oncogenes and abrogation of cellular transformation. BRD4 hyperphosphorylation is also observed in other cancers displaying enhanced BRD4 oncogenic activity. Our study revealed a mechanism that may regulate BRD4 biological function through phosphorylation, which, when dysregulated, could lead to oncogenesis. Our finding points to strategies to target the aberrant BRD4 signaling specifically for cancer intervention.
Assuntos
Carcinoma/genética , Carcinoma/metabolismo , Quinase 9 Dependente de Ciclina/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Fatores de Transcrição/química , Células A549 , Carcinogênese , Carcinoma/tratamento farmacológico , Proteínas de Ciclo Celular , Transformação Celular Neoplásica , Ensaios de Seleção de Medicamentos Antitumorais , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Proteínas de Neoplasias , Proteínas de Fusão Oncogênica/genética , Oncogenes , Fosforilação , Transdução de SinaisRESUMO
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 TandemRESUMO
Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.
Assuntos
Mama/metabolismo , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Mama/citologia , Proteínas de Ciclo Celular , Linhagem Celular , Proteínas de Ligação a DNA/biossíntese , Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , RNA Polimerase II/metabolismo , Transdução de Sinais , Fatores de Transcrição/biossíntese , Transcrição Gênica , Proteínas Supressoras de Tumor/biossínteseRESUMO
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.