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1.
Nat Commun ; 10(1): 2188, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097695

RESUMO

Although promoter-associated CpG islands have been established as targets of DNA methylation changes in cancer, previous studies suggest that epigenetic dysregulation outside the promoter region may be more closely associated with transcriptional changes. Here we examine DNA methylation, chromatin marks, and transcriptional alterations to define the relationship between transcriptional modulation and spatial changes in chromatin structure. Using human papillomavirus-related oropharyngeal carcinoma as a model, we show aberrant enrichment of repressive H3K9me3 at the transcriptional start site (TSS) with methylation-associated, tumor-specific gene silencing. Further analysis identifies a hypermethylated subtype which shows a functional convergence on MYC targets and association with CREBBP/EP300 mutation. The tumor-specific shift to transcriptional repression associated with DNA methylation at TSSs was confirmed in multiple tumor types. Our data may show a common underlying epigenetic dysregulation in cancer associated with broad enrichment of repressive chromatin marks and aberrant DNA hypermethylation at TSSs in combination with MYC network activation.


Assuntos
Cromatina/metabolismo , Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Sítio de Iniciação de Transcrição , Proteína de Ligação a CREB/genética , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Proteína p300 Associada a E1A/genética , Inativação Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Mutação , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/genética
2.
Nat Chem Biol ; 15(5): 519-528, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962627

RESUMO

Silencing of the somatic cell type-specific genes is a critical yet poorly understood step in reprogramming. To uncover pathways that maintain cell identity, we performed a reprogramming screen using inhibitors of chromatin factors. Here, we identify acetyl-lysine competitive inhibitors targeting the bromodomains of coactivators CREB (cyclic-AMP response element binding protein) binding protein (CBP) and E1A binding protein of 300 kDa (EP300) as potent enhancers of reprogramming. These inhibitors accelerate reprogramming, are critical during its early stages and, when combined with DOT1L inhibition, enable efficient derivation of human induced pluripotent stem cells (iPSCs) with OCT4 and SOX2. In contrast, catalytic inhibition of CBP/EP300 prevents iPSC formation, suggesting distinct functions for different coactivator domains in reprogramming. CBP/EP300 bromodomain inhibition decreases somatic-specific gene expression, histone H3 lysine 27 acetylation (H3K27Ac) and chromatin accessibility at target promoters and enhancers. The master mesenchymal transcription factor PRRX1 is one such functionally important target of CBP/EP300 bromodomain inhibition. Collectively, these results show that CBP/EP300 bromodomains sustain cell-type-specific gene expression and maintain cell identity.


Assuntos
Benzimidazóis/farmacologia , Proteína de Ligação a CREB/antagonistas & inibidores , Reprogramação Celular/efeitos dos fármacos , Proteína p300 Associada a E1A/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Isoxazóis/farmacologia , Oxazepinas/farmacologia , Piperidinas/farmacologia , Benzimidazóis/química , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Inibidores Enzimáticos/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Isoxazóis/química , Estrutura Molecular , Oxazepinas/química , Piperidinas/química , Domínios Proteicos/efeitos dos fármacos
3.
Chem Biol Interact ; 306: 54-61, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30958996

RESUMO

In the present study, we investigated the p53-independent mechanism by which quercetin (Q) increased apoptosis in human lung cancer H1299 cells exposed to trichostatin A (TSA), a histone deacetylase inhibitor. We also investigated the role of Q in increasing the acetylation of histones H3 and H4 and the possible mechanism. Q at 5 µM significantly increased apoptosis by 88% in H1299 cells induced by TSA at 72 h. Q also significantly increased TSA-induced death receptor 5 (DR5) mRNA and protein expression as well as caspase-10/3 activities in H1299 cells. Transfection of DR5 siRNA into H1299 cells significantly diminished the enhancing effects of Q on TSA-induced apoptosis. Furthermore, TSA in combination with Q rather than TSA alone significantly increased p300 expression. Transfection of p300 siRNA in H1299 cells significantly diminished the increase of histone H3/H4 acetylation, DR5 protein expression, caspase-10/3 activity and apoptosis induced by Q. In addition, similar effects of Q were observed when Q was combined with vorinostat, another FDA-approved histone deacetylase inhibitor. These data suggest that the up-regulation of p300 expression, which in turn increases histone acetylation and DR5 expression, plays an important role in the enhancing effect of Q on TSA/vorinostat- induced apoptosis in H1299 cells.


Assuntos
Antineoplásicos/farmacologia , Proteína p300 Associada a E1A/genética , Ácidos Hidroxâmicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Quercetina/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteína p300 Associada a E1A/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Vorinostat/farmacologia
4.
Hum Genet ; 138(3): 257-269, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30806792

RESUMO

Rubinstein-Taybi syndrome (RSTS) is an autosomal-dominant neurodevelopmental disease affecting 1:125,000 newborns characterized by intellectual disability, growth retardation, facial dysmorphisms and skeletal abnormalities. RSTS is caused by mutations in genes encoding for writers of the epigenetic machinery: CREBBP (~ 60%) or its homologous EP300 (~ 10%). No causative mutation is identified in up to 30% of patients. We performed whole-exome sequencing (WES) on eight RSTS-like individuals who had normal high-resolution array CGH testing and were CREBBP- and EP300-mutation -negative, to identify the molecular cause. In four cases, we identified putatively causal variants in three genes (ASXL1, KMT2D and KMT2A) encoding members of the epigenetic machinery known to be associated with the Bohring-Opitz, Kabuki and Wiedemann-Steiner syndromes. Each variant is novel, de novo, fulfills the ACMG criteria and is predicted to result in loss-of-function leading to haploinsufficiency of the epi-gene. In two of the remaining cases, homozygous/compound heterozygous variants in XYLT2 and PLCB4 genes, respectively, associated with spondyloocular and auriculocondylar 2 syndromes and in the latter an additional candidate variant in XRN2, a gene yet unrelated to any disease, were detected, but their pathogenicity remains uncertain. These results underscore the broad clinical spectrum of Mendelian disorders of the epigenetic apparatus and the high rate of WES disclosure of the genetic basis in cases which may pose a challenge for phenotype encompassing distinct syndromes. The overlapping features of distinct intellectual disability syndromes reflect common pathogenic molecular mechanisms affecting the complex regulation of balance between open and closed chromatin.


Assuntos
Estudos de Associação Genética , Síndrome de Rubinstein-Taybi/diagnóstico , Síndrome de Rubinstein-Taybi/genética , Sequenciamento Completo do Exoma , Proteína de Ligação a CREB/genética , Criança , Pré-Escolar , Hibridização Genômica Comparativa , Proteína p300 Associada a E1A/genética , Epigênese Genética , Facies , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Fenótipo
5.
Nat Commun ; 10(1): 663, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30737378

RESUMO

The biological role of miR-500a-5p has not yet been reported in the context of colorectal cancer (CRC). Here, we show that miR-500a-5p expression is decreased in CRC tissues compared with adjacent normal tissues. Low miR-500a-5p expression is associated with malignant progression. Moreover, transfection of CRC cells with miR-500a-5p induces G0/G1 cell cycle arrest and inhibits their growth and migration. Mechanistically, miR-500a-5p directly targets HDAC2 and inhibits HDAC2-mediated proliferation in CRC in nude mice. Furthermore, YY1 binds to the promoter of miR-500a-5p and negatively regulates its transcription. Restoration of miR-500a-5p expression is up-regulated via the p300/YY1/HDAC2 complex. Besides, therapeutic delivery of miR-500a-5p significantly suppresses tumour development in a xenograft tumour model and a HDAC2 inhibitor FK228-treated CRC model. Our studies demonstrate that miR-500a-5p functions as a tumour suppressor in CRC by targeting the p300/YY1/HDAC2 axis, which contributes to the development of and provides new potential candidates for CRC therapy.


Assuntos
Neoplasias Colorretais/metabolismo , Proteína p300 Associada a E1A/metabolismo , Histona Desacetilase 2/metabolismo , MicroRNAs/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Neoplasias Colorretais/genética , Proteína p300 Associada a E1A/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Células HCT116 , Histona Desacetilase 2/genética , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Transcrição YY1/genética
6.
BMC Med Genet ; 20(1): 12, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30635043

RESUMO

BACKGROUND: Rubinstein-Taybi syndrome (RSTS) Type 1 (OMIM 180849) is characterized by three main features: intellectual disability; broad and frequently angulated thumbs and halluces; and characteristic facial dysmorphism. CASE PRESENTATION: We report on a Saudi boy with RSTS Type 1 and the following distinct features: a midline notch of the upper lip, a bifid tip of the tongue, a midline groove of the lower lip, plump fingers with broad / flat fingertips, and brachydactyly. The child was found to be heterozygous in the CREBBP gene for a sequence variant designated c.4963del, which is predicted to result in premature protein termination p.Leu1655Cysfs*89. The child and his father were also found to be heterozygous in the EP300 gene for a sequence variant designated c.586A > G, which is predicted to result in the amino-acid substitution p.Ile196Val. CONCLUSION: Our report expands the clinical spectrum of RSTS to include several distinct facial and limb features. The variant of the CREBBP gene is known to be causative of RSTS Type 1. The variant in the EP300 gene is benign since the father carried the same variant and exhibited no abnormalities. However, functional studies are required to investigate if this benign EP300 variant influences the phenotype in the presence of disease-causing CREBBP gene mutations.


Assuntos
Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Estudos de Associação Genética , Síndrome de Rubinstein-Taybi/genética , Pré-Escolar , Éxons , Testes Genéticos , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Fenótipo , Síndrome de Rubinstein-Taybi/fisiopatologia , Arábia Saudita , Análise de Sequência de DNA
7.
J Virol ; 92(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29976669

RESUMO

How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated lacO array of fusions of LacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP, and RNA polymerase II (Pol II) across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.IMPORTANCE Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously (P. Pelka, J. N. G. Ablack, J. Torchia, A. S. Turnell, R. J. A. Grand, J. S. Mymryk, Nucleic Acids Res 37:1095-1106, 2009, https://doi.org/10.1093/nar/gkn1057), and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters.


Assuntos
Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Histonas/metabolismo , Regiões Promotoras Genéticas , Transcrição Genética , Acetilação , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Regulação da Expressão Gênica , Humanos , RNA Polimerase II/metabolismo , Ativação Transcricional
8.
Free Radic Res ; 52(7): 799-807, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29842805

RESUMO

Superoxide dismutase 3 (SOD3) is a SOD isozyme and plays a key role in extracellular redox homeostasis. We previously demonstrated that histone acetylation is involved in 12-O-tetra-decanoylphorbol-13-acetate (TPA)-elicited SOD3 expression in human monocytic THP-1 cells; however, the molecular mechanisms responsible for its expression have not yet been elucidated in detail. The results of the present study demonstrated that the binding of histone deacetylase 1 (HDAC1) to the SOD3 promoter region contributed to SOD3 silencing in basal THP-1 cells. On the other hand, the dissociation of HDAC1 from the SOD3 promoter region and the enrichment of p300, a histone acetyltransferase (HAT), within that region were observed in TPA-induced THP-1 cells. Myocyte enhancer factor 2 (MEF2) functions as a scaffold protein that interacts with histone deacetylases (HDAC) or HAT and regulates gene expression. The present results showed that the MEF2A and MEF2D function as mediators for TPA-elicited SOD3 expression by interacting with HDAC or p300. Additionally, the knockdown of MEF2A or MEF2D in human skin fibroblasts suppressed SOD3 expression at the mRNA and protein levels. Our results provide an insight into epigenetic regulation of redox gene expression, and may ultimately contribute to suppressing the progression of tumours and vascular diseases.


Assuntos
Proteína p300 Associada a E1A/metabolismo , Regulação Leucêmica da Expressão Gênica , Histona Desacetilase 1/metabolismo , Superóxido Dismutase/metabolismo , Acetilação , Proteína p300 Associada a E1A/genética , Histona Desacetilase 1/genética , Histonas , Humanos , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Superóxido Dismutase/genética , Células THP-1 , Transcrição Genética
9.
J Am Chem Soc ; 140(14): 4757-4760, 2018 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-29584949

RESUMO

Protein lysine crotonylation has emerged as an important post-translational modification (PTM) in the regulation of gene transcription through epigenetic mechanisms. Here we introduce a chemical probe, based on a water-soluble phosphine warhead, which reacts with the crotonyl modification. We show that this reagent is complementary to antibody-based tools allowing detection of endogenous cellular proteins such as histones carrying the crotonylation PTM. The tool is also used to show that the histone acylation activity of the transcriptional coactivator, p300, can be activated by pre-existing lysine crotonylation through a positive feedback mechanism. This reagent provides a versatile and sensitive probe for the analysis of this PTM.


Assuntos
Proteína p300 Associada a E1A/análise , Sondas Moleculares/química , Fosfinas/química , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Processamento de Proteína Pós-Traducional
10.
J Virol ; 92(9)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29467311

RESUMO

Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-κB or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs.IMPORTANCE Epstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ∼200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ∼10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.


Assuntos
Proteína p300 Associada a E1A/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Virais/metabolismo , Linfócitos B/virologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Células HEK293 , Herpesvirus Humano 4/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Regiões Promotoras Genéticas/genética , Ativação Transcricional/genética , Proteínas Virais/genética
11.
Gastroenterology ; 154(8): 2209-2221.e14, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29454793

RESUMO

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) contribute to desmoplasia and stiffness of liver metastases by differentiating into matrix-producing myofibroblasts. We investigated whether stiffness due to the presence of tumors increases activation of HSCs into myofibroblasts and their tumor-promoting effects, as well as the role of E1A binding protein p300, a histone acetyltransferase that regulates transcription, in these processes. METHODS: HSCs were isolated from liver tissues of patients, mice in which the p300 gene was flanked by 2 loxP sites (p300F/F mice), and p300+/+ mice (controls). The HSCs were placed on polyacrylamide gels with precisely defined stiffness, and their activation (differentiation into myofibroblasts) was assessed by immunofluorescence and immunoblot analyses for alpha-smooth muscle actin. In HSCs from mice, the p300 gene was disrupted by cre recombinase. In human HSCs, levels of p300 were knocked down with small hairpin RNAs or a mutant form of p300 that is not phosphorylated by AKT (p300S1834A) was overexpressed. Human HSCs were also cultured with inhibitors of p300 (C646), PI3K signaling to AKT (LY294002), or RHOA (C3 transferase) and effects on stiffness-induced activation were measured. RNA sequencing and chromatin immunoprecipitation-quantitative polymerase chain reaction were used to identify HSC genes that changed expression levels in response to stiffness. We measured effects of HSC-conditioned media on proliferation of HT29 colon cancer cells and growth of tumors following subcutaneous injection of these cells into mice. MC38 colon cancer cells were injected into portal veins of p300F/Fcre and control mice, and liver metastases were measured. p300F/Fcre and control mice were given intraperitoneal injections of CCl4 to induce liver fibrosis. Liver tissues were collected and analyzed by immunofluorescence, immunoblot, and histology. RESULTS: Substrate stiffness was sufficient to activate HSCs, leading to nuclear accumulation of p300. Disrupting p300 level or activity blocked stiffness-induced activation of HSCs. In HSCs, substrate stiffness activated AKT signaling via RHOA to induce phosphorylation of p300 at serine 1834; this caused p300 to translocate to the nucleus, where it up-regulated transcription of genes that increase activation of HSCs and metastasis, including CXCL12. MC38 cells, injected into portal veins, formed fewer metastases in livers of p300F/Fcre mice than control mice. Expression of p300 was increased in livers of mice following injection of CCl4; HSC activation and collagen deposition were reduced in livers of p300F/Fcre mice compared with control mice. CONCLUSIONS: In studies of mice, we found liver stiffness to activate HSC differentiation into myofibroblasts, which required nuclear accumulation of p300. p300 increases HSC expression of genes that promote metastasis.


Assuntos
Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/metabolismo , Proteína p300 Associada a E1A/metabolismo , Células Estreladas do Fígado/patologia , Neoplasias Hepáticas/patologia , Miofibroblastos/patologia , Animais , Benzoatos/farmacologia , Tetracloreto de Carbono/toxicidade , Núcleo Celular/metabolismo , Transdiferenciação Celular , Proteína p300 Associada a E1A/genética , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HT29 , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Camundongos , Camundongos Knockout , Camundongos SCID , Miofibroblastos/metabolismo , Fosforilação , Cultura Primária de Células , Pirazóis/farmacologia , RNA Interferente Pequeno/antagonistas & inibidores , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína rhoA de Ligação ao GTP/metabolismo
12.
Biochim Biophys Acta Mol Basis Dis ; 1864(4 Pt A): 1203-1215, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29409755

RESUMO

EP300 is a member of the EP300/CBP family of lysine acetyltransferases (KATs) with multiple roles in development and physiology. Loss of EP300/CBP activity in humans causes a very rare congenital disorder called Rubinstein Taybi Syndrome (RSTS). The zebrafish genome has two co-orthologs of lysine acetyltransferase EP300 (KAT3B) in zebrafish viz. ep300a and ep300b. Chemical inhibition of Ep300 with C646, a competitive inhibitor and morpholino-based genetic knockdown of ep300a and ep300b cause defects in embryonic development reminiscent of the human RSTS syndrome. Remarkably, overexpression of Ep300a KAT domain results in near complete rescue of the jaw development defects, a characteristic feature of RSTS in human suggesting the dispensability of the protein-interaction and DNA-binding domains for at least some developmental roles of Ep300. We also perform a chemical screen and identify two inhibitors of deacetylases, CHIC35 and HDACi III, that can partially rescue the RSTS-like phenotypes. Thus, modeling rare human genetic disorders in zebrafish allows for functional understanding of the genes involved and can also yield small molecule candidates towards therapeutic goals.


Assuntos
Modelos Animais de Doenças , Proteína p300 Associada a E1A , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/genética , Técnicas de Silenciamento de Genes , Síndrome de Rubinstein-Taybi , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Humanos , Síndrome de Rubinstein-Taybi/embriologia , Síndrome de Rubinstein-Taybi/genética , Síndrome de Rubinstein-Taybi/patologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
13.
Biochem J ; 475(2): 477-494, 2018 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-29269396

RESUMO

Oncostatin-M (OSM) is a pleotropic cytokine belonging to the interleukin-6 family. Differential expression of OSM in response to varying stimuli and exhibiting repertoire of functions in different cells renders it challenging to study the mechanism of its expression. Prostaglandin E2 (PGE2) transcriptionally increased osm levels. In silico studies of ∼1 kb upstream of osm promoter region yielded the presence of CRE (cyclic AMP response element)-like sites at the distal end (CREosm). Deletion and point mutation of CREosm clearly indicated that this region imparted an important role in PGE2-mediated transcription. Nuclear protein(s) from PGE2-treated U937 cells, bound to this region, was identified as CRE-binding protein (CREB). CREB was phosphorylated on treatment and was found to be directly associated with CREosm The presence of cofactors p300 and CREB-binding protein in the complex was confirmed. A marked decrease in CREB phosphorylation, binding and transcriptional inhibition on treatment with PKA (protein kinase A) inhibitor, H89 (N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-soquinolinesulfonamide), revealed the role of phosphorylated CREB in osm transcription. Additionally, other nuclear protein(s) were specifically associated with the proximal GC region (GCosm) post PGE2 treatment, later confirmed to be specificity protein 1 (Sp1). Interestingly, Sp1 bound to the proximal osm promoter was found to be associated with phospho-CREB-p300 complex bound to the distal osm promoter. Knockdown of Sp1 abrogated the expression and functionality of OSM. Thus, the present study conclusively proves that these transcription factors, bound at the distal and proximal promoter elements are found to associate with each other in a DNA-dependent manner and both are responsible for the PGE2-mediated transcriptional up-regulation of Oncostatin-M.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dinoprostona/farmacologia , Regulação da Expressão Gênica , Oncostatina M/genética , Fator de Transcrição Sp1/genética , Transcrição Genética , Sítios de Ligação , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Humanos , Isoquinolinas/farmacologia , Mutagênese Sítio-Dirigida , Oncostatina M/metabolismo , Fosforilação/efeitos dos fármacos , Mutação Puntual , Regiões Promotoras Genéticas , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Elementos de Resposta , Transdução de Sinais , Fator de Transcrição Sp1/metabolismo , Sulfonamidas/farmacologia , Células U937
14.
Diabetes ; 67(3): 412-422, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29217654

RESUMO

p300 (EP300) and CBP (CREBBP) are transcriptional coactivators with histone acetyltransferase activity. Various ß-cell transcription factors can recruit p300/CBP, and thus the coactivators could be important for ß-cell function and health in vivo. We hypothesized that p300/CBP contribute to the development and proper function of pancreatic islets. To test this, we bred and studied mice lacking p300/CBP in their islets. Mice lacking either p300 or CBP in islets developed glucose intolerance attributable to impaired insulin secretion, together with reduced α- and ß-cell area and islet insulin content. These phenotypes were exacerbated in mice with only a single copy of p300 or CBP expressed in islets. Removing p300 in pancreatic endocrine progenitors impaired proliferation of neonatal α- and ß-cells. Mice lacking all four copies of p300/CBP in pancreatic endocrine progenitors failed to establish α- and ß-cell mass postnatally. Transcriptomic analyses revealed significant overlaps between p300/CBP-downregulated genes and genes downregulated in Hnf1α-null islets and Nkx2.2-null islets, among others. Furthermore, p300/CBP are important for the acetylation of H3K27 at loci downregulated in Hnf1α-null islets. We conclude that p300 and CBP are limiting cofactors for islet development, and hence for postnatal glucose homeostasis, with some functional redundancy.


Assuntos
Proteína de Ligação a CREB/metabolismo , Proliferação de Células , Proteína p300 Associada a E1A/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Células-Tronco/metabolismo , Acetilação , Animais , Animais Recém-Nascidos , Glicemia/análise , Proteína de Ligação a CREB/genética , Tamanho Celular , Cruzamentos Genéticos , Proteína p300 Associada a E1A/genética , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/patologia , Intolerância à Glucose/sangue , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Histonas/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/patologia , Lisina , Camundongos Knockout , Camundongos Transgênicos , Processamento de Proteína Pós-Traducional , Células-Tronco/citologia , Células-Tronco/patologia
15.
Eur J Med Genet ; 61(3): 125-129, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29133209

RESUMO

Many disease genes are defined by their role in causing specific clinically recognizable syndromes. Heterozygous loss of function of the gene EP300 is responsible for a minority of cases of Rubinstein-Taybi syndrome (RSTS). With the application of whole-exome sequencing and whole-genome sequencing, there is the potential to discover new genotype-phenotype correlations. The purpose of this case series is to describe three unrelated females without classic manifestations of RSTS who were unexpectedly found on genome-wide sequencing to have likely pathogenic variants in EP300. These individuals expand our knowledge of the disease spectrum by virtue of their very rare or novel clinical features. Results are placed within the context of all prior published EP300 cases not ascertained by targeted testing, which are disproportionately female compared with a cohort identified because of a clinical suspicion of RSTS (p = 0.01). There are implications for diagnosis, management, and genetic counselling of individuals with EP300-related disease.


Assuntos
Proteína p300 Associada a E1A/genética , Genoma Humano , Estudo de Associação Genômica Ampla , Mutação , Síndrome de Rubinstein-Taybi/genética , Adulto , Criança , Pré-Escolar , Mapeamento Cromossômico , Feminino , Estudos de Associação Genética , Humanos , Lactente , Masculino , Fenótipo , Síndrome de Rubinstein-Taybi/patologia , Análise de Sequência de DNA
16.
Biochem Biophys Res Commun ; 495(2): 1675-1680, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29217191

RESUMO

Tumor necrosis factor (TNF)-α is responsible for expressions of several clock genes and affects joint symptoms of rheumatoid arthritis (RA) with diurnal fluctuation. We tried to determine the mechanism involved in over-expression of Bmal1, induced by TNF-α, in primary cultured rheumatoid synovial cells. Cells were incubated with intra-cellular Ca2+ chelator BAPTA-AM, calcineurin inhibitor FK506 and p300/CBP (CREB binding protein) inhibitor C646, respectively, or transfected with p300 and CBP small interfering RNA (siRNA) before stimulation with TNF-α. Oscillation phase and amplitude of Bmal1, transcriptional activator Rorα, transcriptional repressor Rev-erbα, and histone acetyltransferases (p300 and Cbp) were evaluated by quantitative real-time PCR. As results, TNF-α did not influence the oscillation phase of Rev-erbα, while enhanced those of Rorα, resulting in over-expression of Bmal1. When Ca2+ influx was inhibited by BAPTA-AM, TNF-α-mediated up-regulation of Rorα was cancelled, however, that of Bmal1 was still apparent. When we further explored another pathway between TNF-α and Bmal1, TNF-α suppressed the expression of Rev-erbα in the absence of Ca2+ influx, as well as those of p300 and Cbp genes. Finally, actions of TNF-α, in increasing Bmal1/Rorα and decreasing Rev-erbα, were cancelled by C646 treatment or silencing of both p300 and Cbp. In conclusion, we determined a novel role of TNF-α in inducing Bmal1 via dual calcium dependent pathways; Rorα was up-regulated in the presence of Ca2+ influx and Rev-erbα was down-regulated in the absence of that. Results proposed that inhibition of p300/CBP could be new therapeutic targets for RA.


Assuntos
Fatores de Transcrição ARNTL/genética , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Sinalização do Cálcio , Relógios Circadianos/genética , Membrana Sinovial/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Artrite Reumatoide/patologia , Benzoatos/farmacologia , Proteína de Ligação a CREB/antagonistas & inibidores , Proteína de Ligação a CREB/genética , Quelantes de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/genética , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Pirazóis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/farmacologia
17.
BMC Cancer ; 17(1): 874, 2017 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-29262808

RESUMO

BACKGROUND: Histones undergo extensive post-translational modifications and this epigenetic regulation plays an important role in modulating transcriptional programs capable of driving cancer progression. Acetylation of histone H3K18, associated with gene activation, is enhanced by P300 and opposed by the deacetylase Sirtuin2 (SIRT2). As these enzymes represent an important target for cancer therapy, we sought to determine whether the underlying genes are altered during prostate cancer (PCa) progression. METHODS: Tissue microarrays generated from 71 radical prostatectomy patients were initially immunostained for H3K18Ac, P300 and SIRT2. Protein levels were quantified using VECTRA automation and correlated with clinicopathologic parameters. The Cancer Genome Atlas (TGCA, n = 499) and Gene Expression Omnibus (n = 504) databases were queried for expression, genomic and clinical data. Statistics were performed using SPSSv23. RESULTS: Nuclear histone H3K18Ac staining increases in primary cancer (p = 0.05) and further in metastases (p < 0.01) compared to benign on tissue arrays. P300 protein expression increases in cancer (p = 0.04) and metastases (p < 0.001). A progressive decrease in nuclear SIRT2 staining occurs comparing benign to cancer or metastases (p = 0.04 and p = 0.03 respectively). Decreased SIRT2 correlates with higher grade cancer (p = 0.02). Time to Prostate Specific Antigen (PSA) recurrence is shorter in patients exhibiting high compared to low H3K18Ac expression (350 vs. 1542 days respectively, P = 0.03). In GEO, SIRT2 mRNA levels are lower in primary and metastatic tumors (p = 0.01 and 0.001, respectively). TGCA analysis demonstrates SIRT2 deletion in 6% and increasing clinical stage, positive margins and lower PSA recurrence-free survival in patients with SIRT2 loss/deletion (p = 0.01, 0.04 and 0.04  respectively). In this dataset, a correlation between decreasing SIRT2 and increasing P300 mRNA expression occurs in tumor samples (R = -0.46). CONCLUSIONS: In multiple datasets, decreases in SIRT2 expression portend worse clinicopathologic outcomes. Alterations in SIRT2-H3K18Ac suggest altered P300 activity and identify a subset of tumors that could benefit from histone deacetylation inhibition.


Assuntos
Biomarcadores Tumorais/genética , Proteína p300 Associada a E1A/metabolismo , Histonas/metabolismo , Recidiva Local de Neoplasia/mortalidade , Neoplasias da Próstata/mortalidade , Processamento de Proteína Pós-Traducional , Sirtuína 2/genética , Acetilação , Idoso , Biomarcadores Tumorais/metabolismo , Progressão da Doença , Proteína p300 Associada a E1A/genética , Epigênese Genética , Seguimentos , Histonas/química , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Deleção de Sequência , Taxa de Sobrevida
18.
Nat Commun ; 8(1): 1286, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-29097680

RESUMO

Chronic kidney disease (CKD) is defined by reduced estimated glomerular filtration rate (eGFR). Previous genetic studies have implicated regulatory mechanisms contributing to CKD. Here we present epigenome-wide association studies of eGFR and CKD using whole-blood DNA methylation of 2264 ARIC Study and 2595 Framingham Heart Study participants to identify epigenetic signatures of kidney function. Of 19 CpG sites significantly associated (P < 1e-07) with eGFR/CKD and replicated, five also associate with renal fibrosis in biopsies from CKD patients and show concordant DNA methylation changes in kidney cortex. Lead CpGs at PTPN6/PHB2, ANKRD11, and TNRC18 map to active enhancers in kidney cortex. At PTPN6/PHB2 cg19942083, methylation in kidney cortex associates with lower renal PTPN6 expression, higher eGFR, and less renal fibrosis. The regions containing the 243 eGFR-associated (P < 1e-05) CpGs are significantly enriched for transcription factor binding sites of EBF1, EP300, and CEBPB (P < 5e-6). Our findings highlight kidney function associated epigenetic variation.


Assuntos
Metilação de DNA/genética , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/fisiopatologia , Idoso , Sítios de Ligação/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ilhas de CpG , Progressão da Doença , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Epigênese Genética , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Taxa de Filtração Glomerular/genética , Humanos , Rim/metabolismo , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Brain ; 140(11): 2860-2878, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29053796

RESUMO

The autosomal dominant cerebellar ataxias, referred to as spinocerebellar ataxias in genetic nomenclature, are a rare group of progressive neurodegenerative disorders characterized by loss of balance and coordination. Despite the identification of numerous disease genes, a substantial number of cases still remain without a genetic diagnosis. Here, we report five novel spinocerebellar ataxia genes, FAT2, PLD3, KIF26B, EP300, and FAT1, identified through a combination of exome sequencing in genetically undiagnosed families and targeted resequencing of exome candidates in a cohort of singletons. We validated almost all genes genetically, assessed damaging effects of the gene variants in cell models and further consolidated a role for several of these genes in the aetiology of spinocerebellar ataxia through network analysis. Our work links spinocerebellar ataxia to alterations in synaptic transmission and transcription regulation, and identifies these as the main shared mechanisms underlying the genetically diverse spinocerebellar ataxia types.


Assuntos
Redes Reguladoras de Genes/genética , Ataxias Espinocerebelares/genética , Animais , Células COS , Caderinas/genética , Cercopithecus aethiops , Proteína p300 Associada a E1A/genética , Exoma/genética , Feminino , Células HEK293 , Humanos , Cinesina/genética , Masculino , Linhagem , Fosfolipase D/genética , Plasmídeos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Transfecção
20.
J Exp Clin Cancer Res ; 36(1): 125, 2017 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-28893318

RESUMO

BACKGROUND: ICG-001, a small molecule, binds CREB-binding protein (CBP) to disrupt its interaction with ß-catenin and inhibits CBP function as a co-activator of Wnt/ß-catenin-mediated transcription. Given its ability to inhibit Wnt/ß-catenin signaling pathway, ICG-001 has been used in some tumor types to exert its anticarcinogenic effect. Here, we examined ICG-001 and its potential role as a therapeutic in gastric cancer (GC). METHODS: The gastric cancer cell lines SGC-7901, MGC-803, BGC-823 and MKN-45 were used in vitro and in vivo. The abilities of cell proliferation, tumor sphere formation, metastasis, tumorgenesis and chemoresistance to chemotherapy drugs in vitro were evaluated by MTT assay, colony formation assay, flow cytometry, migration and invasion assay, and tumor spheres culture. The in vivo experiments were performed using a subcutaneous transplantation tumor model in athymic nude mice. Alterations at RNA and protein levels were followed by qRT-PCR, western blot, coimmunoprecipitations and immunofluorescence assay. RESULTS: In this study, we showed that ICG-001 significantly inhibited growth and metastasis of multiple GC cell lines, induced cell apoptosis, and augmented in vitro tumor spheres suppression when used in combination with chemotherapy drugs probably through robustly blocking association of ß-catenin with CBP and N-cadherin, but promoting association of ß-catenin with P300 and E-cadherin, instead of altering the distribution and expression of ß-catenin. CONCLUSIONS: Our findings suggest that ICG-001 suppresses GC cell line growth, metastasis and reduces its stem cell-like properties and chemoresistance, indicating that ICG-001 is a potentially useful small molecule therapeutic for GC.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Pirimidinonas/administração & dosagem , Neoplasias Gástricas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteína p300 Associada a E1A/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Camundongos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
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