RESUMO
Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , DNA Ligases/genética , Metilação de DNA , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas/genética , Western Blotting , Ciclo Celular , Proliferação de Células , Colo/metabolismo , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , DNA Metiltransferase 3A , Feminino , Inativação Gênica , Humanos , Masculino , Metaloproteinase 9 da Matriz/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
While transcription as regulated by histones and their post-translational modifications has been well described, the function of histone variants in this process remains poorly characterized. Potentially important insight into this process pertain to the frequently occurring mutations of H3.3, leading to G34 substitutions in childhood glioblastoma and giant cell tumor of the bone (GCTB). In this study, we have established primary cell lines from GCTB patients and used them to uncover the influence of H3.3 G34W substitutions on cellular growth behavior, gene expression, and chromatin compaction. Primary cell lines with H3.3 G34W showed increased colony formation, infiltration and proliferation, known hallmarks of tumor development. Isogenic cell lines with H3.3 G34W recapitulated the increased proliferation observed in primary cells. Transcriptomic analysis of primary cells and tumor biopsies revealed slightly more downregulated gene expression, perhaps by increased chromatin compaction. We identified components related to splicing, most prominently hnRNPs, by immunoprecipitation and mass spectrometry that specifically interact with H3.3 G34W in the isogenic cell lines. RNA-sequencing analysis and hybridization-based validations further enforced splicing aberrations. Our data uncover a role for H3.3 in RNA processing and chromatin modulation that is blocked by the G34W substitution, potentially driving the tumorigenic process in GCTB.
Assuntos
Substituição de Aminoácidos , Neoplasias Ósseas/genética , Cromatina/genética , Tumor de Células Gigantes do Osso/genética , Histonas/genética , Mutação , Processamento Pós-Transcricional do RNA , Neoplasias Ósseas/diagnóstico , Proteínas de Transporte , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromatografia Líquida , Biologia Computacional , Feminino , Perfilação da Expressão Gênica , Tumor de Células Gigantes do Osso/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Humanos , Masculino , Modelos Biológicos , Ligação Proteica , Mapeamento de Interação de Proteínas , Espectrometria de Massas em Tandem , TranscriptomaRESUMO
This corrects the article DOI: 10.1038/ng.3889.
RESUMO
Several mechanisms of action have been proposed for DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi), primarily based on candidate-gene approaches. However, less is known about their genome-wide transcriptional and epigenomic consequences. By mapping global transcription start site (TSS) and chromatin dynamics, we observed the cryptic transcription of thousands of treatment-induced non-annotated TSSs (TINATs) following DNMTi and HDACi treatment. The resulting transcripts frequently splice into protein-coding exons and encode truncated or chimeric ORFs translated into products with predicted abnormal or immunogenic functions. TINAT transcription after DNMTi treatment coincided with DNA hypomethylation and gain of classical promoter histone marks, while HDACi specifically induced a subset of TINATs in association with H2AK9ac, H3K14ac, and H3K23ac. Despite this mechanistic difference, both inhibitors convergently induced transcription from identical sites, as we found TINATs to be encoded in solitary long terminal repeats of the ERV9/LTR12 family, which are epigenetically repressed in virtually all normal cells.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Proteínas Quinases Associadas com Morte Celular/genética , Código das Histonas , Inibidores de Histona Desacetilases/farmacologia , Sequências Repetidas Terminais/genética , Sítio de Iniciação de Transcrição/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/fisiologia , Metilação de DNA , Proteínas Quinases Associadas com Morte Celular/antagonistas & inibidores , Repressão Epigenética , Éxons/genética , Feminino , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Ácidos Hidroxâmicos/farmacologia , Íntrons/genética , Camundongos , Camundongos Nus , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , VorinostatRESUMO
Although clonal selection by genetic driver aberrations in cancer is well documented, the ability of epigenetic alterations to promote tumor evolution is undefined. We used 450k arrays and next-generation sequencing to evaluate intratumor heterogeneity and evolution of DNA methylation and genetic aberrations in chronic lymphocytic leukemia (CLL). CLL cases exhibit vast interpatient differences in intratumor methylation heterogeneity, with genetically clonal cases maintaining low methylation heterogeneity and up to 10% of total CpGs in a monoallelically methylated state. Increasing methylation heterogeneity correlates with advanced genetic subclonal complexity. Selection of novel DNA methylation patterns is observed only in cases that undergo genetic evolution, and independent genetic evolution is uncommon and is restricted to low-risk alterations. These results reveal that although evolution of DNA methylation occurs in high-risk, clinically progressive cases, positive selection of novel methylation patterns entails coevolution of genetic alteration(s) in CLL.
Assuntos
Metilação de DNA , Instabilidade Genômica , Leucemia Linfocítica Crônica de Células B/genética , Idoso , Cromossomos Humanos , Ilhas de CpG , Progressão da Doença , Epigênese Genética , Feminino , Regulação Leucêmica da Expressão Gênica , Heterogeneidade Genética , Genoma Humano , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs.
Assuntos
Carcinoma de Células Escamosas/genética , Papillomavirus Humano 16/fisiologia , Neoplasias Orofaríngeas/genética , Infecções por Papillomavirus/genética , Família Aldeído Desidrogenase 1 , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/virologia , Estudos de Casos e Controles , Ilhas de CpG , Metilação de DNA , Intervalo Livre de Doença , Fator de Transcrição GATA4/genética , Regulação Neoplásica da Expressão Gênica , Células HeLa , Proteínas de Homeodomínio/genética , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/genética , Humanos , Estimativa de Kaplan-Meier , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Orofaríngeas/mortalidade , Neoplasias Orofaríngeas/virologia , Infecções por Papillomavirus/mortalidade , Infecções por Papillomavirus/virologia , Regiões Promotoras Genéticas , Modelos de Riscos Proporcionais , Receptores de AMPA/genética , Retinal Desidrogenase/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética , TranscriptomaRESUMO
The scaffold protein A-kinase anchor protein 12 (AKAP12) exerts tumor suppressor activity and is downregulated in several tumor entities. We characterized AKAP12 expression and regulation in astrocytomas, including pilocytic and diffusely infiltrating astrocytomas. We examined 194 human gliomas and 23 normal brain white matter samples by immunohistochemistry or immunoblotting for AKAP12 expression. We further performed quantitative methylation analysis of the AKAP12 promoter by MassARRAY® of normal brain, World Health Organization (WHO) grade I to IV astrocytomas, and glioma cell lines. Our results show that AKAP12 is expressed in a perivascular distribution in normal CNS, strongly upregulated in tumor cells in pilocytic astrocytomas, and weakly expressed in diffuse astrocytomas of WHO grade II to IV. Methylation analyses revealed specific hypermethylation of AKAP12α promoter in WHO grade II to IV astrocytomas. Restoration experiments using 5-aza-2'-deoxycytidine in primary glioblastoma cells decreased AKAP12α promoter methylation and markedly increased AKAP12α mRNA levels. In summary, we demonstrate that AKAP12 is differentially expressed in human astrocytomas showing high expression in pilocytic but low expression in diffuse astrocytomas of all WHO-grades. Our results further indicate that epigenetic mechanisms are involved in silencing AKAP12 in diffuse astrocytomas; however, a tumor suppressive role of AKAP12 in distinct astrocytoma subtypes remains to be determined.