RESUMO
Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation and bone formation. Osx null mice develop a normal cartilage skeleton but fail to form bone and to express osteoblast-specific marker genes. To better understand the control of transcriptional regulation by Osx, we identified Osx-interacting proteins using proteomics approaches. Here, we report that a Jumonji C (JmjC)-domain containing protein, called NO66, directly interacts with Osx and inhibits Osx-mediated promoter activation. The knockdown of NO66 in preosteoblast cells triggered accelerated osteoblast differentiation and mineralization, and markedly stimulated the expression of Osx target genes. A JmjC-dependent histone demethylase activity was exhibited by NO66, which was specific for both H3K4me and H3K36me in vitro and in vivo, and this activity was needed for the regulation of osteoblast-specific promoters. During BMP-2-induced differentiation of preosteoblasts, decreased NO66 occupancy correlates with increased Osx occupancy at Osx-target promoters. Our results indicate that interactions between NO66 and Osx regulate Osx-target genes in osteoblasts by modulating histone methylation states.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Osteoblastos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Dioxigenases , Histona Desmetilases , Histonas/química , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/genética , Lisina/química , Camundongos , Osteoblastos/citologia , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição Sp7 , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Head and neck squamous cell carcinoma (HNSCC) is the fifth most frequent cancer in the US. Several genetic and epigenetic alterations are associated with HNSCC tumorigenesis, including inactivation of CDKN2A, which encodes the p16 tumor suppressor, in cell lines and primary tumors by DNA methylation. Reactivation of tumor suppressor genes by DNA-demethylating agents and histone deacetylase (HDAC) inhibitors shows therapeutic promise for other cancers. Therefore, we investigated the ability of these agents to reactivate p16 in Tu159 HNSCC cells. Treatment of cells with 5-aza-2'deoxycytidine (5-aza-dC) increases CDKN2A expression and slightly increases histone H3 acetylation at this gene. No reactivation of CDKN2A is observed upon treatment with the HDAC inhibitor trichostatin A (TSA), but synergistic reactivation of CDKN2A is observed upon sequential treatment of Tu159 cells with both 5-aza-dC and TSA. Silencing of CDKN2A in Tu159 cells is correlated with increased methylation of histone H3 at lysine 9 and decreased methylation at lysine 4 relative to the upstream p15 gene promoter. Interestingly, global levels of H3-K9 methylation are decreased upon treatment with 5-aza-dC. Together these data indicate that DNA methylation is a dominant epigenetic mark for silencing of CDKN2A in Tu159 tumor cells. Moreover, changes in DNA methylation can reset the histone code by impacting multiple H3 modifications.
Assuntos
Azacitidina/análogos & derivados , Carcinoma de Células Escamosas/genética , Metilação de DNA , Genes p16 , Neoplasias de Cabeça e Pescoço/genética , Histonas/metabolismo , Animais , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Carcinoma de Células Escamosas/metabolismo , Decitabina , Inativação Gênica , Genes p16/efeitos dos fármacos , Genes p16/fisiologia , Neoplasias de Cabeça e Pescoço/metabolismo , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/farmacologiaRESUMO
The genetic events associated with the development and progression of head and neck squamous carcinoma (HNSC) are largely unknown. We analysed 12 matched pairs of histologically normal squamous mucosa and tumor specimens from six conventional and six phenotypic variants HNSC to define the differentially expressed genes in these tumors. Parallel expression analysis of 8055 unique genes was performed, and the level of the hybridization signal for each gene was measured after normalization. Hierarchical cluster analysis of the expressed genes showed distinct inter- and intra-tumoral patterns in and between conventional squamous carcinoma and squamous carcinoma variants. We also identified 26 (0.32%) differentially expressed genes that were consistently different between matched pairs of normal and tumor specimens; a selected set of the overexpressed genes was validated using real-time quantitative RT-PCR. The majority of the genes were associated with differentiation and proliferation. Our study defines a set of genes that could form the basis for the construction of limited HNSC targeted expression array and in-depth studies and further highlights gene profile differences that may be useful in pathobiologic classification of HNSC.