RESUMO
The TAF4b subunit of the transcription factor IID, which has a central role in transcription by polymerase II, is involved in promoter recognition by selective recruitment of activators. The activating protein-1 (AP-1) family members participate in oncogenic transformation via gene regulation. Utilizing immunoprecipitation of endogenous protein complexes, we documented specific interactions between Jun family members and TATA box binding protein-associated factors (TAF) in colon HT29 adenocarcinoma cells. Particularly, TAF4b and c-Jun were found to colocalize and interact in the nucleus of advanced carcinoma cells and in cells with epithelial-to-mesenchymal transition (EMT) characteristics. TAF4b was found to specifically regulate the AP-1 target gene involved in EMT integrin alpha6, thus altering related cellular properties such as migration potential. Using a chromatin immunoprecipitation approach in colon adenocarcinoma cell lines, we further identified a synergistic role for TAF4b and c-Jun and other AP-1 family members on the promoter of integrin alpha6, underlining the existence of a specific mechanism related to gene expression control. We show evidence for the first time of an interdependence of TAF4b and AP-1 family members in cell type-specific promoter recognition and initiation of transcription in the context of cancer progression and EMT.
Assuntos
Integrina alfa6/genética , Invasividade Neoplásica/genética , Neoplasias/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição TFIID/genética , Ativação Transcricional/genética , Células CACO-2 , Desdiferenciação Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Integrina alfa6/metabolismo , Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição TFIID/metabolismoRESUMO
The presence of different forms of histone covalent modifications, such as phosphorylation, acetylation and methylation in localized promoter regions are markers for chromatin packing and transcription. Activation of RAS signalling pathways through oncogenic RAS mutations is a hallmark of colorectal cancer. Overexpression of Harvey-Ras oncogene induces epithelial-mesenchymal transition (EMT) in Caco-2 cells. We focused on the role of epigenetic modifications of histone H3 and its dependence on RAS signal transduction pathways and oncogenic transformation. Using cell lines stably overexpressing oncogenic Harvey-RAS with EMT phenotype, we studied the acquired changes in the H3 histone modification patterns. Two genes show inverse protein expression patterns after Ha-RAS overexpression: Cyclin D1, a cell cycle-related gene, and the EMT marker-gene E-cadherin. We report that these two genes demonstrate matching inverse histone repression patterns on their promoter, while histone markers associated with an active state of genes were affected by the RAS-activated signalling pathway MEK-ERK-MSK1. Furthermore, we show that though the level of methyltransferases enzymes was increased, the status of H3 three-methylation at lysine 27 (H3K27me(3)), associated with gene repression on the promoter of Cyclin D1, was lower. Together, these results suggest that histone covalent modifications can be affected by oncogenic RAS pathways to regulate the expression of target genes like Cyclin D1 or E-cadherin and that the dynamic balance of opposing histone-modifying enzymes is critical for the regulation of cell proliferation.
Assuntos
Neoplasias Colorretais/metabolismo , Células Epiteliais/metabolismo , Histonas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteínas ras/metabolismo , Células CACO-2 , Caderinas/biossíntese , Caderinas/genética , Transformação Celular Neoplásica , Neoplasias Colorretais/patologia , Ciclina D1/biossíntese , Ciclina D1/genética , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , MAP Quinase Quinase Quinases/metabolismo , Células-Tronco Mesenquimais/patologia , Processamento de Proteína Pós-Traducional/genética , Transdução de Sinais , Transgenes , Proteínas ras/genéticaRESUMO
The process of epithelial mesenchymal transition, whereby cells acquire molecular alterations and fibroblastic features, is a fundamental process of embryogenesis and cancer invasion/metastasis. The mechanisms responsible for epithelial mesenchymal transition remain elusive. Human tumors frequently establish constitutively activated RAS signaling, which contributes to the malignant phenotype. In an effort to dissect distinct RAS isoform specific functions, we previously established human colon cell lines stably overexpressing activated Harvey-RAS (Ha-RAS) and Kirsten-RAS (Ki-RAS). Using these, we observed that only oncogenic Ha-RAS overexpression resulted in morphologic and molecular changes suggestive of epithelial to mesenchymal transition. We showed that vimentin, a key molecule of epithelial mesenchymal transition, was differentially regulated between Ha-RAS and Ki-RAS leading to a Ha-RAS specific induction of a migratory phenotype and eventually epithelial to mesenchymal transition. We demonstrated that the AP-1 sites in vimentin promoter could be involved in this regulation. A potential role of FRA-1 was suggested in the regulation of vimentin during the Ha-RAS-induced epithelial to mesenchymal transition, in association with colon cell migration. Our results therefore propose that in colon cells, the induction of epithelial mesenchymal transition by oncogenic Ha-RAS could occur through the overexpression of proteins like FRA-1 and vimentin.
