RESUMO
The C-Circle Assay has satisfied the need for a rapid, robust and quantitative ALT assay that responds quickly to changes in ALT activity. The C-Circle Assay involves (i) extraction or simple preparation (Quick C-Circle Preparation) of the cell's DNA, which includes C-Circles (ii) amplification of the self-primed C-Circles with a rolling circle amplification reaction and (iii) sequence specific detection of the amplification products by native telomeric DNA dot blot or telomeric qPCR. Here we detail the protocols and considerations required to perform the C-Circle Assay and its controls, which include exonuclease removal of linear telomeric DNA, production of the synthetic C-Circle C96 and modulation of ALT activity by γ-irradiation.
Assuntos
Biomarcadores Tumorais/genética , DNA Circular/análise , DNA de Neoplasias/genética , Neoplasias/diagnóstico , Telômero , Humanos , Neoplasias/genéticaRESUMO
TFIIH is a multisubunit factor essential for transcription initiation and promoter escape of RNA polymerase II and for the opening of damaged DNA double strands in nucleotide excision repair (NER). In this study, we have analyzed at which step of the transcription cycle TFIIH is essential for transcription by RNA polymerase I. We demonstrate that TFIIH associates with the rDNA promoter and gene-internal sequences and leaves the rDNA promoter in a complex with RNA polymerase I after start of transcription. Moreover, mutations in the TFIIH subunits XPB and XPD found in Cockayne syndrome impair the interaction of TFIIH with the rDNA, but do not influence initiation complex formation or promoter escape of RNA polymerase I, but preclude the productivity of the enzyme by reducing transcription elongation in vivo and in vitro. Our results implicate that reduced RNA polymerase I transcription elongation and ribosomal stress could be one factor contributing to the Cockayne syndrome phenotype.
Assuntos
RNA Polimerase I/metabolismo , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismo , Células Cultivadas , Síndrome de Cockayne/genética , DNA Helicases/genética , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Imunoprecipitação , Mutação , Regiões Promotoras Genéticas , Fator de Transcrição TFIIH/genética , Proteína Grupo D do Xeroderma Pigmentoso/genéticaRESUMO
Telomerase is activated in the majority of invasive breast cancers, but the time point of telomerase activation during mammary carcinogenesis is not clear. We have recently presented a transgenic mouse model to study human telomerase reverse transcriptase (TERT) gene expression in vivo (hTERTp-lacZ). In the present study, hTERTp-lacZxWAP-T bitransgenic mice were generated to analyze the mechanisms responsible for human and mouse TERT upregulation during tumor progression in vivo. We found that telomerase activity and TERT expression were consistently upregulated in SV40-induced invasive mammary tumors compared to normal and hyperplastic tissues and ductal carcinoma in situ (DCIS). Human and mouse TERT genes are regulated similarly in the breast tissue, involving the CEBP transcription factors. Loss of CEBP-α and induction of CEBP-ß expression correlated well with the activation of TERT expression in mouse mammary tumors. Transfection of CEBP-α into human or murine cells resulted in TERT repression, whereas knockdown of CEBP-α in primary human mammary epithelial cells resulted in reactivation of endogenous TERT expression and telomerase activity. Conversely, ectopic expression of CEBP-ß activated endogenous TERT gene expression. Moreover, ChIP and EMSA experiments revealed binding of CEBP-α and CEBP-ß to human TERT-promoter. This is the first evidence indicating that CEBP-α and CEBP-ß are involved in TERT gene regulation during carcinogenesis.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias Mamárias Experimentais/patologia , Proteínas do Leite/genética , Regiões Promotoras Genéticas/genética , Telomerase/genética , Animais , Western Blotting , Proteínas Estimuladoras de Ligação a CCAAT/genética , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/metabolismo , Ativação Transcricional , Células Tumorais CultivadasRESUMO
The nucleolus has long been considered to be a pure ribosome factory. However, over the last two decades it became clear that the nucleolus is involved in numerous other functions besides ribosome biogenesis. Our experiments indicate that the activity of RNA polymerase I (Pol I) transcription monitors the integrity of the DNA and influences the response to nucleolar stress as well as the rate of survival. Cells with a repressed ribosomal DNA (rDNA) transcription activity showed an increased and prolonged p53 stabilisation after UVC-irradiation. Furthermore, p53 stabilisation after inhibition and especially after UVC-irradiation might be due to abrogation of the HDM2-p53 degradation pathway by ribosomal proteins (RPs). Apoptosis mediated by highly activated p53 is a typical hallmark of Cockayne syndrome cells and transcriptional abnormalities and the following activation of the RP-HDM2-p53 pathway would be a possible explanation.
Assuntos
RNA Polimerase I/metabolismo , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta , Apoptose/efeitos da radiação , Linhagem Celular , Células HCT116 , Humanos , Proteínas Pol1 do Complexo de Iniciação de Transcrição/antagonistas & inibidores , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Estabilidade Proteica/efeitos da radiação , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Interferência de RNA , RNA Polimerase I/genética , RNA Ribossômico/metabolismo , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mutations in the Cockayne syndrome A (CSA) protein account for 20% of Cockayne syndrome (CS) cases, a childhood disorder of premature aging and early death. Hitherto, CSA has exclusively been described as DNA repair factor of the transcription-coupled branch of nucleotide excision repair. Here we show a novel function of CSA as transcription factor of RNA polymerase I in the nucleolus. Knockdown of CSA reduces pre-rRNA synthesis by RNA polymerase I. CSA associates with RNA polymerase I and the active fraction of the rDNA and stimulates re-initiation of rDNA transcription by recruiting the Cockayne syndrome proteins TFIIH and CSB. Moreover, compared with CSA deficient parental CS cells, CSA transfected CS cells reveal significantly more rRNA with induced growth and enhanced global translation. A previously unknown global dysregulation of ribosomal biogenesis most likely contributes to the reduced growth and premature aging of CS patients.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , RNA Polimerase I/metabolismo , Ribossomos/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Ligação a Poli-ADP-Ribose , RNA Polimerase I/genética , Precursores de RNA/biossíntese , Precursores de RNA/metabolismo , RNA Ribossômico/biossíntese , RNA Ribossômico/metabolismo , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
In addition to performing its canonical function, Telomerase Reverse Transcriptase (TERT) has been shown to participate in cellular processes independent of telomerase activity. Furthermore, although TERT mainly localizes to Cajal bodies, it is also present within the nucleolus. Because the nucleolus is the site of rDNA transcription, we investigated the possible role of telomerase in regulating RNA polymerase I (Pol I). Here we show that TERT binds to rDNA and stimulates transcription by Pol I during liver regeneration and Ras-induced hyperproliferation. Moreover, the inhibition of telomerase activity by TERT- or TERC-specific RNA interference, the overexpression of dominant-negative-TERT, and the application of the telomerase inhibitor imetelstat reduce Pol I transcription and the growth of tumour cells. In vitro, telomerase can stimulate the formation of the transcription initiation complex. Our results demonstrate how non-canonical features of telomerase may direct Pol I transcription in oncogenic and regenerative hyperproliferation.