RESUMO
High activity of GLI family zinc finger protein 2 (GLI2) promotes tumor progression. Removal of the repressor domain at the N terminus (GLI2∆N) by recombinant methods converts GLI2 into a powerful transcriptional activator. However, molecular mechanisms leading to the formation of GLI2∆N activator proteins have not been established. Herein we report for the first time that the functional activities of GLI2 are parted into different protein isoforms by alternative promoter usage, selection of alternative splicing, transcription initiation and termination sites. Functional studies using melanoma cells revealed that transcriptional regulation of GLI2 is TGFbeta-dependent and supports the predominant production of GLI2∆N and C-terminally truncated GLI2 (GLI2∆C) isoforms in cells with high migratory and invasive phenotype. Taken together, these results highlight the role of transcription and RNA processing as major processes in the regulation of GLI2 activity with severe impacts in cancer development.
Assuntos
Processamento Alternativo , Melanoma/genética , Invasividade Neoplásica/genética , Proteínas Nucleares/genética , RNA/genética , Ativação Transcricional , Proteína Gli2 com Dedos de Zinco/genética , Linhagem Celular Tumoral , Humanos , Melanoma/metabolismo , Melanoma/patologia , Invasividade Neoplásica/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismoRESUMO
Changes in alternative splicing have been linked to cancer development. We hypothesized that changes occurring in tumor tissue can also be detected in the peripheral blood of cancer patients leading to discovery of blood biomarkers of breast cancer. Alternative splicing profiles of 94 genes were examined in cancerous breast tissue. Discriminating splice variants were analyzed in the peripheral blood of early stage (BCI/II) (stage I-II; n = 26), neoadjuvant receiving locally advanced breast cancer patients (LABC) (stage IIb-IIIa, b; n = 10) and healthy volunteers (n = 26) using qRT-PCR analysis. Changes in marker expression during neoadjuvant therapy were analyzed at 15 timepoints. High expression of REST-N50, the alternatively spliced variant of REST, was detected in the blood of LABC patients but not in BCI/II and healthy controls (p = 0.0032 and p = 0.0029, respectively). Expression levels of DOPEY1v2, the alternative splice variant of DOPEY1, in the blood could differentiate cancer from healthy controls (p = 0.024) and discriminate between patient groups (BCI/II vs LABC, p = 0.002). Positive response to neoadjuvant therapy of REST-N50-positive LABC patients correlated with a decrease in REST-N50 levels (p < 0.0001). Assessment of REST-N50 and DOPEY1v2 may prove useful in diagnostic blood tests of breast cancer. REST-N50 shows a high potential as a blood biomarker for evaluating the effectiveness of therapy in the neoadjuvant setting.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Proteínas do Tecido Nervoso/genética , Proteínas Repressoras/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo/genética , Biomarcadores Tumorais/sangue , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Estadiamento de Neoplasias , Proteínas Repressoras/sangueRESUMO
Transcription factor IID (TFIID) activity can be regulated by cellular signals to specifically alter transcription of particular subsets of genes. Alternative splicing of TFIID subunits is often the result of external stimulation of upstream signaling pathways. We studied tissue distribution and cellular expression of different splice variants of TFIID subunit TAF4 mRNA and biochemical properties of its isoforms in human mesenchymal stem cells (hMSCs) to reveal the role of different isoforms of TAF4 in the regulation of proliferation and differentiation. Expression of TAF4 transcripts with exons VI or VII deleted, which results in a structurally modified hTAF4-TAFH domain, increases during early differentiation of hMSCs into osteoblasts, adipocytes and chondrocytes. Functional analysis data reveals that TAF4 isoforms with the deleted hTAF4-TAFH domain repress proliferation of hMSCs and preferentially promote chondrogenic differentiation at the expense of other developmental pathways. This study also provides initial data showing possible cross-talks between TAF4 and TP53 activity and switching between canonical and non-canonical WNT signaling in the processes of proliferation and differentiation of hMSCs. We propose that TAF4 isoforms generated by the alternative splicing participate in the conversion of the cellular transcriptional programs from the maintenance of stem cell state to differentiation, particularly differentiation along the chondrogenic pathway.
Assuntos
Processamento Alternativo , Diferenciação Celular/genética , Condrogênese/genética , Células-Tronco Mesenquimais/citologia , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/genética , Adipócitos/citologia , Ciclo Celular/genética , Proliferação de Células , Regulação da Expressão Gênica/genética , Humanos , Especificidade de Órgãos , Osteoblastos/citologia , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Proteínas Wnt/metabolismoRESUMO
The SWItch/Sucrose NonFermentable, a nucleosome remodeling complex (SWI/SNF) chromatin-remodelling complexes act upon the nucleosomal structure and regulate transcription, replication, repair of chromatin and splicing. In this study, we present evidence that human, mouse and rat genes encoding one of the SWI/SNF complex subunits, BAF57, undergo neuron-specific splicing of exons II, III and IV. Alternative splicing yields in at least three isoforms of BAF57 protein that have truncated N-termini (N-BAF57s). The transcripts encoding N-BAF57 isoforms are predominantly expressed in the nervous system. The biochemical fractionation data supported by the results of the co-immunoprecipitation analysis show that N-BAF57 isoforms associate into protein complexes together with Brg1, Brm, BAF155 and BAF170. Transient over-expression of N-BAF57 isoforms in non-neural cells affects the level of expression of certain neuron-restrictive silencer element-containing genes. Together these data suggest that neuronal isoforms of BAF57 contribute to functional SWI/SNF complexes regulating neurogenesis.