Publicação
Functional studies on zeb transcription factors in glioblastoma
| Resumo: | Glioblastoma (GBM) remains the deadliest primary brain tumour in adults, in part due to its highly invasive nature. Although not a classical model of epithelial-to-mesenchymal transition (EMT), recent work from our group has implicated the EMT transcription factor (TF) ZEB1 in regulating an "EMT-like" process that contributes to GBM tumour invasion. It is also known that ZEB1 works both as an activator and repressor of gene expression in various gene expression paradigms, including in GBM. Another member of the ZEB family, ZEB2, has also been implicated in GBM pathophysiology, but how its function differs from that of ZEB1, remains unclear. In this work, we focused on the role of ZEB2 in GBM, and how it compares with ZEB1. First, we compared the activity of ZEB proteins in transcriptional assays, performing reporter gene assays in transfected cells. Results show ZEB2 has repressive activity in two gene expression paradigms where ZEB1 functions as a transcriptional activator, revealing distinct features of these TFs. Next, we investigated how the two ZEB TFs compare in correlational studies using transcriptomics data from large cohorts of GBM tumours. We found both TFs to be differently expressed across different GBM subtypes. However, we discovered high inconsistency of results obtained across data sets, highlighting unexpected differences between transcriptomics databases. Last, we compared how ZEB TFs are recruited to regulatory regions of target genes in a cellular model of GBM using chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR). This showed that the opposing transcriptional activities observed are both associated with binding of each TF to regulatory regions. Moreover, the ChIP protocol established was used for preparing a ChIP-sequencing sample to compare the genome-wide binding profiles of both ZEB TFs. |
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| Autores principais: | Ramalho, Nuno Miguel Mendes Ferreira |
| Assunto: | Transcriptómicos Coortes de gbm Tumor cerebral primário Epitélio-mesênquima (emt) Genes-repórter |
| Ano: | 2020 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Algarve |
| Idioma: | inglês |
| Origem: | Sapientia - Universidade do Algarve |
| Resumo: | Glioblastoma (GBM) remains the deadliest primary brain tumour in adults, in part due to its highly invasive nature. Although not a classical model of epithelial-to-mesenchymal transition (EMT), recent work from our group has implicated the EMT transcription factor (TF) ZEB1 in regulating an "EMT-like" process that contributes to GBM tumour invasion. It is also known that ZEB1 works both as an activator and repressor of gene expression in various gene expression paradigms, including in GBM. Another member of the ZEB family, ZEB2, has also been implicated in GBM pathophysiology, but how its function differs from that of ZEB1, remains unclear. In this work, we focused on the role of ZEB2 in GBM, and how it compares with ZEB1. First, we compared the activity of ZEB proteins in transcriptional assays, performing reporter gene assays in transfected cells. Results show ZEB2 has repressive activity in two gene expression paradigms where ZEB1 functions as a transcriptional activator, revealing distinct features of these TFs. Next, we investigated how the two ZEB TFs compare in correlational studies using transcriptomics data from large cohorts of GBM tumours. We found both TFs to be differently expressed across different GBM subtypes. However, we discovered high inconsistency of results obtained across data sets, highlighting unexpected differences between transcriptomics databases. Last, we compared how ZEB TFs are recruited to regulatory regions of target genes in a cellular model of GBM using chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR). This showed that the opposing transcriptional activities observed are both associated with binding of each TF to regulatory regions. Moreover, the ChIP protocol established was used for preparing a ChIP-sequencing sample to compare the genome-wide binding profiles of both ZEB TFs. |
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