Abstract
In normal as in cancerous cells, gene expression is tightly regulated by transcription factors, which are responsible for up- or down-regulation of thousands of targets involved in different cell processes. Transcription factors can directly regulate the expression of genes by binding to specific DNA sequences known as response elements. Identification of these response elements is important to characterize targets of transcription factors in order to understand their contribution to gene regulation. Here, we describe In silico analysis coupled to selected mutagenesis and promoter gene reporter assay procedures to identify and analyze response elements in the proximal promoter sequence of genes.
Keywords: Site-directed mutagenesis, in silico analysis, Gene expression, Promoter gene-reporter assay, Response elements, Transcription factors
Background
The impact of a transcription factor on global gene expression can be studied through its knockdown using shRNA or CRISPR-Cas9 methods followed by microarray analysis which can provide significant data on hundreds of dysregulated genes. This analysis, although very useful, lacks information about the direct control of the dysregulated genes. To further investigate these genes, In silico analysis using bioinformatics provides additional information to identify direct targets of the transcription factor studied. Additionally, the functionality of these response elements can be analyzed using site-directed mutagenesis and promoter-gene-reporter assays. We have shown recently that the oncogenic transcription factor MYC (Cellular Myelocytomatosis Oncogene) controls the expression of the ITGA1 (integrin alpha 1 subunit) gene in colorectal cancer, and that their expression correlates in 72% of colorectal tumors. This protocol describes a procedure for the analysis of the ITGA1 promoter and the identification of response elements for the MYC oncogene. The latter is known to be a member of the MYC/MAX/MAD network. Interactions between these factors lead to gene activation or repression depending on upstream signalization and cell condition.
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Acknowledgments
We thank Elizabeth Herring for reviewing the manuscript. This work was supported by the Canadian Institute of Health Research Grant MOP-123415 (JFB is a member of the Centre de Recherche of the Centre Hospitalier Universitaire de Sherbrooke funded by the Fonds de la Recherche du Québec-Santé. This protocol was adapted from Boudjadi et al. (2016), originally published in Oncogene.
References
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