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In silico Analysis and Site-directed Mutagenesis of Promoters
Published: Vol 7, Iss 6, Mar 20, 2017 DOI: 10.21769/BioProtoc.2181 Views: 10145
Edited by: HongLok Lung Reviewed by: Raghuveer Kavarthapu
Original research article
The authors used this protocol in:
Mar 2016
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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.
Materials and Reagents
- 1.5 ml tubes
- 10 cm dish
- 12-well plates (Corning, Falcon®, catalog number: 353043 )
- White 96-well assay plate (Corning, catalog number: 3912 )
- Ice
- HEK293T cells (ATCC, catalog number: CRL-3216 ) at low passage
- DH5α competent cells
- Primers (Sequences of the oligonucleotide primers used for the site directed mutagenesis):
Forward 5’-CGACTTCACGGTGAATTTGGACAATCCGCAGGGGATGGAAGG-3’
Reverse 5’-CCTTCCATCCCCTGCGGATTGTCCAAATTCACCGTGAAGTCG-3’ - The promoter of interest, as for example here, the ITGA1 proximal promoter sequence inserted in the pLightSwitch_prom plasmid vector (SwitchGear Genomics, catalog number: S706788 )
- Plasmid constructs carrying the activator or repressor to be characterized, as for example here: pcDNA-Empty vector, pcDNA-MYC and pCMV-MAD, as described in Ni et al., 2005
- The pGL4.13 plasmid (luc2/SV40) as a control of transfection efficiency
- GeneArt Site-Directed Mutagenesis Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: A13282 )
- DNase and RNase free water
- Distilled water
- 0.5 M EDTA, pH 8
- SOC medium
- LB agar plates
- Trypan blue
- Effectene transfection reagent (QIAGEN, catalog number: 301425 )
- PBS
- DMEM medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11995073 )
- Fetal bovine serum (FBS) (BOLLE COMMUNICATION, WISENT, catalog number: 080-150 )
- GlutaMAX (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
- HEPES (BOLLE COMMUNICATION, WISENT, catalog number: 330-050-EL )
- Dual Luciferase Reporter Assay System (Promega, catalog number: E1980 )
Luciferase Assay substrate
Luciferase Assay buffer - DMEM culture medium for HEK293T cells (see Recipes)
- LAR II solution (see Recipes)
- 1x Stop & Glo (see Recipes)
Equipment
- Orion Microplate Luminometer (Berthold, Bad Wildbad, Germany)
- MyCycler Personal Thermal Cycler for PCR reactions (Bio-Rad Laboratories)
- Water bath
- Cell culture incubator
Software
- MatInspector web based software. Genomatix (Munich, Germany)
https://www.genomatix.de/online_help/help_matinspector/matinspector_help.html - Blast Global Align
https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=blastn&BLAST_SPEC=GlobalAln&LINK_LOC=BlastHomeLink
Procedure
Category
Molecular Biology > DNA > Mutagenesis
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