Protocol for evaluating the impact of non-coding variants on transcription factor binding and gene expression
Edwin G Peña-Martínez1, Jessica M Rodríguez-Ríos1, Jean L Messon-Bird1
1Department of Biology, University of Puerto Rico-Río Piedras, San Juan, PR 00931, USA.
STAR Protocols
|June 6, 2025
Summary
This protocol evaluates how non-coding variants affect transcription factor (TF)-DNA binding and gene expression. It details methods for assessing TF binding affinity and gene regulation changes.
Area of Science:
- Genetics
- Molecular Biology
- Bioinformatics
Background:
- Non-coding genetic variants can influence gene expression by altering transcription factor (TF) binding to DNA.
- Understanding these regulatory mechanisms is crucial for deciphering disease-associated genetic variations.
Purpose of the Study:
- To present a comprehensive protocol for assessing the functional impact of non-coding variants on TF-DNA interactions.
- To provide a method for evaluating how these variants affect gene expression.
Main Methods:
- Protocol involves expressing and purifying specific DNA-binding proteins.
- Preparation of DNA probes and reporter gene constructs.
- Measurement of alterations in TF-DNA binding affinity and subsequent gene expression changes.
Main Results:
- The protocol enables the quantitative assessment of TF-DNA binding affinity changes.
- It allows for the measurement of gene expression dysregulation caused by non-coding variants.
Conclusions:
- This protocol offers a robust framework for investigating the functional consequences of non-coding variants.
- It facilitates the study of genetic variant impact on gene regulation and TF binding dynamics.
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