Protocol for evaluating the impact of non-coding variants on transcription factor binding and gene expression

Abstract

Non-coding variants can alter transcription factor (TF)-DNA binding and dysregulate gene expression. Here, we present a protocol for evaluating the impact of non-coding variants on TF binding and gene expression. We describe steps for expressing and purifying DNA-binding proteins, preparing DNA probes and reporter constructs, and measuring changes in TF-DNA binding affinity. Finally, we provide instructions for quantification and statistical analysis. For complete details on the use and execution of this protocol, please refer to Peña-Martínez and Messon-Bird et al.¹.

Keywords: Cell Biology; Gene Expression; Genomics; Protein Biochemistry; Protein expression and purification.

Graphical abstract

Figure 1

Purification of recombinant GATA DNA-binding domain (DBD) with hexahistidine tag

Figure 2

Generating fluorescently labeled double-stranded DNA through primer extension reaction

Figure 3

Cloning genomic sequences centered on variant into the reporter construct Steps for (A) Linearization of pGL4.23, (B) amplification of 60 bp variant sequences and addition of homologous regions, and (C) Gibson assembly cloning are detailed in the Step-by-step methods section.

Figure 4

Example gel shift assay to evaluate TF-DNA binding Lanes used for reference DNA are represented in blue, and alternate DNA in orange.

Figure 5

Quantifying TF-DNA binding from gel image Areas used for pixel quantification are represented with blue dashed squares. Bound fractions are indicated with odd numbers and unbound with even numbers. The area used for subtracting background is #15. This figure was generated from Figure 4.

Figure 6

Binding curves of reference and alternate sequences This binding curve was generated using gel from Figure 4. Error bars represent variation from experimental triplicates. Figure re-used from: Peña-Martínez, E.G. et al, Cardiovascular disease-associated non-coding variants disrupt GATA4-DNA binding and regulatory functions, Copyright Elsevier (2025). Permission license number: 6015491253169.

Figure 7

Quantifying luciferase activity on 96-well plates Luminescent measurements are performed in triplicates for reference (blue rectangle) and alternate (orange rectangle) alleles. Experimental design should also include triplicate controls that only contain reporter plasmids for luciferase activity normalization.

Figure 8

Quantifying changes in gene expression between reference and alternate sequences Error bars represent variation from experimental triplicates. Significance was determined by un-paired t-test, ∗∗∗ p<0.001. Figure re-used from: Peña-Martínez, E.G. et al, Cardiovascular disease-associated non-coding variants disrupt GATA4-DNA binding and regulatory functions, Copyright Elsevier (2025). Permission license number: 6015491253169.