Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Abstract

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

The Effect of Single CpG Demethylation on the Pattern of DNA-Protein Binding

“…To further pursue this issue we employed DNA-affinity precipitation assay (DAPA) followed by mass spectrometry analysis to detect proteins that bound differentially to the methylated or unmethylated probes. This assay, followed by Western blot analysis, is routinely used to examine the binding of transcription factors and other proteins of interest to the analyzed sequence [ 27 ]. The combination of DAPA and mass spectrometry has been used earlier, for example, to compare protein binding to hydroxymethylcytosine and formylcytosine [ 28 ].…”

SLE non-coding genetic risk variant determines the epigenetic dysfunction of an immune cell specific enhancer that controls disease-critical microRNA expression

“…DAPA mass spectrometry. Cells (1 × 10 7 ) were first lysed with cytoplasmic extraction buffer with a final concentration of 10 mM HEPES pH 7.9, 10 mM KCl, and 0.1 mM EDTA, then were further lysed with nuclear extraction (NE) buffer with a final concentration of 20 mM HEPES pH 7.9, 0.4 M NaCl, and 1 mM EDTA to get nuclear extract 88 . Protein concentrations of nuclear lysates were detected by Bradford's method.…”

Genetics of eosinophilic esophagitis

“…Indeed, ascertaining causality in noncoding variants is non-trivial and can be dependent on specific cell types and the presence of specific inflammatory signaling pathways. 34 …”