Identification and quantitative characterization of protein-DNA interactions with the native holdup assay: GATA1 regulation of the plasma membrane calcium pump

Polymorphisms in regulatory regions of the genome can alter RNA and protein expression and are implicated in a wide range of diseases. It has been recently discovered that a common haplotype in the erythroid-specific promoter of the ATP2B4 gene alters the expression of Plasma Membrane Calcium ATPase 4 (PMCA4) in red blood cells and reduces malaria susceptibility. Identification of transcription factors that bind to such regions can be challenging and is of particular interest. Here, we introduce the DNA-native holdup assay, a versatile method that allows proteome-wide identification of proteins binding to short DNA fragments. It is a quantitative retention assay that can measure altered binding affinity to different polymorphic DNA sequences. Using this approach, we present direct evidence that GATA1 is the transcription factor whose binding is most affected by the haplotype in the erythroid-specific promoter of the ATP2B4 gene. Furthermore, we demonstrate that this method is suitable for the convenient biophysical characterization of DNA binding of different mutant versions of transcription factors, allowing the rapid evaluation of mutations found in patients. Analyzing the DNA binding properties of six different mutants of the GATA1 protein leading to severe anemia in humans revealed altered DNA-binding properties for R307C and for the short version (GATA1s) lacking the N-terminal transactivation domain (N-TAD) of the protein. This method provides a simple quantitative approach to characterize DNA-protein interactions, and to assess the effect of polymorphic DNA variants or mutations in transcription factors on DNA binding.