Characterization of DNA binding of the two zinc finger domains of transcription factor zBED6

Sammanfattning: The zinc finger protein, zBED6, is a transcriptional regulator of IGF2 along with hundreds of other genes relating to development and growth. Studies on the growth of commercially bred pigs discovered a single nucleotide substitution in the third intron of IGF2 which disrupts the binding of zBED6 and is responsible for the three-fold upregulation of IGF2 in skeletal muscle. The mutation is linked to decreased subcutaneous fat deposition, larger organ size, and increased skeletal muscle mass. Three different constructs of the zBED6 protein made by Björklund 2018 were expressed and purified to characterize their binding affinity, where one contained both zinc finger domains and two of the constructs contained only one zinc finger domain each. Electrophoretic mobility shift assay protocol was optimized to determine the apparent Kd (= 210 ± 31nM) for the full-length construct C13 and to determine which zinc finger domain was sensitive to the mutation in the IGF2 gene. The first zinc finger domain seems to be more specific in its binding target. Preliminary microscale thermophoresis results were highly variable, needing further optimization of the protocol in order to obtain a full binding curve. The next steps involve site directed mutagenesis of residues binding DNA to determine which interactions are the most significant and possibly crystallization studies as well.