Screening Of Proteins Interacting With PITX2by Yeast Two-Hybrid System

Atrial fibrillation (AF) is the most common cardiac arrhythmia seen in hospitals andcauses significant morbidity and mortality worldwide. AF has a high heritability of62%,indicating that genetic factors are critical to the initiation and development of AF. However,the majority of AF-causing genes and AF susceptibility genes have not been uncovered yet.In2007, the first genome-wide association study (GWAS) identified one polymorphic locuson chromosome4q25(SNP rs2200733) associated with AF in several independent studiesof European and Asian populations. This locus is intergenic, close to PITX2gene, whichencodes a homeobox transcription factor of the paired type. Further functional studiessupport the hypothesis that dysregulation of PITX2expression can be responsible forsusceptibility to AF, and firmly establish PITX2as new susceptibility genes for AF.Due to its critical importance in cardiac physiology and human heart disease, studiesto define the regulatory proteins and other components of PITX2would be of great interest.For this purpose, we performed a conventional yeast two-hybrid screen to identify proteinsthat interact with PITX2in the heart. The full-length PITX2cDNA sequence (purchasedfrom ORIGENE Company) was amplified by polymerase chain reaction (PCR) method andwas cloned into the pGBKT7bait plasmid which was used as the bait in yeast two-hybridscreening, After the bait plasmid pGBKT7-PITX2was transformed into yeast to examine itsexpression, toxicity and autoactivation, the plasmid was then transformed into the yeaststrain AH187and was used to mate with pretransformed human heart cDNA library in yeaststrain Y109according to the protocol. Positive clones growth on the auxotrophic mediumwere picked up for X-β-gal chromogenicreaction analysis. Finally we obtained286positiveclones.100potential positive clones were sequenced and characterized by Blast analysisagainst the NCBI database. Two potential positive clones from the screening wereidentified which encode the portion of WDR6(Homo sapiens WD repeat-containing protein6) and ITLN1(Homo sapiens intelectin1(galactofuranose binding)).In conclusion, to investigate the role of PITX2in the heart, we identified two potentialcandidate PITX2-interacting protein by using a yeast two-hybrid screen. Further studies areneeded to confirm the interaction between WDR6/ITLN1and PITX2, if these two proteins are really physically interacting with PITX2, we will also investigate the physiological andthe pathophysiological roles of these specific interactions.