Studies On New Pygo Signaling Pathway In Adult Drosophila

The wingless/Wnt signaling pathway plays critical roles in the regulation of cardiac development and dysfunction. The pygo gene is a new member of the canonical Wnt(wingless) signaling pathway and it was reported to be a regulator in cardiac development through Wnt signaling. Our Research Center showed that knockdown of pygo’s expression severely blocked adult heart physiological activities, however, knockdown of Wnt / beta-catenen-TCF signals was significantly weaker phenotype or even has non- phenotype. Reminding that pygo functions on adult heart were independent of Wnt signaling.But on the other,TCF-like mediators may cooperate with this function, since a TCF dominant negative mutation(TCF-DN) severely blocked adult heart physiological activities and performed the similar phenotype as pygo.Thus, what we focus on is to explore the new molecular regulation of pygo by using the model of drosophila.In order to explore the molecular regulation mechanism of pygo in Drosophila, we mainly used the method of ChIP-Seq for the identification new targets of pygo in the regulation of adult cardiac function. ChIP-Seq is a method which combined the chromatin immunoprecipitation and high-throughput sequencing. Firstly, we verified the important function of pygo in the maintenance of adult cardiac function by using the adult cardiac function analysis system. Secondly we constructed cardiac specificly over-expressed UAS-HA-pygo strain by the driving of hand 4.2 Gal4. Testing HA antibody effectiveness by using immunofluorescence assay method, we detected the expression of pygo in drosophila’s heart. We did chromatin immunoprecipitation assay by a series of procedures, such as stripped drosophila’s heart, cross linked with formaldehyde and ultrasonic, broken into small fragments of a certain length, took a part of chromatin as input, and others for HA antibody and IgG antibody, dispergation, purified of DNA. Also, tested the quality of DNA library and is enough for high-throughput sequence. After sequencing, we disposed the data with multi-level and multi-way, including Peaks detected by MACS, through MACS method we tracked 799 important genome characteristic peak, and classified its genetic structure. Peak structural and functional annotation by using the newest UCSC RefSeq database, promoter-centered annotation, GO analysis. By analysis three duplicate samples intersect promoter peak, we found more than 120 symbol gene. Also we analysis pathway signals of peaks gene from three duplicate samples. Based on those preliminary assessment we decided that more than 20 genes as candidate genes which might regulated by Pygo protein. At present, the further identification of candidate genes is in progress.