Screening And Identification Of The Heart Development Related Gene Foxp4’s Interacting Proteins

Our laboratory has previously obtained 15 candidates interacting with the cardiac development gene Foxp4 by using yeast two hybrid system, and has studied 3 of them. In this article, we continued to identify the Foxp4 interacting proteins from the other 12 proteins, which will provide interesting information for the research of Foxp4 on signal pathway involved in heart development.First, we cloned 12 candidate genes, including Actal, C17orf81, ABHD11, PTP4A3, RMND5B, TNXB, HLA-DPB1, Loc100288161, COL1A1, COL3A1, COL4A and FN1. We prepared cDNA from total RNA extracted from human adult muscle and heart tissues for PCR amplification. The target genes were acquired by nested PCR and cloned into pMD18T vector. After analyzed by restriction enzyme digestion and sequencing, the target fragments were cloned into eukaryotic expression vector pCMV-Myc. The results showed that the 12 genes and their eukaryotic expression plasmids were successfully cloned and constructed.Next, each of the 12 genes’ pCMV-Myc plasmids was co-transfected into Hek293 cells with pCMV-Tag2c-Foxp4 separately for co-immunoprecipitation (CO-IP) experiment undertaken from two sides with anti-Flag and anti-Myc. The results showed that 3 proteins of them, Actal, COL1A1 and COL4A2, interact with Foxp4. We also prepared the polyclonal antibody of zebrafish Foxp4 gene, and detected Foxp4’s expression in zebrafish embryo and adult tissues by western blot. The result indicated that Foxp4 started to express from 1-cell stage and its expression level was gradually increased, while decreased at the blastocyst stage, and increased at 90% epiboly again. In zebrafish adult tissues, Foxp4 was expressed in the brain, heart, muscle, eyes, and gills, with a stronger expression in the brain, muscle and gill, while slightly weaker in the heart and eyes.In addition, we further studied another candidate gene Fbxl5’s expression pattern, and analyzed its effect on other genes when knocking down it in the zebrafish by gene chip. We cloned the zebrafish Fbxl5 gene by overlap PCR, prepared its polyclonal antibody and detected its expression in zebrafish embryo and adult tissues. The results indicated that Fbxl5 stated to express at 1-cell stage, and its expression became stronger, then slightly weaker and stronger again in the Blastocyst stage up to 24 h stage. In zebrafish adult tissues, Fbxl5 expressed in brain, heart, kidney, gill, muscle and intestine, with a stronger expression in gill, while relatively weaker in the muscle and intestinal. Fbxl5’s expression was knocked down by Fbxl5-MO in Zebrafish embryos, and then the mRNA’s expression in the embryos at 48 h were analyzed by gene chip. The results indicated that 1874 out of the 14295 genes were detected with changes at mRNA level, including the cardiac marker genes tnc, vmhc, myl7 and so on.Totally speaking, we have cloned 12 candidate genes and found 3 of them, Actal, COL1A1 and COL4A2 interacted with Foxp4 by CO-IP test. We also prepared a Foxp4 polyclonal antibody and analyzed its expression in zebrafish embryos and adult tissues, which laid a base for further studies on its molecular mechanism. Also the results of the candidate gene Fbxl5 obtained will provide a base for further studies on its function.