| In this paper, we used zebrafish as a model to explore the biological functions of the heart and muscle high-expression gene POP3 in heart development. POP family was first isolated from vertebrates. Their protein size is about 300-360 amino acids, containing a highly conserved transmembrane domain, a highly conservative Popeye domain in the C-terminal, and 1-2 glycosylation sites in the N-terminal. It has been showed that the three members of the family, POP1, POP2 and POP3, are expressed in human, mice, zebrafish and xenopus, while POP2 is not expressed in chick. These genes give a higher expression in adult rat heart and skeletal muscles than that in the lung. Studies have shown that, POP 1 protein may form dimers with itself or other proteins, work as an adhesion molecule involved in cell junction, and play an important role in chick coronary blood vessels development. Although the functional studies for POP2 and POP3 have not reported, their unique expression pattern in heart and muscle suggests that they might play a prevital role in heart development.The human POP3 gene is located in chromosome 6q21, containing 4 exons and 3 introns, and up to 22kb’s wide. There are two copies of transcripts, the first is 1848 bp nucleotides that contain an ORF of 876 bp nucleotides encoding a protein of 291 amino acids with a molecular weight of 34 kDa, and the second is 1389 bp that contain a sequence dificiency in the middle of the third exon, causing nonsense-mediated mRNA degradation and no protein is formed. Bioinformatics analysis shows that POP3 contains three transmembrane domains, a Popeye domain and a cNMP-binding domain.We cloned the cDNA of the first POP3 transcript and generated polyclonal antibodies against pop3. Western blot showed that POP3 gave a high level expression in mouse heart and muscle tissue. Sub-cellular localizaion analysis suggested that POP3 is located mainly in the cytoplasm, and some in the nucleus and cell membrane. POP3 mRNA was detected mainly in heart by whole amount in situ hybridization of zebrafish embryos, and its protein is located in the nucleus, cytoplasm and membrane by the antibody staining.Using morpholino oligo-nucleotide knock-down techniques, we found that knocking down the expression of POP3 led to severe heart malformations in zebrafish embryos, including unlooping heart tube, large pericardial cavity and arrhythmia. We have also used a homozygous heart-specific GFP-positive zebrafish line Tg (nppa:gfp) to analyze heart morphology and physical function. Their heart, even the number and morphology of myocardial cells, may be analysed easily and clearly under microscope. Based on the results of the numbers of the myocardial cells analysed by biometrics software analysis, we found that the number and the measurement of the valve between the atrium and ventricle were abnormal. All these data showed that POP3 was involved in the proliferation of cardiac myocytes. However, when knocking down POP1 or POP2, the zebrafish embryos developed normally, indicating POP1 and POP2 may be of different biological functions from POP3.By immunoprecipitation from 6 month human embryo heart tissue with a polyclonal antibody against human POP3, we obtained the ATF4 protien that interacts with POP3. Although ATF4 was not reported to be involved in signaling pathways of cardiac development, it may be involved in the regulation of cell proliferation. We showed that ATF4 did interact with POP3, but not with POP1 or POP2 by co-immunoprecipitation and mammalian two-hybrid.GATA4 is one of the earliest markers of heart differentiation. Its direct downstream targets are cell cycle factors Cyclin D2 and Cdk4 and it is necessary for the proliferation of heart precursors. Western blot results showed that an obvious down-regulation of cardiac marker proteins GATA4 and NPPA was observed not only in the embryos when knocking down either POP3 or ATF4 by morpholino, but also in the cell lines when interfering by POP3 or ATF4 siRNAs. These results indicate that POP3 interacts with ATF4, and the interacting complex binds to the CRE site in the GATA4 promoter through POP3, which regulate the expression of GATA4 and its involvement in cardiac precursor cells proliferation.The other research projects that I joined during my PhD program include the interaction and expression analysis of ZNF480 and the cloning and identification of a novel gene C6orf142. Through the two-hybrid yeast system, ACTC1 was isolated that interacts with ZNF480. By RT-PCR and proteomics analysis with a C2C12 cell model, the increasing expression of ACTC1 in the ZNF480 stable cell line after induction was observed, indicating that the interaction of ZNF480 with ACTC1 plays an prevital role in muscular differentiation. I also cloned a candidate gene C6orf142. The results analysed by RT-PCR and report genes suggested that C6orf142 might be involved in heart development. |
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