Cloning And Functional Analysis Of Three Human Heart Developmental Candidate Genes

Cardiovascular diseases have become one of most serious diseases that threaten human beings. It is a critical prerequisite to understand the normal progress of heart development and the underlying molecular mechanisms that cause cardiovascular diseases. As it known, mistakes in spatiotemporal expression of critical genes result in congenital cardiovascular diseases. Furthermore, studies on high risk population of cardiovascular disease have also suggested the relationship between noncongenital cardiovascular diseases and genetic factors. In order to facilitate our understanding for cardiovascular development and disclose the molecular regulatory mechanisms during this progress, our laboratory performed a large scale scanning project screening and cloning heart developmental candidate genes.With the aim of identifying genes involved in human heart development and cardiovascular diseases, two zinc finger protein novel genes, ZNF325and ANKZF1b, and a PDZ gene, AHNAKc, were isolated from heart cDNA library. ZNF325 and ANKZFlb map to chromosome 7p22 and 2q36.1, respectively. They all belong to Cys2/His2 zinc finger protein family. Northern Blot indicated that ZNF325 gene is expressed in all tissues from human embryos, which is similar to the expression pattern in adult human tissues, while ZNF325 gives an increasing expression level in heart tissue. Reverse-transcription PCR analysis with human embryonic tissues shows that ANKZFlb is expressed only in heart, brain, kidney and stomach. It suggested that these two genes may have important function in heart development. In addition, pEGFP-ZNF325 and pEGFP-ANKZF1b fusion mammalian expression protein were constructed and introduced into the COS-7 cells. Observation under the fluorescent microscopy showed that both pEGFP-ZNF325 and pEGFP-ANKZF1 were expressed and existed mainly in the nucleus. Reporter gene assays showed that ZNF325 and ANKZF1b both are transcriptional repressors because overexpression of ZNF325 and ANKZF1b in the COS-7 cells inhibit the transcriptional activities of AP-1 and SRE. Further, the deletion and RNAi analysis for ZNF325 indicate that the C2H2 zinc finger motifs represent the basal transcriptional repressive activity. It is suggested that ZNF325 and ANKZF1b may probably involve in MAPK signaling pathway. Gene AHNAKc maps to chromosome 11q12.2. Observation under the fluorescent microscopy showed that pEGFP-AHNAKc was expressed and existed in the nuclear membrane as well as cytoplasm. AHNAKc belongs to a protein family containing PDZ domain which potentially engaged in protein-protein interactions and intracellular signallings. AHNAK tanscript variant 1, AHNAKa, induces actin bundling and stabilizes muscle contraction, so it is essential to address whether it involves in human heart development and cardiovascular diseases.Taken together, ZNF325, ANKZF1b and AHNAKc are three candidate genes that may involve in heart development, which need to be further performed.