Analysis Of Zinc Finger Protein Binding Sites Of Special-Primate Gene ZNF480 On Myogenin Promoter

The zinc finger motifs of KRAB-related zinc finger constitute the largest individual family of transcription factors in mammals as a result of rapid increasing and also frequently lost. The repetitive characterize and the structural flexibility of the zinc finger motifs makes these zinc finger proteins recognize the DNA binding sites much more precisely and specifically, thus zinc finger proteins fuction diversity in adaptive evolution.According to statistics, in 742 human distinct proteins and 341 pseudogene sequences, 423 proteins are KRAB-related zinc finger proteins, approximately 30% of KRAB-related zinc finger proteins are primate-specific. We identified 159 primate-specific KRAB-related zinc finger proteins in public databases. According to every motif figure analysis of these primate-specific zinc finger proteins, KRAB-A-B occupies the biggest part in these KRAB-related zinc finger proteins, and the rest KRAB- related zinc finger proteins contian KRAB-B, KRAB-BL, KRAB-b, KRAB-C motifs or SCAN motifs.Compared the activity of every motifs in transcriptional activity and MAPK signal pathway, results indicate that KRAB motifs is always conserved in its repressive role, while C2H2 motifs shows diversity role both in primate-specific and non primate-specific KRAB-related zinc finger proteins. Both KRAB motifs and C2H2 motifs can function independently, and the coordinated function of these motifs make the whole protein functions diversity.Our previous results showed ZNF480 regulated myogenin in dose-dependent manner.But how did ZNF480 regulated myogenin is unknown. Analysis myogenin promoter by Genomatix wedsite,we find 7 potential zinc finger protein binding sites(named ZBPF1～7) in human myogenin promoter,and 6 in ratter myogenin peomoter,5 in mus myogenin promoter. The position of 5 mus ZBPF sites is at the same sites in human and ratter myogenin promoter. However, human ZBPF1 site have no corresponding sites in ratter and mus myogenin promoter, this binding site shows low conserved in 3 species. However, human ZBPF3, ratter ZBPF2 and mus ZBPF1 site is highly conserved in 3 species.Our EMS A assay show, ZNF480 could bind to human ZBPF 1 and ZBPF2 sites. According to the nucleotides differences of human ZBPF1 in 3 species, we further analysis the binding effect of human ZNF480 to every nucleotide mutation of human ZBPF 1 site. EMSA assay shows,3 nucleotides are conserved nucleotides, whichever mutation of 3 nucleotides lead to human ZNF480 could not bind to human ZBPF1 sites.HEK293 line and ratter C2C12 line EMSA assay show, human ZNF480 could not bind to human ZBPF3, human ZBPF4, ratter ZBPF2 and mus ZBPF1 sites.To sum up, primate-specific human zinc finger protein ZNF480 may regulate myogenin throung binding to 2 zinc finger binding sites in myogenin promotor.