Studies On Interaction Between HPRG1 Protei N And The TGF? Signaling Members In Zebrafish

Heart,which plays a very important role,is one of the earliest organs of vertebrate development.Its development process is often controlled by a variety of genes.The occurrence of heart disease is often associated with the abnormal expression of these genes.In cardiac development,TGF? signaling pathway plays a crucial role.Previous studies show that the zebrafish HPRG1 gene plays an important role in early morphogenesis of heart and has mutual effect with Smad protein.This suggests that HPRG1 may be regulated by the TGF? / Smad signaling pathway for cardiac development.As its mechanism still being unclear till now,And most studies have shown that the promoter region of the gene is located within the first 3 kb sequence of the start ATG of a gene(with a few gene exceptions).this paper explores the interaction between zebrafish HPRG1 protein and the TGF? signaling member,and the effect on transcriptional expression of the TGF? signaling member.In order to analyze the interaction and transcriptional of zebrafish HPRG1 protein to TGF? family members,this paper has conducted following researches:(1)Firstly,collect about 200 zebrafish embryos that developed for 24 hours,isolate the chromatin,and conducte experiment of Chromatin immunoprecipitation analysis of the interaction situation with TGF?1a,TGF?2,TGF?3,TGF?r1a,TGF?r2,TGF?r3,Smad1,Smad2,Smad3 a,Smad4,Smad5,Smad6 a,bmp2b,bmp4.And the cardiac development transcription factor:nkx2.5,GATA4,ISL1 gene promoter.The preliminary results shows that all of them attached.(2)Using the MEME website to analyze the conserved binding domains on the 3Kb promoter of each gene: The above-mentioned chromatin immunoprecipitation(CHIP)technique showed that the HPRG1 protein could bind to the 3Kb promoter of each member of the TGF? signaling family The promoters of these members present a conserved binding region of the HPRG1 protein.In order to prove this hypothesis,we used the MEME website to analyze the common conserved binding domain on the 3Kb promoter of each gene.It was found that there were three common conserved binding domains on the 3Kb promoter of each member of the TGF? signaling family,Suggesting that HPRG1 protein may be able to bind to the conserved binding domain of each member of the TGF? signaling family,but its true conserved binding domain remains to be further studied.(3)Build the HPRG1 overexpressing vector pcmv-myc-HPRG1 and fluorescent reporter plasmid :PGL3-TGF?1a?PGL3-TGF?r1a-1?PGL3-TGF ? r1a-2 ? PGL3-TGF ? r2 ? PGL3-Smad1 ? PGL3-Smad2 ?PGL3-Smad3a? PGL3-Smad6a?PGL3-bmp4?PGL3-GATA4,Put the Fluorescent Reporter Plasmids,and HPRG1 overexpressing vector plasmids Co-transfection into HEK293 T cells.after 48 hrs,when each group plasmids was transfected with liposome transfection reagent,the cells are collected.Cleaving cells with lysis buffer and analyzing performed the single luciferase reporter.The results show HPRG1 gene can be inhibited the express of TGF?1a?TGF?r2?Smad3a?GATA4?bmp4?TGF?r1a-2,while it can activate the expression of TGF?r1a-1.In conclusion,the above experiments show that HPRG1 gene can interaction with majority of members of TGF? signal family And inhibits or activates the transcriptional expression of the TGF? family members.