The Role Of Zfp296 In Pluripotent Stem Cell Maintenance

Zinc finger gene family is the largest one of human gene families,and also widely distributes in plants,animals,and microbiology,participating in biological processes like gene expression regulation,cell differentiation and embryonic development,ect.Recently,many zinc finger proteins related to somatic reprogramming have gradually been found,such as Zfp281,Zfp42 and Zfp57,etc.Zfp296 belongs to Krüppel C2H2 zinc finger protein family,which is composed of 445 amino acids,including two zinc finger elements.Zfp296 is expressed in pluripotent cells and germ cells specifically,acting as an essential component of pluripotent transcriptional networks.Zfp296 thus is often used as a marker of undifferentiated ESCs and reprogrammed cells.On this basis,this paper aims to further explore the role of Zfp296 in pluripotent stem cell maintenance.The main contents are listed as follows:1.Primarily,we proved that Zfp296 is localized to the nucleus by carrying out immunofluorescence staining in NIH/3T3 and F9 cell lines.Next,via q RT-PCR analysis,we detected the effect of Zfp296 expression change on key pluripotent factors,epigenetic factors and three germ layer marker factors in F9 EC cells.We discovered that overexpression Zfp296 could promote the expression levels of pluripotent factors like Klf4,Sox2,Oct4 and Nanog;influence the expression of epigenetic factors,for instance,Tet1,Tet2 and Dnmt3a;enhance the expression level of mesodermal marker factors,such as T and Bmi1;reduce the expression levels of endodermal marker factor,ectodermal marker factor and trophic layer marker facor,such as Gata4,Fgf5 and Cdx2.Besides,knockdown of Zfp296 can depress the expression levels of pluripotent factors like Eras,Oct4 and Klf4;promote the expression levels of endodermal marker factor,ectodermal marker factor and trophic layer marker factor,for example,Gata4,Fgf5 and Cdx2.2.Dual Luciferase Reporter Assay,q RT-PCR and Western Blotting were utilized to explore the upstream regulation of Zfp296 gene.Finally,we found some reprogramming-related molecules and factors could affect the expression of Zfp296 at levels of m RNA transcription and protein translation,as well as the acticity of Zfp296 promoter.Based on these,we furtherdiscovered that Zfp296 is involved in LIF/STAT3 and Wnt/β-catenin signaling pathways,exhibiting a novel role as their common downstream target.3.Subsequently,we investigate the effect of Zfp296 on the core reprogramming factors,we focused on looking for pluripotent factors which have specific regulation relationship with Zfp296,especially for protein factors interacted with Zfp296.Through searching for the GEO Profiles,we found Zfp296 is highly expressed in the period of zygotic activation.Accordingly,we further screened some pluripotent factors whose high expression period is same with Zfp296,such as Nanog,Oct4,Dppa3 and Tet3;we also screened some factors that exhibit adnormal localization and functional deficits in the process of zygotic activation,such as Dnmt1,Uhrf1 and G9a.Indeed,q RT-PCR and Western blot experiments supported the conclusion that Zfp296 enhances the expression of Nanog at levels of m RNA transcription and protein translation.Additionally,the zinc finger protein reduced the expression of Uhrf1 at levels of m RNA transcription and protein translation.Subsequently,we demonstrated that ectopic overexpression of Zfp296 can significantly enhance the activity of Nanog promoter through Dual Luciferase Reporter assay.Co-immunoprecipitation also suggested Zfp296 inherently interacted with Nanog and Uhrf1,respectively.Results aboved show that Zfp296 is one of the key Nanog-interacted proteins with functions as its positive regulator,but the biological significance of the interaction between Zfp296 and Uhrf1 also remains to be further digging.In summary,this study demonstrated that Zfp296 acts multiple roles in the pluripotent stem cell networks.Zfp296 is downstream target of both LIF/STAT3 and Wnt/β-catenin signaling pathways;on the other hand,it is significantly associated with Nanog and Uhrf1.Moreover,our study extends the knowledge of pluripotent stem cell networks,providing new cues and insight for the researches of pluripotency regulation of embryonic stem cells.