Construction And Applications Of Stable Cell Line Expressing Subunits Of Chromatin Remodeling Complex HINO80 With Flp-In System Combined With HaloTag Technology

Epigenetic refers to stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.In epigenetic studies,chromatin remodeling,which works mainly by chromatin remodeling complexes,is one of most important mechanisms that can regulate and effect the phenotype.In eukaryotic cells,these complexes can be classified into four super-families: SWI/SNF,ISWI,CHD and INO80.The INO80 chromatin remodeling complex was first identified in Saccharomyces cerevisiae,where it was shown to be composed of roughly 15 subunits.Similar with yINO80,hINO80 is an ATP-dependent multi-subunit complex belongs to INO80 superfamily.It can be divided into three modules: the Ino80 amino-terminal domain(NTD),the helicase–SANT-associated(HSA)domain and a SNF-2 ATPase included Insertion domain.The INO80 complex is able to regulate the chromatin status in two ATP-dependent ways: histone variant exchange and chromosome sliding.These can lead to changes in chromatin structure and its accessibility,which result in regulation of gene expression.In addition,INO80 can be recruited to the double strand break(DSB)sites and exchange histone variant H2 A.Z with H2 A to promote the presynaptic filament formation during homologous recombination and promote the DSB repair as a result.The Flp-In system is a system that allows integration and stable expression of your genes of interest into genome of mammalian cell lines at a specific FRT site.The system introduces a Flp recombination site(Flp Recombination Target,FRT)into genome,followed by expression vector containing genes of interest transfected.Then the vector will be integrated into genome of host cells by recombinase Flp at FRT site.Finally,in the new open reading frame,the gene of interest can be transcripted and expressed stably.Compared with other methods,the Flp-In system,it can be more efficient and stable to get the stable cell line,and permits polyconal selection of stable cell lines as well.HaloTag is a protein tag of 33 KD and is initially derived from Rhodococcus rhodochrous as dehalogenase.The HaloTag is capable of binding the alkyl residue to proteins in a covalent manner rapidly and stably.Because of the characteristics,the HaloTag can be bound to ligands for applications: bind to ligands of different colors or biotin for cell imaging;bind to biotin or affined to magnetic beads for protein purification,co-localization and chromatin immunoprecipitation(ChIP);bind to reporter or solid surface for fluorescent detection or affinity.According to previous data from our lab,hINO80 modules were raised and analysed.The data showed that knock down of several subunits that belong to HSA domain and Insertion domain respectively led to defective in ATPase and chromosome-remodeling activity or decreased chromosome recognition.But the interaction among the hINO80 subunits,their characteristics and the INO80 distribution genome-wide are still not clear,according to recent researches.In addition,the expression level of the complex is quite low in vivo,which increases the difficulty of INO80 study.Here,we used the 293 FRT cell line to construct stable cell line expressing hINO80 subunits with Flp-In and HaloTag system.The stable cell lines were then confirmed with antibiotic confirmation,western blot and immunoprecipitation(IP).Moreover,the HaloTag system was compared with the Flag-tag system in IP and was found more efficient with better purity.The project illustrated a novel method to construct stable cell line and protein purification,which provides basic methods and foundation for studying INO80 distribution genome-wide and subunits interactions.