Three-dimensional Chromatin Structure Regulates The Expression Of Interferon-induced Genes

With the invention and wide application of Chromosome Conformation Capture(3C)and its derivative technologies in the study of chromosome conformation,a better understanding of the three-dimensional chromatin structure has been gained.People recoganize that the three-dimensional architecture of chromatin is not random and messy,but organized and hierarchical,with fine organization at different levels such as chromosome territories,open/ closed compartments,topological associated domains and chromatin loops.Further investigation reveals that three-dimensional chromatin structure is widely involved in gene expression regulation.Gene loop structure between the promoter and terminator of certain genes ensures the transcription direction and recycling of RNA polymerase.Through long-range interactions with promoters,distal enhancers or repressors activate or repress transcritption of target genes respectively.Coodinated expression and regulation of function-related genes is mainly achieved through by the sharing of transcription factories.Topological associated domains and interactions between two insulators bracket and isolate regulatory elements from affecting the expression of genes outside.Interferons(IFNs)are broad-spectrum antiviral proteins.However,interferons themselves do not directly kill viruses,but activate the expression of enormous interferon-induced genes.Proteins expressed by interferon-induced genes inhibit various steps of virus infection including entrance,replication,transcription,translation,release etc.Though many researches have indicated that three-dimensional chromatin structure plays an important role in the transcriptional regulation of interferon genes,studies on the regulation of interferon-induced genes expression by three-dimensional chromatin structure remains elusive.Therefore,our scientific question is what roles the three-dimensional chromatin structure play in the regulation of interferon-induced genes expression.To answer the question,we focused on interferon-induced genes IFITM(Interferon-Induced Transmembrane protein)and MX1(Myxovirus resistance protein 1),and resolved their local chromosomal conformation or genomewide interactome in order to dissect their gene regulation mechanism:Part ?: Coordinated regulation of IFITM1,2 and 3 genes by an IFN-responsive enhancer through long-range chromatin interactionsHuman IFITM1,2 and 3 genes reside in chromosome 11 as a cluster.They are expressed basally in primary tissues and cell lines and robustly induced by interferons.IFITM proteins inhibit the entry of viruses into cells by block the fusion of endosomal membrane with vial envelope.They are broad-spectrum antiviral protein which inhibit many kinds of viruses such as infuenza virus,dengue virus,Ebola virus,Marburg virus,West Nile virus and severe acute respiratory syndrome coronavirus.Additionally,polymorphisms within coding and regulatory regions of IFITM genes might lead to differences in host susceptibility to disease occurance or severity of disease development.Therefore,it is of vital importance to study the transcriptional regulation of IFITM genes.However,researches on the regulation of IFITM genes expression by enhancers,which are an important class of cis-acting regulatory elements,remain exlusive.By integrating datasets of RNA polymerase II Ch IA-PET interaction,histone modification H3K4me1 and H3K27 ac,DNase I hypersensitivity sites,Ch IP-seq of transcription factor P300,STAT1 and STAT2 etc from ENCODE and other public resources and our experiment results of luciferase reporter assay,for the first time,we identified an interferon-responsive enhancer named E2-3 about 35 kb upstream of the IFITM3 promoter.After truncation of the enhancer in HEK293 cells by CRISPR,the interferon-induced expression of IFITM1,2 and 3 genes was decreased at both m RNA and protein levels,which strongly indicated IFITM1,2 and 3 were the target genes of enhancer E2-3.Normaly,interferons induce the expression of genes through JAK-STAT pathway.Using chromatin immunoprecipitation technique,we found that interferon signalling pathway related transcription factor STAT1 bound on E2-3 upon interferon treatment.In addition,luciferase reporter assay revealed that mutation of STAT1 binding motif decreased the enhancer activity of E2-3,suggesting the binding of STAT1 was essential to the function of E2-3.As the enhancer E2-3 was located 35 kb away from the IFITM locus,we speculated that it might exert its function through long-range chromatin interactions with the promoters of IFITM genes.Indeed 3C assay revealed E2-3 formed constitutive interactions with the promoters of IFITM1,2 and 3 in both HEK293 and A549 cells. Further study showed that chromatin interactions existed among the promoters of IFITM1,2 and 3,suggesting IFITM1,2 and 3 genes clusterd together and coodinatedly regulated by E2-3 through long-range interactions.Surprisingly,these interactions still existed even in E2-3-truncated HEK293 cells,indicating that the factors mediating these interactions might bind on the regions outside E2-3.Influenza virus infection triggers the expression of interferons,which then induces the expression of IFITM genes to resist the infection.Therefore,we assessed the function mediated by enhancer E2-3 in the influenza infection model.Infecting wild type and E2-3-truncted HEK293 cells by influenza virus respectively,we found that though the expression of IFITM1,2 and 3 was upregulated in both groups,the expression of IFITM1,2 and 3 genes was lower in E2-3-truncated cells than that in wild type cells.This again validated the regulation of IFITM genes by enhancer E2-3.Furthermore,when cells were treated by interferons before the infection of influenza viruses,viruses replicated more efficiently in E2-3-truncated cells,suggesting that truncation of enhancer E2-3 decreased interferon-induced resistance against influenza viruses.Collectively,our study reported the first enhancer regulating the expression of IFITM genes.This remote enhancer coordinately upregulated IFN-induced expression of IFITM1,2 and 3 genes through constitutive long-range interactions and IFN-induced recruitment of STAT1,thus contributing to IFN-induced resistance to IAV infection.These findings expanded our insights into the roles and mechanisms of enhancers in transcriptional regulation of IFITM genes and IFN-induced resistance to virus infection,which might contribute to our understanding of the complicated mechanisms underlying innate immunity,and may shed new light on the characterization of novel disease susceptibility loci and medicine targets.Part ?: Involement of the three dimensional chromatin structure in the transcriptional regulation of MX1 geneHuman MX1 gene reside in chromosome 21,and its expression can be induced by interferons.The protein encoded by MX1 gene is a GTPase and has a broad-spectrum antiviral activity.Viruses inhibited by MX1 include influenza virus,Thogoto virus,La Crosse virus,vesicular stomatitis virus,Hanta virus,hepatitis B virus and so on.Though MX1 plays such an important role in antiviral immunity,little has been studied about the transcriptional regulation mechanism of MX1,expecillay the role of the three-dimensional chromatin structure which no research have ever been conducted to our knowledge.Using Circular Chromosome Conformation Capture combined high-throughput sequencing(4C-seq)technology,we obtained the interaction profiles of MX1 promoter before and after the treatment of interferons for the first time.We found that most interactions were cis interactions,in accordance with chromosome territory.We also observed that more than half of interactions were constitutive interactions,that is,they interacted with MX1 promoter whether treatment of interferons or not.To investigate the epigenetic characteristics of the MX1 interactome,we downloaded Ch IP-seq datasets of histone modifications,histone variant and transcription factors in A549 cells,and analyzed their enchriment on the interaction fragments.Active histone modifications and variant,together with activating transcription factors were found to be highly enriched on the MX1 interaction fragments both before and after the treatment of interferons,while repressive histone modifications and transcritption factors were not.These results suggested that the promoter of MX1 gene constitutively colocalized with open chromatin.Further analyzing the MX1 interaction fragments after the treatment of interferons,we identified two fragments 3.3Mb and 210 kb upstream of MX1 gene respectively that harbored enhancer-like epigenetic characteristics.Using luciferase reporter assay,we validated the two fragments were enhancers,indicating they might increased the expression of MX1 through long-range interactions upon the interferon treatment.Furthermore,when the two enhancers were tandomly ligated together,the enhancer activity increased,suggesting that the two enhancers might function in a coordinated manner.It have been well documented that function-related genes were coodinatedly expressed by sharing the common transcription factories.Among genes interacting with MX1 identifeed from our 4C-seq data,interferon-induced genes MX2 and ITSN1 and interferon receptor genes IFNAR1,IFNAR2,IFNGR2 and IL10 RB were observed,suggesting MX1 might also colocalized with other interferon-related genes sharing transcription factories to achieve coordinated expression and regulation.Taken together,we obtained the interactomes of MX1 promoter before and after the treatment of interferons for the first time and found most interactions were cis interactions and resided in active open chromatin region,in accordance with the presence of chromosome territory and open compartment.Furthermore,we screened and identified enhancers,interferon-induced genes and interferon receptor genes from interaction profiles,suggesting three-dimensional chromatin structure like promoter-enhancer loop and transcription factory might play an important role in the transcriptional regulation of MX1 gene.All these studies will provide a clue and paradigm for further investigation of the involvement of three-dimensial chromatin structure in the transcriptional regulation of MX1 and other interferon-induced genes.