Functional & Mechanistic Study Of NcRNA-chromatin Interactions By PIRCh-seq

Non-coding RNAs(ncRNAs)are the RNA molecules that are transcribed but not translated into proteins.With the development of high-throughput sequencing technology,we have identified hundreds of thousands of non-coding RNAs expressed in mammalian cells and other species as important regulators for different biological processes.To study the functions and mechanisms of ncRNAs is one of the most frontier and hot area in biological research nowadays.In previous studies on ncRNAs' function,many ncRNAs were found regulating gene transcription through chromatin association,such as XIST,HOTAIR,MALAT1 and so on.This is considered as one the major mechanisms through which ncRNAs,especially long noncoing RNAs(IncRNAs)perform their funcions.LncRNAs can either recruit chromatin modifiers to regulate the chromatin states or directly regulate of transcription through chromosome looping to bridge distal enhancer elements to promoters.On the other hand,since the function of more than 90%of ncRNAs remain unclear,the field is also committed to developing novel ncRNA functional screening techniques at the genome-wide scale.For example,CRISPRi-based method was used to screen for functional IncRNAs in cell growth and proliferation.Despite this,systematic studies of the ncRNA-chromatin association are still lacking.To address these questions,we have developed a new method named PIRCh-seq(Profiling Interacting RNAs on Chromatin followed by sequencing).This technique can utilize different histone or histone modifier antibodies,such as H3,H3K4me3,H3K27ac,to enrich chromatin associated ncRNAs,and then using high-throughput sequencing technology to qualitatively and quantitatively analyze chromatin associated ncRNAs at the epigenomic level.With PIRCh-seq,we screen chromatin,especially histone modification specific chromatin associated ncRNAs in different cell types,systematically describe the characteristics of chromatin-ncRNA association,and in the meantime,further study the function and transcriptional regulatory mechnisms of those chromatin associated ncRNAs through novel bioinformatics algorithms and modeling.We generated and analyzed PIRCh-seq profiles in 5 different cell types(namely,HFF,H9,mESC,MEF and NPC)using antibodies targeting histone H3 and 6 distinct histone modifications(H3,H3K4me1,H3K4me3,H3K9me3,H3K27me3,H3K27ac,and H4K16ac).Compared to the Input controls without antibody,we found PIRCh-seq can accurately capture the chromatin associated ncRNAs,and different ncRNAs were enriched on histone with distinct chemical modifications.For example,XIST is one of the most critical IncRNAs in silence the tranacription of one of the chromasomes through X chromosome inactivation only significantly enriched on chromatin with H3K27me3 in female cells.By further comparing other state-of-the-art techniques in detecting chromatin-RNA association,PIRCh-seq is significantly superior in terms of reducing the primary noise signal from nascent transcript,and is very reliable with different histone modifer antibodies.Further analysis showed that,compare with mRNAs,ncRNAs were indeed highly enriched on chromatin,which account for a major proportion(more than 90%).Though a series of bioinformatics filtering,we identified 258 chromatin associated ncRNAs in mESC V6.5 cell line,most of which had not been functionally annotated.In order to study the patterns of ncRNA-chromatin association and their related functions,we performed a dimensional reduction and unsupervised clustering on top of the normalized enrichment score matrix.The results showed that there were 6 different ncRNA-chromatin association clusters,which represent 6 distinct chromatin states,such as ehancers marked by H3K4mel and H3K27ac,promoters makred by H3K4me3 and H3K27ac,and heterochromatin regions marked with H3K27me3.This result indicates that the regulatory functions of IncRNAs were realized by associating with chromatin under different states.By further comparing the expression levels of adjacent coding genes,we observed their gene expressions were highly correlated with the pattern of the corresponding lncRNA-chromatin assocaiton,suggesting that most of the lncRNAs regulating gene expression in cis.By integrating the PIRCh-seq profiles from different cell lines in mice,we found that ncRNAs associated with chromatin at a cell-type specific manner,and there were only a few ncRNAs enriched on chromatin in multiple cell lines.Interestingly,we found some ncRNAs,such as ub008bcq.1,who were enriched on both active and respressive chromatin(namely bi-chromatin enriched)in mESC,suggesting they may function as an enhancer and represser in embryonic stem cells,but were enriched on either active or repressive chromatin(namely mono-chromatin enriched)on NPC and MEF,suggesting they may function as either an enhancer or represser in more differentiated cells.In order to futher study the mechnisms of IncRNA-chromatin association,we integrated published RNA secondary structure data by icSHAPE and RNA m6A methylation profiles,and found that single stranded regions were more likely to bind to chromatin compare with double stranded regions,and RNA SNPs that alter the local RNA secondary structures will affect their abilities to associate with chromatin.In the end,to further confirm the biological function of the PIRCh-seq detected chromatin associated ncRNAs,we selected one of the ncRNAs screened from mouse neural progenitor cells,named lnc-Nr2f1.We performed Inc-Nr2fl ChIRP-seq experiment and identified its genomic biding siets on chromatin.The ChIRP-seq results and histone modification ChIP-seq results indicated that lnc-Nr2f1 does bind to the chromatin with H3K4me3 modification,which was highly consistent with our PIRCh-seq results.Further analysis of the lnc-Nr2f1 biding sited identified in ChIRP-seq suggested that it regulates neural developments,which was under further investigation.In summary,the PIRCh-seq technology developed in this study can effectively enrich chromatin-associated ncRNAs at a histone modification specific manner.This technique has important scientific significance for systematically revealing the mechanisms of ncRNA-chromatin associaiton,and provides new methods to further study the regulation mechanisms of ncRNA on gene transcription,may have broad application prospects in the field of RNA research.