The Study Of RNA Mediated Chromatin Interaction

Nucleus is the largest organelle in eukaryotic cells,and is the storage site of genome,and it is a kind of subcellular structure with strict regionalization and high heterogeneity.Chromatin is the form of genome,the largest complex structure in cells,and the common carrier of genetic and epigenetic information.Chromatin is mainly composed of DNA,RNA,histone and non histone.The chromatin,which is about two meters long,is compressed into the nucleus with diameter of about 10?m after a series of folding.The folding process of chromatin is highly ordered.Firstly,DNA entangled histone octamer forms nucleosomes,then four nucleosomes are one unit and folded to form left-hand spiral structure.The spiral structure is further orderly compressed,and finally forms the primary structure of 10 nm chromatin,the second-order structure of30 nm chromatin and the staining of MB scale,including different types of structural regions Quality level 4 or advanced structure.3C(chromosome formation)technology invented by Job Dekker in 2002 provides us with strong support for us to understand the advanced structure and interaction of chromatin under higher resolution conditions.Then,a series of 3C derivations 4C,5C,Hi-C and other technologies have been developed,and more comprehensive technical techniques are provided for the study of chromatin advanced structure.It has been found that chromatin has more fine high-level conformation,which successively provides chromatin interaction ring(LOOP),sub topology associated domain,structural domain(including topological associated domain),lamina associated domain,nuclear nucleolus associated domain,chromatin domain,etc Territories,etc.It is found that non coding RNA plays an important role in the formation of chromatin three-dimensional structure.Long-chain noncoding lncrna can mediate the interaction between chromatin and protein or other RNA.For example,the noncoding RNA Xist is tightly wrapped on the X chromosome expressing itself,and then it is condensed and compressed into a close inactivation state by a series of DNA methylation and histone modification grade linkage reaction.In order to explore the overall effect of noncoding RNA on chromatin interaction,we designed different strategies to remove RNA by artificial addition of RNase in vitro and transient induction of RNase expression in vivo.Meanwhile,Hi-C technology was combined to systematically and comprehensively explore the role of RNA,especially lncrna in chromatin interaction and the formation of three-dimensional advanced structure.In the strategy of RNase removal in vitro,after cell cross-linking,cleavage and Hi-C experiment,the effects of RNA on chromatin interaction were investigated by comparing the data of RNase A treatment and Hi-C data.Gm12878 was used as the material,and the whole genome interaction(rank sum test P < 0.05),the decay index IDE,the chromatin interaction difference,TAD and boundary strength,and the room and difference of a and B were carried out respectively by Hi-C experiment and a series of quality control analysis of high-throughput sequencing data(Wilcox test).The results showed that the Hi-C library was of good quality whether RNase A was treated or not,and the proportion of the end matched fragment,effective action segment,effective comparison fragment and unique matched fragment were 97.92% vs.98.43%,95.35% vs.95.09%,82.09% vs.74.10%,74.65% vs.73.52%,which met the quality control requirements.After RNase treatment,the long-distance chromatin interactions increased,and the short-distance interactions decreased.This result suggests that RNA participates in the construction and maintenance of the local structure of chromatin,and may also participate in the regulation of gene expression;considering the looseness of the local structure of chromatin,the strength of long-distance chromatin interactions will increase,which may influence the whole structure of chromatin.This implies that nuclear RNA plays an important role in maintaining specific long-distance interactions and the overall structure of the genome.Our results also show that although RNA in the nucleus has an effect on the three-dimensional high-level chromatin structure in RNase treatment in vitro,the effect is relatively weak in general,but it is only on X chromosome.Because our experiment is to cross-link,then use RNase treatment in vitro,degrade RNA coupled with chromatin to investigate the effect of chromatin coupled RNA on chromatin interaction.Essentially,it is an in vitro experiment.Most of the chromatin interactions in the cross-linking process,especially those near chromatin interactions,have been fixed.So before and after RNase treatment,the near-range chromatin interaction should be studied Not much has changed.On the contrary,some long-distance,RNA mediated interactions are affected,so chromosomes generally become loose.Because RNA plays an important role in the highly condensed and compressed inactivated X-chromosome,even after cross-linking,RNase treatment will still lead to obvious changes in its interaction.Therefore,our experimental results are in line with the expectations and are consistent with the reports of foreign countries.After RNase treatment in vitro,we found that chromatin coupled RNA plays an important role in chromatin interaction.Then we try to establish a genetic system in vivo which can be adjusted by RNase activity in vivo,so as to activate RNase activity and degrade most of the RNA in cell before cross-linking.Then,we can cross-link and fix it again,so as to investigate the effect of RNA on chromatin structure in vivo.Based on this,we successfully constructed the eukaryotic expression vector pteone DD RNase A,which is regulated by small molecule shld-1 and derived from FK506,and expressed by tetracycline.In this way,DD RNase A can be expressed,stable and play the role of RNA degradation only in the presence of DOX and shld-1,so that the r before cross-linking can be realized Na degradation was instantaneous.On this basis,the construction of cell line was carried out by the slow virus transfection.At present,the multiclonal strains have been obtained.The expression of RNase A and cell activity were detected by q PCR,CCK8 was used to detect the cell viability,and the apoptosis of each group was detected by flow cytometry.The final results showed that RNase A m RNA expression in DOX and DOX + shield I treatment group was up regulated;the apoptosis of cells in DOX + shield I treatment group increased,cell activity decreased,and the cell line was successfully constructed.