Study On The Effect Of HiGC Motif On Gene Cooperative Expression In CYHR1 And JAG2 Regions

The research team used a large amount of transcriptome data of disease populations in the early stage and found that some regions of the human genome have the phenomenon of simultaneous expression of neighboring genes.Since then,bioinformatics prediction and analysis of different cancer genomes have been carried out.By setting strict screening methods and standards,Coordinately Regulated Regions(CRRs)that are widely present on each chromosome have been obtained.The research team conducted a correlation analysis between CRRs in different cancer tissues,and screened out members that may share regulatory mechanisms from the cooperative expression regions of endometrial cancer.We categorized the latter and named them sub-sets of cooperative expression regions(Sub CRRs).The study uses MEME suit,GLAM2 and other tools to predict and enrich conservative sequences in endometrial cancer Sub CRRs,and finally obtain a class of significantly enriched high GC conservative sequences,which are named hiGC motifs by our team.Previous studies have confirmed that the HSF1-hiGC motif in the 8q24.3 region of endometrial cancer exerts its regulatory function by recruiting CTCF to influence chromatin conformation.The expression differences of Sub CRRs containing hiGC motifs can better classify cancer into different subtypes,suggesting that the regional cooperative expression mediated by the hiGC motif may be of great significance for cancer prevention and treatment.Therefore,in this study,in order to verify whether the hiGC motif exerts the same biological regulatory function in other CRRs,two cooperative expression regions of CYHR1(8q24.3)and JAG2(14q32.33)have been newly identified in Sub CRRs of endometrial cancer.In this study,a gene editing system based on the CRISPR-Cas9 lentiviral system was designed and constructed to knock out the hiGC motifs in the two regions.Quantitative PCR(q RT-PCR)was used to detect the changes of gene expression in the regions before and after the conservative motifs were knocked out.Studies have shown that in endometrial cancer cells,the CYHR1-hiGC motif can regulate the coordinated expression of genes in the region;After the hiGC motif was deleted,the up-regulation of genes in the region before and after the induction of starvation increased,and the up-regulation of multiple gene loci was significantly higher than that of the wild-type control group.Subsequently,the regulatory mechanism mediated by the CYHR1-hiGC motif was studied.Ch IP-PCR experiments confirmed that the conserved motif can influence the chromatin conformation by recruiting CTCF to regulate the cooperative expression of genes in the region;knock out the hiGC motif or increasing the level of histone acetylation at hiGC motif sites can reduce the combination of hiGC motifs and CTCF,so that the chromatin packaging in the region tends to be looser,and the up-regulation of gene expression in the region increases significantly.In addition,the study also evaluated the cell proliferation and migration ability before and after the CYHR1-hiGC motif knockout,and found that the conservative motif knockout can significantly reduce the proliferation and migration ability of cancer cell lines.In the study of the JAG2 cooperative expression region,since this sequence occupies part of the exons,we hope that the function of the gene will not be affected after gene editing.Hi GC motif was selected to be partially knocked out.Quantitative PCR was also used to detect the conservative motif before and after knockout.The results showed that regardless of whether the hiGC motif was knocked out or not,the gene expression in the JAG2 region changed very little before and after the induction of starvation,and there was no synergistic up-regulation.Ch IP-PCR was used to detect the binding of this conserved motif to CTCF,and the results showed that in wild-type Ishikawa cells,hiGC motifs can also recruit CTCF to exert its regulatory function on genes in the region,while the partial knockout of the JAG2-hiGC motif does not affect the recruitment of CTCF,which may be the reason why partial knockout of this motif does not cause the synergistic up-regulation of gene expression in the region.In summary,based on the Sub CRRS database of previous endometrial cancer,the coexpression regions of CYHR1 and JAG2 were newly screened in this study,revealing the function and mechanism of the effect of HIGC motif in the region on gene co-expression in the region regulated by chromatin conformation by recruiting CTCF.The regulatory functions performed in the genome range provide strong evidence.