Research On Regional Gene Co-expression Regulatory Element CRE30 And Its Regulatory Pattern In Cancer Genomes

Evidences emerged to support the non-random gene order in human genome,linking to gene co-expression in clusters,which is called regional co-expression.Except for sharing of transcriptional factors among paralogous gene clusters in few regions,the regulatory mechanisms in most co-expression regions remain unclear.There are also co-expression regions identified in cancer genomes,which contained large numbers of differentially expressed genes and suggested its functional roles in global dysregulation.Expression of genes in co-expression regions was significantly correlated with cancer staging and case survival,indicating the effect of such regulatory mechanism in carcinogenesis and cancer development.Exploration of the regulatory mechanism and functional element in co-expression regions will be valuable for understanding of the global expression disorder in cancer genome,as well as provide new targets for survival prediction and tumor therapy.Studies on few regions showed that long-range epigenetic modification and high-order chromatin organization might contribute to this regional regulation.However,the regulatory mechanism is still obscure,especially still lacking of evidence about functional DNA elements and multi-genome analysis.Based on transcriptome data of cancer cases in TCGA database,expressional correlation between adjacent genes was calculated and co-regulated regions(CRRs)were obtained in 8 cancers in this study.Then the inter-regional correlations were calculated among CRRs and the sub-groups of CRRs(sub-CRR)which exhibited high inter-regional correlations were classified.The similar expressional patterns among sub-CRRs indicated possible regulation mechanism might be shared.We also observed significant correlations between gene expression patterns in sub-CRRs and cancer survival,suggesting its functional roles in biology process of cancer.Recurring DNA element candidates were obtained within the sub-CRRs through sequence conservation prediction,among which the CRE30 element was significantly enriched in sub-CRR region.Segment of CRE30 was knocked-out by CRISPR-cas9 in sub-CRR95 region in endometrial and cervical cancer cell lines.CRE30 deletion resulted in significant increase of up-regulation range in sub-CRR95 under starvation stress,indicating that CRE30,as an essential element,restricted the regulatory amplitude of genes in such region.CRE30 deletion also reduced the proliferation and migration of cancer cells,suggesting the regulatory function of CRE30 in carcinogenesis progress.The CRE30 binding protein candidates were obtained by DNA pull-down assay.The chromatin conformation related factor CTCF was identified to be involved in the regulation process by protein interaction and functional enrichment analysis.Ch IP experiments were used to confirm its binding on CRE30 locus,then3 C and 3D-FISH experiments were performed and indicated that CRE30 mediates the establishment of chromatin loops and 3D architecture in sub-CRR95.While CRE30 deletion results in chromatin loops disruption and chromatin decondensation.These results suggested that CRE30 could recruit CTCF and maintain a compact chromatin architecture,which associated with gene regulation restriction in sub-CRR95.Considering the effect of epigenetic modification on chromatin structure,DNA methylation and histone modification status were altered to explore if gene expression pattern in the region was also changed accordingly.Histone acetylation was found to significantly increase the up-regulation range of genes response to starvation.The expressional changes were depended on the existence of CRE30.3C and 3D-FISH experiments confirmed that histone acetylation could lead to disruption of chromatin loops and compact chromatin structure that mediated by CRE30.Ch IP experiments also indicated that histone acetylation induced CTCF dropping on multiple locus in sub-CRR95 including CRE30 site.Considering the similar effect in gene expression and chromatin conformation that induced by CRE30 deletion and histone acetylation,such results suggested that through CTCF recruiting,CRE30 mediated the compact chromatin architecture which associated with restrict gene regulation.Histone acetylation interfered the regulatory function of CRE30 by inducing of CTCF dropping and chromatin decondensation.In conclusion,this research classified a group of co-regulation regions(defined as sub-CRRs here)exhibiting similar regulation patterns in cancer genomes.A conserved DNA element(CRE30)was identified to enrich in sub-CRRs and proved to affect regional gene expression as well as cancer cell proliferation and migration.Mechanistic analysis showed that through CTCF recruiting,CRE30 acts as an essential element to maintain chromatin loops,which restricts gene regulation in compact chromatin regions.Meanwhile,the regulatory restriction is relieved when histone acetylation disrupts CTCF binding and chromatin loops.Our research observed a new regulation pattern for regional gene co-expression that contributed by CRE30.