Multi-omics-based Identification And Characterization Of Tumor Extrachromosomal DNA And Construction Of A Tumor Transcription Factor Association Database

EcDNA（Extrachromosomal DNA）is an extrachromosomal circular DNA whose discovery dates back to 1965,when researchers observed extrachromosomal pairs of ecDNAs in tumor cells.With the development of science and technology,it is found that ecDNA hardly exists in normal tissues,and ecDNA mainly exists in tumor cells or tissues.In earlier studies of Ec DNA,researchers demonstrated that ecDNA can promote tumor resistance and heterogeneity through the amplification of carried oncogenes and drug resistance genes.In a recent study,researchers found in glioblastoma GBM and prostate cancer PC3 that the Enh_ec（Enhancer on ecDNA）can affect oncogenes on other chromosomes,thereby promoting tumor development.However,the characteristics of ecDNA and Enh_ec in tumors and their regulatory mechanisms on genes have not been clearly studied.To this end,in this paper,the human breast cancer cell line MCF7,human large cell lung cancer cell line H460 and human primary myeloid leukemia cell line HL60 were used as the research objects,and multi-omics analysis was carried out on ecDNA and enhancers on ecDNA in these three types of tumors.First,through whole genome sequencing,we obtained the local amplicons in MCF7,H460,and HL60,as well as the genomic regions constituting ecDNA,and found that the ecDNAs in the three types of tumors have the characteristics of complex structures composed of multichromosomal regions.Further combining the DNase-Seq and Hi-c data of the three types of tumors and corresponding normal cell lines,we found that the genomic regions with ecDNA structure have higher chromatin openness and stronger and broader chromosomal interactions.At the same time,using h3k27ac Chip-Seq data,we identified and matched Enh_ec in tumors,and used the AME algorithm to perform differential analysis of enriched transcription factor（TF）motifs for Enh_ec and Enh_noec（enhancers on non-ecDNA）in cancer,found that 8 specific TF motifs in MCF7 were specifically enriched on Enh_ec,and 1 TF motif was specifically enriched in H460.Due to missing GRO-Seq sequencing data for H460 and HL60,we only performed e RNA expression analysis on Enh_ec and Enh_noec in MCF7 cell line,and found that Enh_ec had significantly higher levels of unstable e RNA expression in MCF7 than Enh_noec.This suggests that ecDNA may promote cancer by increasing the expression of e RNA through active enhancers.Finally,through the POLR2A Ch IA-PET data,we identified 69 target genes of Enh_ec in MCF7,and found that the expression of target genes was significantly increased compared with other genes,indicating that ecDNA in MCF7 may play a"mobile enhancer"role,promoting abnormal expression of genes.Previous studies and our results confirmed that transcription factors are involved in the regulation of ecDNA and the aggregation of ecDNA,and transcription factors have an inseparable role in tumor formation.In order to mine the transcription factors involved in tumor regulation,and to further identify the transcription factors potentially regulating ecDNA in different tumors,we developed TFcancer,a database of transcription factor expression changes,molecular structure changes,targeted gene regulation changes,and cancer progression.The above results provide reference data and theoretical methods for the study of ecDNA and ecDNA-related enhancers in cancer,as well as the study of the association between transcription factors and cancer.Of course,there are still many shortcomings in this paper.For example,the above analysis results need further experimental verification.However,because the existing functional experimental methods（such as interference or gene knockout methods）cannot effectively verify the function of elements in the ecDNA region rather than the corresponding linear chromosome region.This has also become one of the major difficulties in the experimental study of functional elements in ecDNA.To this end,this study will continue to track new ecDNA experimental methods in the future and look forward to further verification of the above results through effective experimental methods.