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• W3146078726 abstract "•ecDNAs show intense chromatin connectivity and are in contact with chromosomal DNA•Chromosomal ecDNA contacts are associated with transcriptional activity•Oncogenes are co-localized within the multivalent, aggregated ecDNA-chromatin hubs•ecDNA functions as mobile regulatory elements leading to synthetic aneuploidy Extrachromosomal, circular DNA (ecDNA) is emerging as a prevalent yet less characterized oncogenic alteration in cancer genomes. We leverage ChIA-PET and ChIA-Drop chromatin interaction assays to characterize genome-wide ecDNA-mediated chromatin contacts that impact transcriptional programs in cancers. ecDNAs in glioblastoma patient-derived neurosphere and prostate cancer cell cultures are marked by widespread intra-ecDNA and genome-wide chromosomal interactions. ecDNA-chromatin contact foci are characterized by broad and high-level H3K27ac signals converging predominantly on chromosomal genes of increased expression levels. Prostate cancer cells harboring synthetic ecDNA circles composed of characterized enhancers result in the genome-wide activation of chromosomal gene transcription. Deciphering the chromosomal targets of ecDNAs at single-molecule resolution reveals an association with actively expressed oncogenes spatially clustered within ecDNA-directed interaction networks. Our results suggest that ecDNA can function as mobile transcriptional enhancers to promote tumor progression and manifest a potential synthetic aneuploidy mechanism of transcription control in cancer. Extrachromosomal, circular DNA (ecDNA) is emerging as a prevalent yet less characterized oncogenic alteration in cancer genomes. We leverage ChIA-PET and ChIA-Drop chromatin interaction assays to characterize genome-wide ecDNA-mediated chromatin contacts that impact transcriptional programs in cancers. ecDNAs in glioblastoma patient-derived neurosphere and prostate cancer cell cultures are marked by widespread intra-ecDNA and genome-wide chromosomal interactions. ecDNA-chromatin contact foci are characterized by broad and high-level H3K27ac signals converging predominantly on chromosomal genes of increased expression levels. Prostate cancer cells harboring synthetic ecDNA circles composed of characterized enhancers result in the genome-wide activation of chromosomal gene transcription. Deciphering the chromosomal targets of ecDNAs at single-molecule resolution reveals an association with actively expressed oncogenes spatially clustered within ecDNA-directed interaction networks. Our results suggest that ecDNA can function as mobile transcriptional enhancers to promote tumor progression and manifest a potential synthetic aneuploidy mechanism of transcription control in cancer. Extrachromosomal, circular DNA (ecDNAs) are extrachromosomal circular chromatin elements that frequently carry oncogenes (Cox et al., 1965Cox D. Yuncken C. Spriggs A.I. Minute chromatin bodies in malignant tumours of childhood.Lancet. 1965; 1: 55-58Abstract PubMed Scopus (151) Google Scholar; Spriggs et al., 1962Spriggs A.I. Boddington M.M. Clarke C.M. Chromosomes of human cancer cells.Br. Med. J. 1962; 2: 1431-1435Crossref PubMed Scopus (67) Google Scholar; Turner et al., 2017Turner K.M. Deshpande V. Beyter D. Koga T. Rusert J. Lee C. Li B. Arden K. Ren B. Nathanson D.A. et al.Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.Nature. 2017; 543: 122-125Crossref PubMed Scopus (257) Google Scholar; Verhaak et al., 2019Verhaak R.G.W. Bafna V. Mischel P.S. Extrachromosomal oncogene amplification in tumour pathogenesis and evolution.Nat. Rev. Cancer. 2019; 19: 283-288Crossref PubMed Scopus (105) Google Scholar; Wu et al., 2019Wu S. Turner K.M. Nguyen N. Raviram R. Erb M. Santini J. Luebeck J. Rajkumar U. Diao Y. Li B. et al.Circular ecDNA promotes accessible chromatin and high oncogene expression.Nature. 2019; 575: 699-703Crossref PubMed Scopus (136) Google Scholar). First described as “double-minutes” in the karyotypes of cancer cells by microscopic imaging (Cox et al., 1965Cox D. Yuncken C. Spriggs A.I. Minute chromatin bodies in malignant tumours of childhood.Lancet. 1965; 1: 55-58Abstract PubMed Scopus (151) Google Scholar), ecDNAs exist as extrachromosomal, histone-packaged chromatin bodies and are thought to be a mode of gene amplification associated with in vitro drug resistance (Alt et al., 1978Alt F.W. Kellems R.E. Bertino J.R. Schimke R.T. Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells.J. Biol. Chem. 1978; 253: 1357-1370Abstract Full Text PDF PubMed Google Scholar; deCarvalho et al., 2018deCarvalho A.C. Kim H. Poisson L.M. Winn M.E. Mueller C. Cherba D. Koeman J. Seth S. Protopopov A. Felicella M. et al.Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.Nat. Genet. 2018; 50: 708-717Crossref PubMed Scopus (111) Google Scholar; Nathanson et al., 2014Nathanson D.A. Gini B. Mottahedeh J. Visnyei K. Koga T. Gomez G. Eskin A. Hwang K. Wang J. Masui K. et al.Targeted therapy resistance mediated by dynamic regulation of extrachromosomal mutant EGFR DNA.Science. 2014; 343: 72-76Crossref PubMed Scopus (313) Google Scholar; Xu et al., 2019Xu K. Ding L. Chang T.C. Shao Y. Chiang J. Mulder H. Wang S. Shaw T.I. Wen J. Hover L. et al.Structure and evolution of double minutes in diagnosis and relapse brain tumors.Acta Neuropathol. 2019; 137: 123-137Crossref PubMed Scopus (37) Google Scholar). More recently, ecDNAs have been found to be common in primary cancers (Kim et al., 2020Kim H. Nguyen N.P. Turner K. Wu S. Gujar A.D. Luebeck J. Liu J. Deshpande V. Rajkumar U. Namburi S. et al.Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers.Nat. Genet. 2020; 52: 891-897Crossref PubMed Scopus (79) Google Scholar; Turner et al., 2017Turner K.M. Deshpande V. Beyter D. Koga T. Rusert J. Lee C. Li B. Arden K. Ren B. Nathanson D.A. et al.Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.Nature. 2017; 543: 122-125Crossref PubMed Scopus (257) Google Scholar) and to constitute a bona fide mechanism and adaptive reservoir for oncogene amplification (Kohl et al., 1983Kohl N.E. Kanda N. Schreck R.R. Bruns G. Latt S.A. Gilbert F. Alt F.W. Transposition and amplification of oncogene-related sequences in human neuroblastomas.Cell. 1983; 35: 359-367Abstract Full Text PDF PubMed Scopus (488) Google Scholar). ecDNAs can rapidly accumulate in cancer cells through uneven segregation (Verhaak et al., 2019Verhaak R.G.W. Bafna V. Mischel P.S. Extrachromosomal oncogene amplification in tumour pathogenesis and evolution.Nat. Rev. Cancer. 2019; 19: 283-288Crossref PubMed Scopus (105) Google Scholar), which offers a competitive advantage in response to selective pressures in the tumor microenvironment and in response to cytotoxic therapeutic agents (deCarvalho et al., 2018deCarvalho A.C. Kim H. Poisson L.M. Winn M.E. Mueller C. Cherba D. Koeman J. Seth S. Protopopov A. Felicella M. et al.Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.Nat. Genet. 2018; 50: 708-717Crossref PubMed Scopus (111) Google Scholar; Xue et al., 2017Xue Y. Martelotto L. Baslan T. Vides A. Solomon M. Mai T.T. Chaudhary N. Riely G.J. Li B.T. Scott K. et al.An approach to suppress the evolution of resistance in BRAF(V600E)-mutant cancer.Nat. Med. 2017; 23: 929-937Crossref PubMed Scopus (94) Google Scholar). Rapid fluctuation in ecDNA levels as a result of disjointed inheritance patterns (deCarvalho et al., 2018deCarvalho A.C. Kim H. Poisson L.M. Winn M.E. Mueller C. Cherba D. Koeman J. Seth S. Protopopov A. Felicella M. et al.Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.Nat. Genet. 2018; 50: 708-717Crossref PubMed Scopus (111) Google Scholar) likely contributes to the mechanism of tumor evolution. While the presence of ecDNAs and their structure information are extensively characterized (deCarvalho et al., 2018deCarvalho A.C. Kim H. Poisson L.M. Winn M.E. Mueller C. Cherba D. Koeman J. Seth S. Protopopov A. Felicella M. et al.Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.Nat. Genet. 2018; 50: 708-717Crossref PubMed Scopus (111) Google Scholar; Sanborn et al., 2013Sanborn J.Z. Salama S.R. Grifford M. Brennan C.W. Mikkelsen T. Jhanwar S. Katzman S. Chin L. Haussler D. Double minute chromosomes in glioblastoma multiforme are revealed by precise reconstruction of oncogenic amplicons.Cancer Res. 2013; 73: 6036-6045Crossref PubMed Scopus (67) Google Scholar; Turner et al., 2017Turner K.M. Deshpande V. Beyter D. Koga T. Rusert J. Lee C. Li B. Arden K. Ren B. Nathanson D.A. et al.Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.Nature. 2017; 543: 122-125Crossref PubMed Scopus (257) Google Scholar; Xu et al., 2019Xu K. Ding L. Chang T.C. Shao Y. Chiang J. Mulder H. Wang S. Shaw T.I. Wen J. Hover L. et al.Structure and evolution of double minutes in diagnosis and relapse brain tumors.Acta Neuropathol. 2019; 137: 123-137Crossref PubMed Scopus (37) Google Scholar), the mechanism(s) by which ecDNAs are deployed to modulate tumor growth and to contribute to cancer drug resistance is not yet well understood. Their open and accessible chromatin features, together with co-amplified enhancers demonstrated by recent studies (Koche et al., 2020Koche R.P. Rodriguez-Fos E. Helmsauer K. Burkert M. MacArthur I.C. Maag J. Chamorro R. Munoz-Perez N. Puiggros M. Dorado Garcia H. et al.Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma.Nat. Genet. 2020; 52: 29-34Crossref PubMed Scopus (84) Google Scholar; Morton et al., 2019Morton A.R. Dogan-Artun N. Faber Z.J. MacLeod G. Bartels C.F. Piazza M.S. Allan K.C. Mack S.C. Wang X. Gimple R.C. et al.Functional enhancers shape extrachromosomal oncogene amplifications.Cell. 2019; 179: 1330-1341.e13Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar; Wu et al., 2019Wu S. Turner K.M. Nguyen N. Raviram R. Erb M. Santini J. Luebeck J. Rajkumar U. Diao Y. Li B. et al.Circular ecDNA promotes accessible chromatin and high oncogene expression.Nature. 2019; 575: 699-703Crossref PubMed Scopus (136) Google Scholar), implicate a regulatory function beyond serving as vehicles for oncogene amplifications. Inside the nucleus, chromosomes are extensively folded into chromatin loops that occupy distinct chromatin territories (Cremer and Cremer, 2010Cremer T. Cremer M. Chromosome territories.Cold Spring Harb. Perspect. Biol. 2010; 2: a003889Crossref PubMed Scopus (730) Google Scholar). Such highly organized three-dimensional (3D) chromatin conformation provides a topological basis for many genome functions, including transcription, by bringing distal regulatory elements and their targeted genes into close spatial proximity (Sexton and Cavalli, 2015Sexton T. Cavalli G. The role of chromosome domains in shaping the functional genome.Cell. 2015; 160: 1049-1059Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). The spatiotemporal organization of these chromatin interactions is critical in maintaining normal cell state and function (Bertolini et al., 2019Bertolini J.A. Favaro R. Zhu Y. Pagin M. Ngan C.Y. Wong C.H. Tjong H. Vermunt M.W. Martynoga B. Barone C. et al.Mapping the global chromatin connectivity network for Sox2 function in neural stem cell maintenance.Cell Stem Cell. 2019; 24: 462-476.e466Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar; Ngan et al., 2020Ngan C.Y. Wong C.H. Tjong H. Wang W. Goldfeder R.L. Choi C. He H. Gong L. Lin J. Urban B. et al.Chromatin interaction analyses elucidate the roles of PRC2-bound silencers in mouse development.Nat. Genet. 2020; 52: 264-272Crossref PubMed Scopus (38) Google Scholar). Our existing knowledge of 3D chromatin organization has been largely restricted to the 23 pairs of chromosomes. Little is known about the chromatin organization of extrachromosomal DNA elements and its impact on genome-wide expression regulation. Previous analysis of a set of unique glioblastoma (GBM)-derived neurosphere cultures using whole-genome sequencing (WGS), and computational and cytogenetic image approaches detected multiple ecDNAs harboring oncogenes including EGFR, MYC and CDK4 (deCarvalho et al., 2018deCarvalho A.C. Kim H. Poisson L.M. Winn M.E. Mueller C. Cherba D. Koeman J. Seth S. Protopopov A. Felicella M. et al.Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.Nat. Genet. 2018; 50: 708-717Crossref PubMed Scopus (111) Google Scholar). ecDNAs are also frequently observed in many cancer cell models including prostate cancer cell line PC3 (Wu et al., 2019Wu S. Turner K.M. Nguyen N. Raviram R. Erb M. Santini J. Luebeck J. Rajkumar U. Diao Y. Li B. et al.Circular ecDNA promotes accessible chromatin and high oncogene expression.Nature. 2019; 575: 699-703Crossref PubMed Scopus (136) Google Scholar). Here, we applied the ChIA-PET (chromatin interaction analysis by paired-end tag sequencing) and ChIA-Drop (chromatin interaction analysis with droplet sequencing) technologies (Tang et al., 2015Tang Z. Luo O.J. Li X. Zheng M. Zhu J.J. Szalaj P. Trzaskoma P. Magalska A. Wlodarczyk J. Ruszczycki B. et al.CTCF-mediated human 3D genome architecture reveals chromatin topology for transcription.Cell. 2015; 163: 1611-1627Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar; Zheng et al., 2019Zheng M. Tian S.Z. Capurso D. Kim M. Maurya R. Lee B. Piecuch E. Gong L. Zhu J.J. Li Z. et al.Multiplex chromatin interactions with single-molecule precision.Nature. 2019; 566: 558-562Crossref PubMed Scopus (98) Google Scholar) to characterize both general spatial chromatin organization and RNA polymerase II (RNAPII)-mediated long-range chromatin interactions on the same ecDNAs. We demonstrate that known ecDNAs are readily recognizable through a pattern of dense intra- and intermolecular genome-wide chromatin contacts. Importantly, in deciphering the RNAPII-mediated ecDNA connectomes and their chromosomal partners, we discovered an association between ecDNA and actively expressed chromosomal genes. Their contact regions share the key characteristics of super-enhancers known to drive high-level transcription of oncogenes in many tumor cells (Loven et al., 2013Loven J. Hoke H.A. Lin C.Y. Lau A. Orlando D.A. Vakoc C.R. Bradner J.E. Lee T.I. Young R.A. Selective inhibition of tumor oncogenes by disruption of super-enhancers.Cell. 2013; 153: 320-334Abstract Full Text Full Text PDF PubMed Scopus (1702) Google Scholar). Our data suggest that ecDNAs, beyond manifestation of oncogene amplification, function as mobile transcription-amplifying elements in human cancers. We reasoned that, in contrast to the subnuclear compartments occupied by large chromosomes, the small and circular nature of an extrachromosomal chromatin body enables its mobility within the nucleus and potentially establishes chromosomal interactions. To explore ecDNA-associated chromatin conformation, we performed a chromatin immunoprecipitation (ChIP)-free ChIA-PET analysis, similar to the Hi-C procedure (Dixon et al., 2012Dixon J.R. Selvaraj S. Yue F. Kim A. Li Y. Shen Y. Hu M. Liu J.S. Ren B. Topological domains in mammalian genomes identified by analysis of chromatin interactions.Nature. 2012; 485: 376-380Crossref PubMed Scopus (3743) Google Scholar), on three GBM-patient-derived neurosphere cell lines (Figure 1A). Two of the three neurosphere lines were ecDNA(+) (HF-2354, HF-2927) and one line was ecDNA(−) (HF-3035) (deCarvalho et al., 2018deCarvalho A.C. Kim H. Poisson L.M. Winn M.E. Mueller C. Cherba D. Koeman J. Seth S. Protopopov A. Felicella M. et al.Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.Nat. Genet. 2018; 50: 708-717Crossref PubMed Scopus (111) Google Scholar). We previously reported that HF-2927 harbored a chr7p11/EGFR containing ecDNA (deCarvalho et al., 2018deCarvalho A.C. Kim H. Poisson L.M. Winn M.E. Mueller C. Cherba D. Koeman J. Seth S. Protopopov A. Felicella M. et al.Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.Nat. Genet. 2018; 50: 708-717Crossref PubMed Scopus (111) Google Scholar), referred to as ecEGFR, whereas HF-2354 contained a chr8q24/MYC ecDNA, referred to as ecMYC. Genomic regions amplified on these ecDNAs and their defined copy numbers in their respective cell lines are summarized in Table S1. Hi-C revealed general chromatin contacts within spatial topologically chromatin-associated domains in these cell lines (Figure S1A). Moreover, from the chromatin contact maps, chromosomal structural variants frequently observed in GBM such as deletions of PTEN, and CDKN2A and CDKN2B, on chromosomes 10q23 and 9p21, were readily visualized as an elimination of chromatin interactions (Figure S1B). We also detected a 600-kb deletion of the chrX:31.4–32 Mb common fragile site (Ma et al., 2012Ma K. Qiu L. Mrasek K. Zhang J. Liehr T. Quintana L.G. Li Z. Common fragile sites: genomic hotspots of DNA damage and carcinogenesis.Int. J. Mol. Sci. 2012; 13: 11974-11999Crossref PubMed Scopus (46) Google Scholar) involving the DMD gene in HF-2927, a 15-Mb extensive rearrangement of chr3:168–183 Mb, and a double translocation event of 3.5 and 11.5 Mb between chr3 and chr6 in HF-2354 genomes (Figure S1C). As shown on the chromatin contact heatmaps, both ecDNA loci exhibited extensive contacts across the entire genome (Figure 1B), suggesting a widespread ecDNA connectivity from the extrachromosomal genetic elements. To quantify the degree of chromatin contacts between chromosomal regions, we developed a metric that faithfully describes the average genome-wide trans-chromosomal interaction frequencies (nTIF) normalized across all 23 chromosomes at 50-kb resolution. We observed a significant enrichment (one-sided Wilcoxon rank-sum test with p values <5 × 10−9 in both lines) of the nTIFs specifically in the ecDNA-amplified regions (Table S2). To remove the effects resulted from the high number of copies and verify that the elevated nTIFs were specific to the circular and extrachromosomal conformation nature of the ecDNAs, we used an established linear regression model (Seaman et al., 2017Seaman L. Chen H. Brown M. Wangsa D. Patterson G. Camps J. Omenn G.S. Ried T. Rajapakse I. Nucleome analysis reveals structure-function relationships for colon cancer.Mol. Cancer Res. 2017; 15: 821-830Crossref PubMed Scopus (20) Google Scholar; Wu and Michor, 2016Wu H.J. Michor F. A computational strategy to adjust for copy number in tumor Hi-C data.Bioinformatics. 2016; 32: 3695-3701Crossref PubMed Scopus (27) Google Scholar) to adjust for the impact of copy number (CN) on nTIFs (adjusted nTIFs as adjnTIFs). We found that the adjnTIFs mediated by ecMYC and ecEGFR remained significantly higher after CN normalization (one-sided Wilcoxon rank-sum test with p values <5 × 10−9 in both lines) (Figures 1B and 1C). The adjnTIFs contributed by each ecDNA copy (adjnTIFs/CN) were also significantly higher (median adjnTIFs/CN were ∼0.3–1.1) than per copy of each chromosome (median adjnTIFs/CN were ∼0.2) in both ecDNA(+) lines (one-sided Wilcoxon rank-sum test with p values 1 × 10−3 to 6 × 10−32) (Figure S1D). Therefore, we conclude that ecDNAs exhibit extensive chromatin connectivity and that the elevated contact frequency of ecDNA across the genome is driven by its autonomous capacity. Based on the high level of RNA expressed from genes amplified within the ecDNAs (Figure S2A), we reasoned that ecDNA is highly associated with the RNAPII complex within the active chromatin domains and establishes specific chromosomal interactions which may exert unique regulatory activities that contribute to gene transcription regulation. Therefore, we applied the ChIA-PET assays to pull down RNAPII-associated chromatin and characterized ecDNA-associated chromatin interactomes, including both intra-ecDNA and ecDNA-chromosome interactions (Figures 1A and S2B). In addition to the above three lines, we also included HF-3016 and HF-3177, two neurosphere lines derived from a primary and a recurrent GBM from the same patient (deCarvalho et al., 2018deCarvalho A.C. Kim H. Poisson L.M. Winn M.E. Mueller C. Cherba D. Koeman J. Seth S. Protopopov A. Felicella M. et al.Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.Nat. Genet. 2018; 50: 708-717Crossref PubMed Scopus (111) Google Scholar). In both lines, three genes were found to be amplified extrachromosomally (Figure S2C), of which chr7p11/EGFR and ch12q14.1/CDK4 were demonstrated to be co-amplified on ecDNA while chr8q24/MYC was also found on ecDNA (referred to respectively as ecEGFR, ecCDK4, and ecMYC). We detected sharp elevation in ecMYC, ecEGFR, and ecCDK4 adjnTIF levels as well as cross-interactions between the three loci in HF-3016 and HF-3177 (Figures 2A and 2B ), suggesting that the dominant ecDNA in these lines carries all three oncogenes or that they have a close intermolecular proximity. Similar to the adjnTIFs observed in the Hi-C data, we observed significantly higher adjnTIFs in the ecDNA regions (median adjnTIFs were ∼ 4–24) than the chromosomes (median adjnTIFs were ∼0.2–0.4) in HF-3016 and HF-3177 as well as the HF-2927 and HF-2354 ecDNA(+) lines (one-sided Wilcoxon rank-sum test with p values 5 × 10−9 to 9 × 10−76) but not in the HF-3035 ecDNA(−) cells (median adjnTIF was 0.2) (Figure 2C). Compared with the chromosomal amplified regions (CNs ranged from 5 to 15), which are expected to be constrained within the chromosomal territories, these extrachromosomally amplified regions also exhibited significantly higher adjnTIFs (median adjnTIFs between 4 and 24 versus 0.7–1.8, one-sided Wilcoxon rank-sum test with p values <8 × 10−8 in all four lines). Table S2 summarizes CN-adjusted ecDNA-chromosomal interaction frequencies and statistical analyses. The enrichment in the chromosomal interactions from ecDNA elements associated with RNAPII binding suggests that ecDNA molecules have an important transcriptionally regulatory function. To address how RNAPII-mediated ecDNA-chromatin interactions associated with transcriptional regulation, we performed RNAPII binding and H3K27ac modification ChIP-sequencing (ChIP-seq) profiling to mark active promoters and transcriptional enhancers in these cells. To correct the bias resulting from CN variation, we generated all ChIP-seq data through a unique molecular identifier approach (Kivioja et al., 2011Kivioja T. Vaharautio A. Karlsson K. Bonke M. Enge M. Linnarsson S. Taipale J. Counting absolute numbers of molecules using unique molecular identifiers.Nat. Methods. 2011; 9: 72-74Crossref PubMed Scopus (553) Google Scholar). Furthermore, sequencing data from input (no-ChIP) libraries was used to normalize the CN variation in each of the four ecDNA(+) cells. The sequencing summary and resulting peaks are summarized in Table S1 and Figure S2B. From the RNAPII tethered and CN-normalized chromatin contacts mapped within the known ecDNA regions, we observed that ecDNA regions exhibit 5- to 17-fold enrichment in CN-normalized cis-interaction frequencies compared with the cis-interaction frequencies of their corresponding native chromosomal regions in ecDNA(−) cells (Figure 3A and Table S2). These high-frequency intra-ecDNA interactions including distinct pairs of loops and foci of intense contacts were observed in the 530-kb ecEGFR region in HF-2927, and between the two segments of the ecMYC region in HF-2354 (Figure 3B) as well as in the ecMYC, ecEGFR, and ecCDK4 from the paired primary HF-3016 and recurrent HF-3177 lines (Figure S3A). We confirmed that such high intra-ecDNA interaction frequencies were not caused by potential tandem duplications in the native chromosomal loci. Based on the optical mapping of ultra-long DNA molecules through the BioNano Saphyr platform (Mak et al., 2016Mak A.C. Lai Y.Y. Lam E.T. Kwok T.P. Leung A.K. Poon A. Mostovoy Y. Hastie A.R. Stedman W. Anantharaman T. et al.Genome-wide structural variation detection by genome mapping on nanochannel arrays.Genetics. 2016; 202: 351-362Crossref PubMed Scopus (89) Google Scholar), these loci showed no evidence of local amplification (data not shown). Thus, such intense cis-chromatin interaction patterns are collectively derived from contacts between the native chromosomal loci, different ecDNA molecules, and folding within individual ecDNAs. While we cannot unambiguously differentiate their origins due to their near-identical sequences, we expect that most of the interactions observed were derived from the ecDNA molecules because their CNs far exceed the copies of chromosomal alleles and likely reflected the clustering of ecDNA molecules mediated by RNAPII binding. The vast majority (92% and 89%) of the intra-ecDNA loops detected in HF-3016 and HF-3177 were unique, presumably because the structures subjected to extrachromosomal amplification were highly variable (Figure S2C). In total, there were 220, 271, 587, and 455 RNAPII-mediated chromatin interactions between ecDNAs and their chromosomal partners defined in HF-2354, HF-2927, HF-3016, and HF-3177, respectively (Figure S2B). The contact sites on ecDNAs and their chromosomal targets were mostly enriched at promoters (defined as transcription start site ±2.5 kb) (Figures 3C and S3B). The affinity between ecDNAs and chromosomal promoters was also independently confirmed by Hi-C, which detected significant contacts between ecDNAs and the chromosomal gene promoters (binomial test p value <3 × 10−30). We next examined the potential trans-regulation of the three oncogenes amplified on ecDNA by evaluating their chromosomal interaction regions. From the promoters amplified on the ecDNAs, we detected 9, 20, 68, and 67 non-coding interacting regions on chromosomes in HF-2354, HF-2927, HF-3016, and HF-3177 cell lines, respectively. They exhibited high levels of H3K27ac enrichment and overlaps (56%–95%) with H3K27ac peaks in their respective cell lines (Figure 4A), suggesting that the transcription of the oncogenes on ecDNAs is further enhanced by engaging chromosomal enhancers through chromatin contacts. Similarly, we also observed co-occurrence of high-frequency contact foci and H3K27ac peaks within ecDNAs (Figures 3B and S3A). Within the 530-kb ecEGFR in HF-2927, H3K27ac peaks aligned consistently with high interaction frequency regions (Figure 4B), suggesting enhancer signals accumulated on ecDNA-chromatin contact sites. In comparison with H3K27ac peaks of the chromosomal EGFR region in HF-3035 ecDNA(−) cells, the H3K27ac peaks on ecDNAs exhibited higher enrichment and broader spans. H3K27ac immunostaining on metaphase HF-2927 cells also confirmed overlapping H3K27ac and DAPI (4′,6-diamidino-2-phenylindole) ecDNA signals, further validating the association between enhancer function and ecDNA (Figure S3C). These findings confirm previous report of an enhancer function for ecDNA sequences (Morton et al., 2019Morton A.R. Dogan-Artun N. Faber Z.J. MacLeod G. Bartels C.F. Piazza M.S. Allan K.C. Mack S.C. Wang X. Gimple R.C. et al.Functional enhancers shape extrachromosomal oncogene amplifications.Cell. 2019; 179: 1330-1341.e13Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar; Wu et al., 2019Wu S. Turner K.M. Nguyen N. Raviram R. Erb M. Santini J. Luebeck J. Rajkumar U. Diao Y. Li B. et al.Circular ecDNA promotes accessible chromatin and high oncogene expression.Nature. 2019; 575: 699-703Crossref PubMed Scopus (136) Google Scholar) and extend those by providing their regulatory genes targeted by the active ecDNA-chromosome interactions. We next investigated the chromosomal contact regions on the ecDNA molecules. We found that the majority of the contact sites on the ecDNAs were converged onto only a few distinct (7–26) loci and shared high overlaps with H3K27ac peaks in each of the four ecDNA(+) lines (Figure S3D). These few regions, while only accounting for 1%–2.4% of the ecDNA sizes, mediated 17%–59% of total chromosomal interactions. Such a distinct contact pattern indicates the highly selective nature of ecDNA-chromosomal interactions. To quantify the H3K27ac signal associated with ecDNA-mediated chromatin interactions, we compared fold enrichment and span size from the H3K27ac peaks detected in these high-frequency interaction foci on the ecDNAs (Group A), their corresponding interacting chromosomal partners (Group B), and the genome-wide chromosomal H3K27ac peaks that have no contact with ecDNA (Group C) in each of the four ecDNA(+) cell lines (Table S3). As expected, Group A showed significantly higher enrichment compared with Group C (median value 11–31 versus 6–7, p values 1 × 10−2 to 5 × 10−6, one-sided Wilcoxon rank-sum test) (Figure 4C). Group B, reflecting chromosomal ecDNA anchors, also showed a significant increase in H3K27ac signal compared with Group C (median: 12–20, p values 7 × 10−111 to 5 × 10−264, one-sided Wilcoxon rank-sum test), suggesting that ecDNA-chromosome connectivity converges on transcriptionally active regions. The enhancement of H3K27ac signal was ecDNA specific, as Group A and Group B showed higher fold enrichment compared with the ecMYC, ecEGFR, and ecCDK4 equivalent regions in the ecDNA(−) HF-3035 neurospheres (median value: 7). ecDNA H3K27ac peaks also showed significantly larger spans than the chromosomal H3K27 peaks with no ecDNA contact (median spans of 2.3–4.1 kb in Group A v" @default.
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• W3146078726 title "Oncogenic extrachromosomal DNA functions as mobile enhancers to globally amplify chromosomal transcription" @default.
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• W3146078726 doi "https://doi.org/10.1016/j.ccell.2021.03.006" @default.
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