Biology subjects

Regenberg, B.

Publications and source records attributed to Regenberg, B..

10 recordsLinked to original sources

bioRxiv2025

Functional Role of Small Extrachromosomal Circular DNA in Colorectal Cancer

Extrachromosomal circular DNA (eccDNA) are circular DNA molecules that originate from chromosomal DNA but exist independently. While large eccDNA (ecDNA) contributes to tumorigenesis, the role of smaller eccDNA (<100,000 base pairs) in cancer remains unclear. Our analysis of 25 colorectal cancer (CRC) tumors and adjacent non-tumorous tissues revealed that eccDNA is significantly more abundant in tumor tissues, correlating strongly with chromosomal amplifications. The presence of whole intact genes on 1.29% of eccDNA was non-random. We identified 84 genes that recurred across tumors of multiple patients when present on eccDNA with 19% of genes being cancer-associated. eccDNA-borne genes were often accompanied by increased expression, and their contribution to expression was much larger than that from linear amplifications and the larger ecDNA. The cytokine gene CXCL5 exemplified this phenomenon, showing substantial copy-number increase and upregulation when present on eccDNA. Functional validation in cell lines showed that CXCL5 eccDNA enhanced transcriptional output and immune cell recruitment function. The recurrence and overexpression of CRC-related genes on eccDNA indicate their selection in tumors, suggest eccDNA can serve as a novel mechanism for dynamically influencing gene expression and is capable of conferring cancer phenotypes to cells. Analysis of chromatin landscapes revealed that eccDNA preferentially forms at sites of open chromatin and active transcription, with architectural boundaries marked by CTCF protein. Clinically, patients with higher eccDNA levels showed poorer relapse-free survival. These findings suggest that circular DNA elements across the entire size spectrum participate in cancer evolution, positioning eccDNA as a potential therapeutic target and prognostic biomarker.

cancer biology↗

bioRxiv2025

An Enhancement of Extrachromosomal Circular DNA Enrichment and Amplification to Address the Extreme Low Overlap Between Replicates

Extrachromosomal circular DNA (eccDNA) of chromosomal origin is present in all eukaryotic organisms and tissues that have been tested. Populations of eccDNA exhibit immense diversity and a characteristically low degree of overlap between samples, suggesting low inheritance of eccDNA between cells or a deficiency in the methods by which eccDNA is detected. This study revisits the Circle-seq approach for enrichment of eccDNA to address these limitations, hypothesizing that experimental procedures significantly contribute to the observed low eccDNA overlap. We optimized the protocol by reducing the time needed to complete the procedure. Linear DNA is digested by increasing Exonuclease V activity. We employed CRISPR-Cas9 for mitochondrial linearization, which proved superior to using restriction enzymes. A key finding is the critical role of random hexamer primer concentration and genomic DNA input in Rolling circle amplification (RCA) for generating high-quality long amplicons from eccDNA (concatemeric tandem copy [CTC]), essential for confident de novo eccDNA construction from long-read sequencing data. Lower primer concentrations substantially increased the percentage of CTC-derived eccDNA and improved the overlap of identified eccDNAs in technical replicates. Applying this revised approach to human myeloma and breast cancer cell lines, as well as xenograft models, demonstrated >50% overlap in detected eccDNA, a substantial improvement over the <1% overlap observed in previous studies. Additionally, the oncogenic signature of eccDNAs can be identified across all replicates. These findings provide guidelines for developing standardized procedures for eccDNA profiling, advancing our understanding of eccDNA biology, and its potential clinical applications.

genomics↗

bioRxiv2024

Generative Modelling of Oncogene-carrying Extrachromosomal Circular DNA Biogenesis and Dynamics in Cells

Extrachromosomal circular DNAs (ecDNA) are focal gene amplifications frequently associated with cancer development and often indicating a poor prognosis. To understand the early dynamics of oncogene-carrying ecDNAs, we previously developed CRISPR-C, a tool for precise ecDNA generation by deleting specific chromosomal regions. Here, we adapted CRISPR-C to recreate tumor ecDNAs. This method also allowed us to enhance ecDNA generation efficiency by directly delivering Cas9 protein and sgRNAs as a ribonucleoprotein complex. By using the modified CRISPR-C, we successfully generated ecDNAs carrying oncogenes (EGFR, CDK4, MDM2, MYC, MYCN, FGFR2, ABCB1, and DHFR) in various human cell types. Furthermore, we demonstrated that our method could generate chimeric ecDNAs composed of target sequences from distant intra or inter-chromosomal regions. Using these generative ecDNA cell models, we studied the oncogene ecDNA expression and stability. The MDM2 expression was increased after CRISPR-C, while CDK4 was decreased indicating genomic-context dependent effect. The copy number of CRISPR-C generated CDK4 was ecDNA increased in cells after a long period of treatment with the CDK4 inhibitor palbociclib. Unlike CDK4, the CRISPR-C generated ABCB1 ecDNA was unstable in cells under normal growth conditions, but is stably retained when the cells were treated with colcemid, a recognized substrate for ABCB1. We thus provide valuable tools and an attractive platform for studying ecDNA biogenesisy and in vitro drug screening on ecDNA stability.

cancer biology↗

bioRxiv2024

Technical and biological variations in the purification of extrachromosomal circular DNA (eccDNA) and the finding of more eccDNA in the plasma of lung adenocarcinoma patients compared with healthy donors

Human plasma DNA originates from all tissues and organs, holding the potential as a versatile marker for diseases such as cancer, as fragments of cancer-specific alleles can be found circulating in the blood. While linear DNA has been studied intensely as a liquid biomarker, the role of circular circulating DNA in cancer is more unknown due, in part, to a lack of comprehensive testing methods. Our developed method profiles extrachromosomal circular DNA (eccDNA) in plasma, integrating Solid-Phase Reversible Immobilization (SPRI) bead purification, the removal of linear DNA and mitochondrial DNA, and DNA sequencing. As an initial assessment, we examined the method, biological variations, and technical variations using plasma samples from four patients with lung adenocarcinoma and four healthy and physically fit individuals. Despite the small sample group, we observed a significant eccDNA increase in cancer patients in two independent laboratories and that eccDNA covered up to 0.4 % of the genome/mL plasma. We found a subset of eccDNA from recurrent genes present in cancer samples but not in every control. In conclusion, our data reflect the large variation found in eccDNA sequence content and show that the variability observed among replicates in eccDNA stems from a biological source and can cause inconclusive findings for biomarkers. This suggests the need to explore other biological markers, such as epigenetic features on eccDNA.

molecular biology↗

bioRxiv2022

Did circular DNA shape the evolution of mammalian genomes?

Extrachromosomal circular DNA of chromosomal origin (eccDNA) can rapidly shape the evolution and adaptation of mitotically dividing cells such as tumor cells. However, whether eccDNA has a permanent impact on genome evolution through the germline is largely unexplored. Here, we propose that a large fraction of the syntenic changes that are found between mammalian species are caused by germline transposition of eccDNA. We have previously shown the existence of eccDNA in mammalian meiotic cells. By reanalysis of available synteny maps, we now find that up to 6% of mammalian genomes might have rearranged via a circular DNA intermediate. Hence, eccDNA in the germline is expected to have large effects on evolution of gene order. HighlightsO_LIExtrachromosomal circular DNA (eccDNA) is present in mammalian germline cells showing that eccDNAs are not excluded, repressed or eliminated during meiosis C_LIO_LILarge eccDNA reinsertions into the genome can change gene synteny in a recognizable pattern based on its circular junction and integration breakpoint. C_LIO_LIBy reanalyzing synteny maps form 8 mammalian species, we show that 6% of genome of the ungulates cow and sheep can be explained by eccDNA insertions. C_LIO_LIWe propose that reinsertion of large eccDNA that are fixed in germline cells may have contributed to speciation barriers and evolution of new species C_LI

genetics↗

bioRxiv2022

Accurate Identification of Extrachromosomal Circular DNA from Long-read Sequences.

Extrachromosomal circular DNA (eccDNA) of chromosomal origin is found in a range of eukaryotic species and cell type including cancer where eccDNA with oncogenes appear to drive tumorigenesis. Most studies of eccDNA employ short-read sequencing to identify for their identification. However, short-read sequencing cannot resolve the complexity of genomic repeats, which can lead to missing eccDNA identification. An alternative is the long-read sequencing technologies that can potentially be used to construct complete eccDNA. We present a software suite, Construction-based Rolling-circle amplification for eccDNA Sequence Identification and Location (CReSIL) 2.0, to identify and characterize eccDNA from long-read sequences. CReSILs performance in the identification of eccDNA, with a minimum F1 score of 0.98, is superior to the other bioinformatic tools based on simulated data. CReSIL provides many useful features for genomic annotation, which can used to infer eccDNA function and Circos visualization for eccDNA architecture investigation. We demonstrated CReSILs capability in many of the long-read sequencing datasets. This includes datasets enriched for eccDNA as well as whole genome datasets from many cells that contained large eccDNA. CReSIL suite software will be a versatile tool to deeply investigate eccDNA in biological samples.

bioinformatics↗

bioRxiv2021

Cell free extrachromosomal circular DNA is common in human urine

Cell free extrachromosomal circular DNA (eccDNA) is evolving as a potential biomarker in liquid biopsies for disease diagnosis. In this study, an optimized next generation sequencing-based Circle-Seq method was developed to investigate urinary cell free eccDNA (ucf-eccDNA) from 28 adult healthy volunteers (mean age = 28, 19 males/ 9 females). The genomic distributions and sequence compositions of ucf-eccDNAs were comprehensively characterized. Approximately 1.2 million unique ucf-eccDNAs are identified, covering 14.9% of the human genome. Comprehensive characterization of ucf-eccDNAs show that ucf-eccDNAs contain higher GC content than flanking genomic regions. Most eccDNAs are less than 1000 bp and present four pronounced peaks at 203, 361, 550 and 728 bp, indicating the association between eccDNAs and the numbers of intact nucleosomes. Analysis of genomic distribution of ucf-eccDNAs show that eccDNAs are found in all chromosomes but enriched in chromosomes i.e. chr.17, 19 and 20 with high density of protein-codding genes, CpG islands, SINE and simple repeat elements. Lastly, analysis of sequence motif signatures at eccDNA junction sites reveal that direct repeats (DRs) are commonly found, indicating a potential role of DRs in eccDNA biogenesis. This work underscores the deep sequencing analysis of ucf-eccDNAs and provides a valuable reference resource for exploring potential applications of ucf-eccDNA as diagnostic biomarkers of urogenital disorders in the future. Significance StatementExtrachromosomal circular DNA (eccDNA) is an important genetic element and a biomarker for disease diagnosis and treatment. In this study, we conduct a comprehensive characterization of urinary cell free eccDNA (ucf-eccDNA) in 28 heathy subjects. Over one million ucf-eccDNAs are identified. Ucf-eccDNAs are characterized as high GC content. The size of most ucf-eccDNAs is less than 1000 bp and enriched in four peaks resembling the size of single, double, triple, and quadruple nucleosomes. The genomic distribution of ucf-eccDNAs is enriched in generic regions, protein-coding genes, Alu, CpG islands, SINE and simple repeats. Sequence motif analysis of ucf-eccDNA junctions identified simple direct repeats (DRs) commonly presented in most eccDNAs, suggesting potential roles of DRs in eccDNA biogenesis.

genomics↗

bioRxiv2020

Replicative aging is associated with loss of genetic heterogeneity from extrachromosomal circular DNA in Saccharomyces cerevisiae

Circular DNA of chromosomal origin form from all parts of eukaryotic genomes. In yeast, circular rDNA accumulates as cells divide, contributing to replicative aging. However, little is known about how other chromosome-deri ved circles segregate and contribute to geneticvariation as cells age. We identified circular DNA across the genome of young S. cerevisiae populations and their aged descendants. Young cells had highly diverse circular DNA populations, but lost 94% of the different circular DNA after 20 divisions. Circles present in both young and old cells were characterized by replication origins and included circles from unique regions of the genome, rDNA circles and telomeric Y circles. The loss in genetic heterogeneity in aged cells was accompanied by massive accumulation of rDNA circles >95% of all circular DNA. We discovered circles had flexible inherence patterns. Glucose limited conditions selected for cells with glucose-transporter gene circles, [HXT6/7circle], and up to 50% of cells in a population carried them. [HXT6/7circle] cells were eventually substituted by cells carrying stable chromosomal HXT6 HXT6/7 HXT7 amplifications, suggesting circular DNA were intermediates in chromosomal amplifications. In conclusion, DNA circles can offer a flexible adaptive solution but cells lose genetic heterogeneity from circular DNA as they undergo replicative aging.

cell biology↗

bioRxiv2019

Sensitive detection of circular DNA at single-nucleotide resolution using guided realignment of partially aligned reads

Circular DNA has recently been identified across different species including human normal and cancerous tissue, but short-read mappers are unable to align many of the reads crossing circle junctions and hence limits their detection from short-read sequencing data. Here, we propose a new method, Circle-Map, that guides the realignment of partially aligned reads using information from discordantly mapped reads. We demonstrate how this approach dramatically increases sensitivity for detection of circular DNA on both simulated and real data while retaining high precision.

bioinformatics↗

bioRxiv2018

Circularization of genes and chromosome by CRISPR in human cells

Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders and diseases such as cancer. However, there is a lack of genetic engineering tool to recapitulate these features. Here, we report the discovery that delivery of pairs of CRISPR/Cas9 guide RNAs into human cells generate functional eccDNAs and ring chromosomes. We generated a dual-fluorescence eccDNA biosensor system, which allows us to study CRISPR deletion, inversion, and circularization of genes inside cells. Analysis after CRISPR editing at intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts reveal that CRISPR deleted DNA readily form eccDNA in human cells. DNA in sizes from a few hundred base pairs up to a 47.4 megabase-sized ring chromosome (chr18) can be circularized. Our discoveries advance and expand CRISPR-Cas9 technology applications for genetic engineering, modeling of human diseases, and chromosome engineering.\n\nOne Sentence Summary: CRISPR circularization of DNA offers new tools for studying eccDNA biogenesis, function, chromosome engineering, and synthetic biology.

genomics↗