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Circular DNA can arise from all parts of eukaryotic chromosomes. In yeast, circular ribosomal DNA (rDNA) accumulates dramatically as cells age, however little is known about the accumulation of other chromosome-derived circles or the contribution of such circles to genetic variation in aged cells. We profiled circular DNA in Saccharomyces cerevisiae populations sampled when young and after extensive aging. Young cells possessed highly diverse circular DNA populations but 94% of the circular DNA were lost after ∼15 divisions, whereas rDNA circles underwent massive accumulation to >95% of circular DNA. Circles present in both young and old cells were characterized by replication origins including circles from unique regions of the genome and repetitive regions: rDNA and telomeric Y' regions. We further observed that circles can have flexible inheritance patterns: [HXT6/7circle] normally segregates to mother cells but in low glucose is present in up to 50% of cells, the majority of which must have inherited this circle from their mother. Interestingly, [HXT6/7circle] cells are eventually replaced by cells carrying stable chromosomal HXT6 HXT6/7 HXT7 amplifications, suggesting circular DNAs are intermediates in chromosomal amplifications. In conclusion, the heterogeneity of circular DNA offers flexibility in adaptation, but this heterogeneity is remarkably diminished with age., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Extrachromosomal circular DNA (eccDNA) elements of chromosomal origin are known to be common in a number of eukaryotic species. However, it remains to be addressed whether genomic features such as genome size, the load of repetitive elements within a genome, and/or animal physiology affect the number of eccDNAs. Here, we investigate the distribution and numbers of eccDNAs in a condensed and less repeat-rich genome compared with the human genome, using Columba livia domestica (domestic rock pigeon) as a model organism. By sequencing eccDNA in blood and breast muscle from three pigeon breeds at various ages and with different flight behavior, we characterize 30,000 unique eccDNAs. We identify genomic regions that are likely hotspots for DNA circularization in breast muscle, including genes involved in muscle development. We find that although eccDNA counts do not correlate with the biological age in pigeons, the number of unique eccDNAs in a nonflying breed (king pigeons) is significantly higher (9-fold) than homing pigeons. Furthermore, a comparison between eccDNA from skeletal muscle in pigeons and humans reveals ∼9-10 times more unique eccDNAs per human nucleus. The fraction of eccDNA sequences, derived from repetitive elements, exist in proportions to genome content, that is, human 72.4% (expected 52.5%) and pigeon 8.7% (expected 5.5%). Overall, our results support that eccDNAs are common in pigeons, that the amount of unique eccDNA types per nucleus can differ between species as well as subspecies, and suggest that eccDNAs from repeats are found in proportions relative to the content of repetitive elements in a genome., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Chromosome-derived extrachromosomal circular DNA elements (eccDNAs) are detected in all eukaryotes examined so far. Here I describe the Circle-Seq protocol, applicable for physical enrichment of eccDNAs of a broad size range, combined with sequence confirmation of circular structures.Briefly, by concise alkaline treatment and gentle gravity flow-through an ion-exchange column, eccDNAs are enriched in the eluate fraction. EccDNAs are enzymatically isolated by extensive Plasmid-Safe DNase digestion of linear chromosomes and further enriched by φ29 rolling circle amplification. By means of high throughput sequencing of amplified eccDNA and custom eccDNA mapping software, around ten-thousand unique eccDNA types could be detected at nucleotide resolution in a million human muscle nuclei by this method.

Lifelong regular physical activity is associated with reduced risk of type 2 diabetes (T2D), maintenance of muscle mass and increased metabolic capacity. However, little is known about epigenetic mechanisms that might contribute to these beneficial effects in aged individuals. We investigated the effect of lifelong physical activity on global DNA methylation patterns in skeletal muscle of healthy aged men, who had either performed regular exercise or remained sedentary their entire lives (average age 62 years). DNA methylation was significantly lower in 714 promoters of the physically active than inactive men while methylation of introns, exons and CpG islands was similar in the two groups. Promoters for genes encoding critical insulin-responsive enzymes in glycogen metabolism, glycolysis and TCA cycle were hypomethylated in active relative to inactive men. Hypomethylation was also found in promoters of myosin light chain, dystrophin, actin polymerization, PAK regulatory genes and oxidative stress response genes. A cluster of genes regulated by GSK3β-TCF7L2 also displayed promoter hypomethylation. Together, our results suggest that lifelong physical activity is associated with DNA methylation patterns that potentially allow for increased insulin sensitivity and a higher expression of genes in energy metabolism, myogenesis, contractile properties and oxidative stress resistance in skeletal muscle of aged individuals.

Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders. However, there is a lack of genetic engineering tools to recapitulate and study the biogenesis of eccDNAs. Here, we created a dual-fluorescence biosensor cassette, which upon the delivery of pairs of CRISPR/Cas9 guide RNAs, CRISPR-C, allows us to study the biogenesis of a specific fluorophore expressing eccDNA in human cells. We show that CRISPR-C can generate functional eccDNA, using the novel eccDNA biosensor system. We further reveal that CRISPR-C also can generate eccDNAs from intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts. EccDNAs mainly forms by end-joining mediated DNA-repair and we show that CRISPR-C is able to generate endogenous eccDNAs in sizes from a few hundred base pairs and ranging up to 207 kb. Even a 47.4 megabase-sized ring chromosome 18 can be created by CRISPR-C. Our study creates a new territory for CRISPR gene editing and highlights CRISPR-C as a useful tool for studying the cellular impact, persistence and function of eccDNAs.

Exercise training has been reported to prevent the age-induced decline in muscle mass and fragmentation of mitochondria, as well as to affect autophagy and mitophagy. The interaction between these pathways during aging as well as the similarity between such changes in human and mouse skeletal muscle is however not fully understood. Therefore the aim of the present study was to test the hypothesis that cellular degradation pathways, including apoptosis, autophagy and mitophagy are coordinately regulated in mouse and human skeletal muscle during aging and lifelong exercise training through a PGC-1α-p53 axis. Muscle samples were obtained from young untrained, aged untrained and aged lifelong exercise trained men, and from whole-body PGC-1α knockout mice and their littermate controls that were either lifelong exercise trained or sedentary young and aged. Lifelong exercise training prevented the aging-induced reduction in PGC-1α, p53 and p21 mRNA as well as the increase in LC3II and BNIP3 protein in mouse skeletal muscle, while aging decreased the BAX/Bcl-2 ratio, LC3I and BAX protein in mouse skeletal muscle without effects of lifelong exercise training. In humans, aging was associated with reduced PGC-1α mRNA as well as decreased p62 and p21 protein in skeletal muscle, while lifelong exercise training increased BNIP3 protein and decreased p53 mRNA. In conclusion, there was a divergent regulation of autophagy and apoptosis in mouse muscle with aging and lifelong exercise training, whereas healthy aged human skeletal muscle seemed rather robust to changes in apoptosis, autophagy and mitophagy markers compared with mouse muscle at the investigated age., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The human genome is generally organized into stable chromosomes, and only tumor cells are known to accumulate kilobase (kb)-sized extrachromosomal circular DNA elements (eccDNAs). However, it must be expected that kb eccDNAs exist in normal cells as a result of mutations. Here, we purify and sequence eccDNAs from muscle and blood samples from 16 healthy men, detecting ~100,000 unique eccDNA types from 16 million nuclei. Half of these structures carry genes or gene fragments and the majority are smaller than 25 kb. Transcription from eccDNAs suggests that eccDNAs reside in nuclei and recurrence of certain eccDNAs in several individuals implies DNA circularization hotspots. Gene-rich chromosomes contribute to more eccDNAs per megabase and the most transcribed protein-coding gene in muscle, TTN (titin), provides the most eccDNAs per gene. Thus, somatic genomes are rich in chromosome-derived eccDNAs that may influence phenotypes through altered gene copy numbers and transcription of full-length or truncated genes.

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment was required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by φ29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 10(10) cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30, HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

Extrachromosomal circular DNA (eccDNA) derived from chromosomal Ty retrotransposons in yeast can be generated in multiple ways. Ty eccDNA can arise from the circularization of extrachromosomal linear DNA during the transpositional life cycle of retrotransposons, or from circularization of genomic Ty DNA. Circularization may happen through nonhomologous end-joining (NHEJ) of long terminal repeats (LTRs) flanking Ty elements, by Ty autointegration, or by LTR-LTR recombination. By performing an in-depth investigation of sequence reads stemming from Ty eccDNAs obtained from populations of Saccharomyces cerevisiae S288c, we find that eccDNAs predominantly correspond to full-length Ty1 elements. Analyses of sequence junctions reveal no signs of NHEJ or autointegration events. We detect recombination junctions that are consistent with yeast Ty eccDNAs being generated through recombination events within the genome. This opens the possibility that retrotransposable elements could move around in the genome without an RNA intermediate directly through DNA circularization., (Copyright © 2016 by the Genetics Society of America.)

Examples of extrachromosomal circular DNAs (eccDNAs) are found in many organisms, but their impact on genetic variation at the genome scale has not been investigated. We mapped 1,756 eccDNAs in the Saccharomyces cerevisiae genome using Circle-Seq, a highly sensitive eccDNA purification method. Yeast eccDNAs ranged from an arbitrary lower limit of 1 kb up to 38 kb and covered 23% of the genome, representing thousands of genes. EccDNA arose both from genomic regions with repetitive sequences ≥ 15 bases long and from regions with short or no repetitive sequences. Some eccDNAs were identified in several yeast populations. These eccDNAs contained ribosomal genes, transposon remnants, and tandemly repeated genes (HXT6/7, ENA1/2/5, and CUP1-1/-2) that were generally enriched on eccDNAs. EccDNAs seemed to be replicated and 80% contained consensus sequences for autonomous replication origins that could explain their maintenance. Our data suggest that eccDNAs are common in S. cerevisiae, where they might contribute substantially to genetic variation and evolution.

Microbial populations adapt to environmental fluctuations through random switching of fitness-related traits in individual cells. This increases the likelihood that a subpopulation will be adaptive in a future milieu. However, populations are particularly challenged when several environment factors change simultaneously. We suggest that a population can rapidly adapt to multiple environmental changes if individual members stochastically flip a hub-switch that controls a set of adaptive phenotypes in a single event. This mechanism of coupling phenotypic outcomes via a hub-switch can protect a population against large fluctuations in size. Here we report that the general amino acid transporter Gap1 is a potential hub-switch. The GAP1 gene is flanked by two direct repeats that can lead to GAP1 deletions (∆gap1) and a self-replicating GAP1 circle. Thus, an isogenic GAP1 population can differentiate into two variant, reversible genotypes, ∆gap1 or GAP1 (circle). These subpopulations have different phenotypic advantages. A ∆gap1 population has a selective advantage on allantoin or ammonium as a nitrogen source and high stress tolerance. Advantages of the GAP1 population include amino acid uptake, fast energy recruitment by trehalose mobilization, and in some cases, adherent biofilm growth. Our proposed model of a hub-switch locus enhances the bet-hedging model of population dynamics.

Amino acids can induce yeast cell adhesion but how amino acids are sensed and signal the modulation of the FLO adhesion genes is not clear. We discovered that the budding yeast Saccharomyces cerevisiae CEN.PK evolved invasive growth ability under prolonged nitrogen limitation. Such invasive mutants were used to identify amino acid transporters as regulators of FLO11 and invasive growth. One invasive mutant had elevated levels of FLO11 mRNA and a Q320STOP mutation in the SFL1 gene that encodes a protein kinase A pathway regulated repressor of FLO11. Glutamine-transporter genes DIP5 and GNP1 were essential for FLO11 expression, invasive growth and biofilm formation in this mutant. Invasive growth relied on known regulators of FLO11 and the Ssy1-Ptr3-Ssy5 complex that controls DIP5 and GNP1, suggesting that Dip5 and Gnp1 operates downstream of the Ssy1-Ptr3-Ssy5 complex for regulation of FLO11 expression in a protein kinase A dependent manner. The role of Dip5 and Gnp1 appears to be conserved in the S. cerevisiae strain ∑1278b since the dip5 gnp1 ∑1278b mutant showed no invasive phenotype. Secondly, the amino acid transporter gene GAP1 was found to influence invasive growth through FLO11 as well as other FLO genes. Cells carrying a dominant loss-of-function PTR3(647::CWNKNPLSSIN) allele had increased transcription of the adhesion genes FLO1, 5, 9, 10, 11 and the amino acid transporter gene GAP1. Deletion of GAP1 caused loss of FLO11 expression and invasive growth. However, deletions of FLO11 and genes encoding components of the mitogen-activated protein kinase pathway or the protein kinase A pathway were not sufficient to abolish invasive growth, suggesting involvement of other FLO genes and alternative pathways. Increased intracellular amino acid pools in the PTR3(647::CWNKNPLSSIN)-containing strain opens the possibility that Gap1 regulates the FLO genes through alteration of the amino acid pool sizes.

The tumor microenvironment is now recognized as a major factor in determining the survival and growth of disseminated tumor cells at potential metastatic sites. Tumor cells send signals to stroma cells and stimulate them to produce factors that in turn create favorable conditions for tumor cell metastasis. Activated fibroblasts constitute an important component of the tumor-associated stroma. We have previously shown that S100A4 protein produced by stromal fibroblasts in the primary tumor stimulates metastasis formation. Here we show that activated fibroblasts also stimulate the formation of metastases independently of S100A4 expression during organ colonization. To identify genes that could potentially interfere with fibroblast-driven metastasis, we used gene expression profiling of S100A4-deficient fibroblasts treated with and without tumor cell-conditioned media. Five differentially expressed genes encoding cell surface and secreted proteins with potential metastasis-modulating activity were selected. Expression of lymphocyte antigen 6 complex (Ly6c) and matrix metalloproteinase 3 (Mmp3) was upregulated in fibroblasts in response to tumor-conditioned medium, whereas expression of cadherin-16 (Cdh16), Ccn2, and fibulin-5 (Fbln5) was downregulated. Further analysis showed that Fibulin-5 is able to suppress the metastatic colonization of lungs and liver. Additional studies suggest a mechanism in which Fibulin-5 suppresses metastasis formation by inhibiting production of matrix metalloproteinase 9 (MMP9) and reducing the invasive behavior of fibroblasts. Together our data are consistent with the notion that tumors secrete factors that downregulate expression of Fbln5 in fibroblasts at sites of metastatic colonization, in turn upregulating Mmp9 expression and stimulating metastatic organ colonization.

Background: The tumor microenvironment has been described as a critical milieu determining tumor growth and metastases. A pivotal role of metastasis-inducing S100A4 in the development of tumor stroma has been proven in animal models and verified in human breast cancer biopsies. Expression and release of S100A4 has been shown in various types of stroma composing cells, including fibroblasts and immune cells. However, the events implicated in upstream and downstream pathways regulating the activity of the extracellular S100A4 protein in the tumor milieu remain unsolved., Methodology/principal Findings: We studied the interplay between the tumor cell-derived cytokine regulated-upon-activation, normal T-cell expressed and secreted (RANTES; CCL5) and S100A4 which were shown to be critical factors in tumor progression. We found that RANTES stimulates the externalization of S100A4 via microparticle shedding from the plasma membrane of tumor and stroma cells. Conversely, the released S100A4 protein induces the upregulation of fibronectin (FN) in fibroblasts and a number of cytokines, including RANTES in tumor cells as well as stimulates cell motility in a wound healing assay. Importantly, using wild type and S100A4-deficient mouse models, we demonstrated a substantial influence of tumor cell-derived RANTES on S100A4 release into blood circulation which ultimately increases the metastatic burden in mice., Conclusions/significance: Altogether, the data presented strongly validate the pro-metastatic function of S100A4 in the tumor microenvironment and define how the tumor cell-derived cytokine RANTES acts as a critical regulator of S100A4-dependent tumor cell dissemination. Additionally, for the first time we demonstrated the mechanism of S100A4 release associated with plasma membrane microparticle shedding from various cells types.

The function of S100A4, a member of the calcium-binding S100 protein family, has been associated with tumor invasion and metastasis. Although an essential pro-metastatic role of extracellular S100A4 in tumor progression has been demonstrated, the identification of the precise underlying mechanisms and protein partners (receptors) has remained elusive. To identify putative targets for extracellular S100A4, we screened a phage display peptide library using S100A4 as bait. We identified three independent peptide motifs with varying affinities for the S100A4 protein. Sequence analyses indicated that the most abundant peptide mimicked the F/YCC motif present in the epidermal growth factor domain of ErbB receptor ligands. S100A4 selectively interacted with a number of epidermal growth factor receptor (EGFR) ligands, demonstrating highest affinity for amphiregulin. Importantly, we found that S100A4 stimulated EGFR/ErbB2 receptor signaling and enhanced the amphiregulin-mediated proliferation of mouse embryonic fibroblasts. S100A4-neutralizing antibodies, as well as EGFR- and ErbB2 receptor-specific tyrosine kinase inhibitors, blocked these effects. The present results suggest that extracellular S100A4 regulates tumor progression by interacting with EGFR ligands, thereby enhancing EGFR/ErbB2 receptor signaling and cell proliferation. Structured digital abstract: * MINT-7256556: EGF (uniprotkb:P01133) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256512: BC (uniprotkb:P35070) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256485, MINT-7256618, MINT-7256636: AR (uniprotkb:P15514) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256494: HB-EGF (uniprotkb:Q99075) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256502: P53 (uniprotkb:P04637) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256654: S100A2 (uniprotkb:P29034) binds (MI:0407) to AR (uniprotkb:P15514) by far western blotting (MI:0047) * MINT-7256693: S100A5 (uniprotkb:P33763) binds (MI:0407) to AR (uniprotkb:P15514) by far western blotting (MI:0047) * MINT-7256593: S100A4 (uniprotkb:P26447) binds (MI:0407) to BC (uniprotkb:P35070) by pull down (MI:0096) * MINT-7256567: S100A4 (uniprotkb:P26447) binds (MI:0407) to AR (uniprotkb:P15514) by pull down (MI:0096).