CircleBase: an integrated resource and analysis platform for human eccDNAs.

Rapid advances in high-throughput sequencing technologies have led to the discovery of thousands of extrachromosomal circular DNAs (eccDNAs) in the human genome. Loss-of-function experiments are difficult to conduct on circular and linear chromosomes, as they usually overlap. Hence, it is challenging to interpret the molecular functions of eccDNAs. Here, we present CircleBase (http://circlebase.maolab.org), an integrated resource and analysis platform used to curate and interpret eccDNAs in multiple cell types. CircleBase identifies putative functional eccDNAs by incorporating sequencing datasets, computational predictions, and manual annotations. It classifies them into six sections including targeting genes, epigenetic regulations, regulatory elements, chromatin accessibility, chromatin interactions, and genetic variants. The eccDNA targeting and regulatory networks are displayed by informative visualization tools and then prioritized. Functional enrichment analyses revealed that the top-ranked cancer cell eccDNAs were enriched in oncogenic pathways such as the Ras and PI3K-Akt signaling pathways. In contrast, eccDNAs from healthy individuals were not significantly enriched. CircleBase provides a user-friendly interface for searching, browsing, and analyzing eccDNAs in various cell/tissue types. Thus, it is useful to screen for potential functional eccDNAs and interpret their molecular mechanisms in human cancers and other diseases.

Progress on the role of extrachromosomal DNA in tumor pathogenesis and evolution.

The amplification of oncogenes on extrachromosomal DNA (ecDNA) provides a new mechanism for cancer cells to adapt to the changes in the tumor microenvironment and accelerate tumor evolution. These extrachromosomal elements contain oncogenes, and their chromatin structures are more open than linear chromosomes and therefore have stronger oncogene transcriptional activity. ecDNA always contains enhancer elements, and genes on ecDNA can be reintegrated into the linear genome to regulate the selective expression of genes. ecDNA lacks centromeres, and the inheritance from the parent cell to the daughter cell is uneven. This non-Mendelian genetic mechanism results in the increase of tumor heterogeneity with daughter cells that can gain a competitive advantage through a large number of copies of oncogenes. ecDNA promotes tumor invasiveness and provides a mechanism for drug resistance associated with poorer survival outcomes. Recent studies have demonstrated that the overall proportion of ecDNA in tumors is approximately 40%. In this review, we summarize the current knowledge of ecDNA in the field of tumorigenesis and development.

Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma.

Extrachromosomal circularization of DNA is an important genomic feature in cancer. However, the structure, composition and genome-wide frequency of extrachromosomal circular DNA have not yet been profiled extensively. Here, we combine genomic and transcriptomic approaches to describe the landscape of extrachromosomal circular DNA in neuroblastoma, a tumor arising in childhood from primitive cells of the sympathetic nervous system. Our analysis identifies and characterizes a wide catalog of somatically acquired and undescribed extrachromosomal circular DNAs. Moreover, we find that extrachromosomal circular DNAs are an unanticipated major source of somatic rearrangements, contributing to oncogenic remodeling through chimeric circularization and reintegration of circular DNA into the linear genome. Cancer-causing lesions can emerge out of circle-derived rearrangements and are associated with adverse clinical outcome. It is highly probable that circle-derived rearrangements represent an ongoing mutagenic process. Thus, extrachromosomal circular DNAs represent a multihit mutagenic process, with important functional and clinical implications for the origins of genomic remodeling in cancer.

Neurobiological disease etiology and inheritance: an epigenetic perspective.

Epigenetic marks in mammals are essential to properly control the activity of the genome. They are dynamically regulated during development and adulthood, and can be modulated by environmental factors throughout life. Changes in the epigenetic profile of a cell can be positive and favor the expression of advantageous genes such as those linked to cell signaling and tumor suppression. However, they can also be detrimental and alter the functions of important genes, thereby leading to disease. Recent evidence has further highlighted that some epigenetic marks can be maintained across meiosis and be transmitted to the subsequent generation to reprogram developmental and cellular features. This short review describes current knowledge on the potential impact of epigenetic processes activated by environmental factors on the inheritance of neurobiological disease risk. In addition, the potential adaptive value of epigenetic inheritance, and relevant current and future questions are discussed.

Exceptional inheritance of plastids via pollen in Nicotiana sylvestris with no detectable paternal mitochondrial DNA in the progeny.

Plastids and mitochondria, the DNA-containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co-transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild-type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS-92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid-transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.

CorA, the magnesium/nickel/cobalt transporter, affects virulence and extracellular enzyme production in the soft rot pathogen Pectobacterium carotovorum.

Pectobacterium carotovorum (formerly Erwinia carotovora ssp. carotovora) is a phytopathogenic bacterium that causes soft rot disease, characterized by water-soaked soft decay, resulting from the action of cell wall-degrading exoenzymes secreted by the pathogen. Virulence in soft rot bacteria is regulated by environmental factors, host and bacterial chemical signals, and a network of global and gene-specific bacterial regulators. We isolated a mini-Tn5 mutant of P. carotovorum that is reduced in the production of extracellular pectate lyase, protease, polygalacturonase and cellulase. The mutant is also decreased in virulence as it macerates less host tissues than its parent and is severely impaired in multiplication in planta. The inactivated gene responsible for the reduced virulent phenotype was identified as corA. CorA, a magnesium/nickel/cobalt membrane transporter, is the primary magnesium transporter for many bacteria. Compared with the parent, the CorA(-) mutant is cobalt resistant. The mutant phenotype was confirmed in parental strain P. carotovorum by marker exchange inactivation of corA. A functional corA(+) DNA from P. carotovorum restored exoenzyme production and pathogenicity to the mutants. The P. carotovorum corA(+) clone also restored motility and cobalt sensitivity to a CorA(-) mutant of Salmonella enterica. These data indicate that CorA is required for exoenzyme production and virulence in P. carotovorum.

Effects of cellular differentiation, chromosomal integration and 5-aza-2'-deoxycytidine treatment on human papillomavirus-16 DNA methylation in cultured cell lines.

Human papillomavirus-16 (HPV-16) genomes in cell culture and in situ are affected by polymorphic methylation patterns, which can repress the viral transcription. In order to understand some of the underlying mechanisms, we investigated changes of the methylation of HPV-16 DNA in cell cultures in response to cellular differentiation, to recombination with cellular DNA, and to an inhibitor of methylation. Undifferentiated W12E cells, derived from a precancerous lesion, contained extrachromosomal HPV-16 DNA with a sporadically methylated enhancer-promoter segment. Upon W12E cell differentiation, the viral DNA was demethylated, suggesting a link between differentiation and the epigenetic state of HPV-16 DNA. The viral genomes present in two W12I clones, in which individual copies of the HPV-16 genome have integrated into cellular DNA (type 1 integrants), were unmethylated, akin to that seen in the cervical carcinoma cell line SiHa (also a type 1 integrant). This finding is consistent with hypomethylation being necessary for continued viral gene expression. In contrast, two of three type 2 integrant W12I clones, containing concatemers of HPV-16 genomes integrated into the cellular DNA contained hypermethylated viral DNA, as observed in the cervical carcinoma cell line CaSki (also a type 2 integrant). A third, type 2, W12I clone, interestingly with fewer copies of the viral genome, contained unmethylated HPV-16 genomes. Epithelial differentiation of W12I clones did not lead to demethylation of chromosomally integrated viral genomes as was seen for extrachromosomal HPV-16 DNA in W12E clones. Hypomethylation of CaSki cells in the presence of the DNA methylation inhibitor 5-aza-2'-deoxycytidine reduced the cellular viability, possibly as a consequence of toxic effects of an excess of HPV-16 gene products. Our data support a model wherein (i) the DNA methylation state of extrachromosomal HPV16 replicons and epithelial differentiation are inversely coupled during the viral life cycle, (ii) integration of the viral genome into the host chromosome events leads to an alteration in methylation patterns on the viral genome that is dependent upon the type of integration event and possibly copy number, and (iii) integration universally results in the viral DNA becoming refractory to changes in methylation state upon cellular differentiation that are observed with extrachromosomal HPV-16 genomes.

Reflex sympathetic dystrophy: complex regional pain syndrome type I in children with mitochondrial disease and maternal inheritance.

OBJECTIVE: Complex regional pain syndrome type I (CRPS-I), previously known as reflex sympathetic dystrophy (RSD), is an idiopathic condition characterised by localised, abnormally intense and prolonged pain, allodynia and autonomic nervous system changes (ie, swelling, skin colour and temperature changes and altered perspiration) that usually appear following a "noxious" trigger such as trauma or surgery. The objective of this report is to demonstrate that children with CRPS-I can have additional dysautonomic conditions secondary to an underlying maternally inherited mitochondrial disease, an association not previously published. METHODS: Medical records of about 500 patients seen by one paediatric metabolic geneticist were reviewed to identify children meeting established CRPS diagnostic criteria. RESULTS: CRPS-I was present in eight children in seven families, each of which also had additional functional/dysautonomic conditions, the most common (> or = 4 cases per condition) being gastrointestinal dysmotility, migraine, cyclic vomiting and chronic fatigue. All seven probands studied met Nijmegen (2002) diagnostic criteria for definite mitochondrial disease on the basis of the clinical signs and symptoms and biochemical analyses. Six of the seven families met our pedigree-based criteria for probable maternal inheritance. CONCLUSION: In one tertiary-care paediatric genetics practice, children meeting the CRPS-I diagnostic criteria frequently had additional autonomic-related conditions secondary to maternally inherited mitochondrial disease, suggesting that mitochondrial DNA sequence variants can predispose children towards the development of CRPS-I and other dysautonomias. CRPS-I should be considered in patients with mitochondrial disease who complain of idiopathic pain. Maternally inherited mitochondrial disease may not be a rare cause of CRPS-I, especially in children who present with other manifestations of dysautonomia.

The plasmid replicon of Epstein-Barr virus: mechanistic insights into efficient, licensed, extrachromosomal replication in human cells.

The genome of Epstein-Barr Virus (EBV) and plasmid derivatives of it are among the most efficient extrachromosomal replicons in mammalian cells. The latent origin of plasmid replication (oriP), when supplied with the viral Epstein-Barr Nuclear Antigen 1 (EBNA1) in trans, provides efficient duplication, partitioning and maintenance of plasmids bearing it. In this review, we detail what is known about the viral cis and trans elements required for plasmid replication. In addition, we describe how the cellular factors that EBV usurps are used to complement the functions of the viral constituents. Finally, we propose a model for the sequential assembly of an EBNA1-dependent origin of DNA synthesis into a pre-Replicative Complex (pre-RC), which functions by making use only of cellular enzymatic activities to carry out the replication of the viral plasmid.

Retroviral E-DNA: persistence and gene expression in nondividing immune cells.

Following retroviral infection of cells, not only is the proviral DNA integrated into the host genome, but there is also an accumulation of unintegrated extrachromosomal DNA (E-DNA), both linear and circular. Although the integrated DNA is responsible for the production of viral proteins and new viral progeny, the role of E-DNA has remained uncertain. Several reports have shown that E-DNA is transcriptionally active producing both RNA, as well as viral proteins and that circular E-DNA can persist in nondividing cells, raising questions regarding the potential consequences of this reservoir. Furthermore, integrase inhibitors, presently in clinical trials, shifts the balance of proviral DNA to the E-DNA form. This review is focused on recent work in this field with an emphasis on exploring the potential role of E-DNA in both pathogenesis of retroviral infections, especially HIV-1, and as a tool to deliver and express genes.

Lack of evidence for cytoplasmic effects for four traits of Polypay sheep.

Analyses of birth and weaning weights, fleece weights of ewes, and number born per litter of Polypay sheep collected at the U.S. Sheep Experimental Station from 1978 through 1998, confirmed previous analyses of three other dual-purpose breeds that cytoplasmic effects do not contribute to variation in these four traits. In general, estimates of genetic parameters that would be needed for national genetic evaluation were similar to previous estimates for Columbia, Rambouillet, and Targhee sheep, although estimates of direct heritability for Polypay were somewhat less for birth weight, slightly greater for weaning weight, significantly greater for fleece weight, and the same for number born as for those three breeds. For birth weight only, evidence was found for important dam x year or dam x number born interactions, which are essentially litter effects, as was found for the other dual-purpose breeds. There were 11,896, 11,104, 7,748, and 7,831 records for birth and weaning weights, fleece weight, and number born per litter, with 255 to 316 sires of animals with records. There were 260 and 261 cytoplasmic lines for fleece weight and number born, and 861 and 882 for weaning and birth weights.

The B12/23 restriction is critically dependent on recombination signal nonamer and spacer sequences.

Ag receptor variable region gene assembly is initiated through the formation of a synaptic complex which minimally includes the recombination-activating gene (RAG) 1/2 proteins and a pair of recombination signals (RSs) flanking the recombining gene segments. RSs are composed of conserved heptamer and nonamer sequences flanking relatively nonconserved spacers of 12 or 23 bp. RSs regulate variable region gene assembly within the context of the 12/23 rule which mandates that recombination only occurs between RSs of dissimilar spacer length. RSs can exert additional constraints on variable region gene assembly beyond imposing spacer length requirements. At a minimum this restriction, termed B12/23, is imposed on the Vbeta to DJbeta rearrangement step by the 5' Dbeta RS and is enforced at or before the DNA cleavage step of the V(D)J recombination reaction. In this study, the components of the 5' Dbeta RS required for enforcing the B12/23 rule are assessed on chromosomal substrates in vivo in the context of normal murine thymocyte development and on extrachromosomal substrates induced to undergo recombination in nonlymphoid cell lines. These analyses reveal that the integrity of the nonamer sequence as well as the highly conserved spacer nucleotides of the 5' Dbeta1 RS are critical for enforcing the B12/23 restriction. These findings have important implications for understanding the B12/23 restriction and the manner in which RS synaptic complexes are assembled in vivo.

Extrachromosomal circular DNA of tandemly repeated genomic sequences in Drosophila.

One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA). This DNA is found in various eukaryotes from yeast to humans, and its levels are elevated by exposure to carcinogens. eccDNA is heterogeneous in size and composed of chromosomal sequences. In this study we used two-dimensional gel electrophoresis to detect and characterize eccDNA in Drosophila. We found eccDNA throughout the fly's life cycle. These molecules comprise up to 10% of the total repetitive DNA content, and their size ranges from <1 kb to >20 kb. The eccDNA population contains circular multimers of tandemly repeated genes such as histones, rDNA, Stellate, and the Suppressor of Stellate. Multimers of centromeric heterochromatin sequences are included in eccDNA as well. Our findings are consistent with the hypothesis that intramolecular homologous recombination between direct tandem repeats is a favorite mechanism for eccDNA formation. The level of eccDNA increased following MMS treatment of wild-type larvae, consistent with phenomena observed in cultured mammalian cells. This shows mutagen-induced eccDNA formation in the context of the whole organism for the first time. Mutations in the genes okra, mus309, and mei41 did not affect eccDNA under normal conditions or following mutagen treatment, implying that eccDNA formation is different from known pathways of DNA repair.

[A new planarian extrachromosomal virus-like element revealed by subtraction hybridization]. / Novyi ékstrakhromosomnyi virusopodobnyi élement planarii obnaruzhen metodom vychitaiushchei gibridizatsii.

A combination of suppression subtraction hybridization (SSH) and a new technique of mirror orientation selection (MOS) was used to compare the total DNA for two, sexual (SR) and asexual (AR), races of freshwater planarian Giradia tigrina. Several race-specific DNA fragments were found. A new element termed planarian extrachromosomal virus-like element (PEVE) was revealed in AR. The PEVE genome contains two unique regions, Ul and Us, which are flanked by inverted repeats. Two variants observed for the PEVE genome differ in combination of single- and double-stranded regions corresponding to Ul and Us. The PEVE genome codes for two helicases, one homologous to the circovirus replication initiation protein (Rep) and one corresponding to the helicase domain of papillomavirus E1. PEVE is nonuniformly distributed though the planarian body and is possibly replicated only in certain parenchymal cells.

Evolution of large extrachromosomal elements in HL-60 cells during culture and the associated phenotype alterations.

In the HL-60 sublines that were isolated after a long-term continuous culture, abnormally stained or abnormally banded regions on chromosomes replaced extrachromosomal double minutes. The c-MYC gene is amplified in these structures. We followed the c-MYC gene loci during a consecutive passage by using FISH, and have found a large extrachromosomal element (LEE) that preexisted at the earliest passage in a very small fraction of cells. No chromosomal integration of c-MYC sequences was observed in up to 225 passages. The LEEs persistently evolved during culture and were not excluded from the nucleus. In the LEE-positive cells, the spontaneous differentiation was blocked and the granulocytic differentiation that was induced by treatment with dimethyl sulfoxide was reversed by withdrawal of the drug. The c-MYC gene integration into LEEs is unlikely to lead to these phenotypes. The reversibility might be related to the reversible c-MYC down-regulation during the early phase of the drug treatment of HL-60 cells at early cell passages.

Classification and genetic features of neonatal haemochromatosis: a study of 27 affected pedigrees and molecular analysis of genes implicated in iron metabolism.

Neonatal haemochromatosis (NH) is a severe and newly recognised syndrome of uncertain aetiology, characterised by congenital cirrhosis or fulminant hepatitis and widespread tissue iron deposition. NH occurs in the context of maternal disease including viral infection, as a complication of metabolic disease in the fetus, and sporadically or recurrently, without overt cause, in sibs. Although an underlying genetic basis for NH has been suspected, no test is available for predictive analysis in at risk pregnancies. As a first step towards an understanding of the putative genetic basis for neonatal haemochromatosis, we have conducted a systematic study of the mode of transmission of this disorder in a total of 40 infants born to 27 families. We have moreover carried out a molecular analysis of candidate genes (beta(2)-microglobulin, HFE, and haem oxygenases 1 and 2) implicated in iron metabolism. No pathogenic mutations in these genes were identified that segregate consistently with the disease phenotype in multiplex pedigrees. However, excluding four pedigrees with clear evidence of maternal infection associated with NH, a pedigree showing transmission of maternal antinuclear factor and ribonucleoprotein antibodies to the affected infants, and two families with possible matrilineal inheritance of disease in maternal half sibs, a large subgroup of the affected pedigrees point to the inheritance of an autosomal recessive trait. This included 14 pedigrees with affected and unaffected infants and a single pedigree where all four affected infants were the sole offspring of consanguineous but otherwise healthy parents. We thus report three distinct patterns of disease transmission in neonatal haemochromatosis. In the differentiation of a large subgroup showing transmission of disease in a manner suggesting autosomal recessive inheritance, we also provide the basis for further genome wide studies to define chromosomal determinants of iron storage disease in the newborn.

Deconstructing Mendel: new paradigms in genetic mechanisms.

As knowledge of the mechanisms of genetic action expands, this new information must be incorporated into the whole. The result is that old concepts are modified or deleted or new paradigms are created. The authors review advances in the understanding of traditional and nontraditional inheritance, including genomic imprinting and mitochondrial inheritance.

Protein-only inheritance in yeast: something to get [PSI+]-ched about.

Recent work suggests that two unrelated phenotypes, [PSI+] and [URE3], in the yeast Saccharomyces cerevisiae are transmitted by non-covalent changes in the physical states of their protein determinants, Sup35p and Ure2p, rather than by changes in the genes that encode these proteins. The mechanism by which alternative protein states are self-propagating is the key to understanding how proteins function as elements of epigenetic inheritance. Here, we focus on recent molecular-genetic analysis of the inheritance of the [PSI+] factor of S. cerevisiae. Insights into this process might be extendable to a group of mammalian diseases (the amyloidoses), which are also believed to be a manifestation of self-perpetuating changes in protein conformation.