Mechanisms Of Genome Rearrangements Research Articles

R-loops and regulatory changes in chronologically ageing fission yeast cells drive non-random patterns of genome rearrangements.

Aberrant repair of DNA double-strand breaks can recombine distant chromosomal breakpoints. Chromosomal rearrangements compromise genome function and are a hallmark of ageing. Rearrangements are challenging to detect in non-dividing cell populations, because they reflect individually rare, heterogeneous events. The genomic distribution of de novo rearrangements in non-dividing cells, and their dynamics during ageing, remain therefore poorly characterized. Studies of genomic instability during ageing have focussed on mitochondrial DNA, small genetic variants, or proliferating cells. To characterize genome rearrangements during cellular ageing in non-dividing cells, we interrogated a single diagnostic measure, DNA breakpoint junctions, using Schizosaccharomyces pombe as a model system. Aberrant DNA junctions that accumulated with age were associated with microhomology sequences and R-loops. Global hotspots for age-associated breakpoint formation were evident near telomeric genes and linked to remote breakpoints elsewhere in the genome, including the mitochondrial chromosome. Formation of breakpoint junctions at global hotspots was inhibited by the Sir2 histone deacetylase and might be triggered by an age-dependent de-repression of chromatin silencing. An unexpected mechanism of genomic instability may cause more local hotspots: age-associated reduction in an RNA-binding protein triggering R-loops at target loci. This result suggests that biological processes other than transcription or replication can drive genome rearrangements. Notably, we detected similar signatures of genome rearrangements that accumulated in old brain cells of humans. These findings provide insights into the unique patterns and possible mechanisms of genome rearrangements in non-dividing cells, which can be promoted by ageing-related changes in gene-regulatory proteins.

Partially local three-way alignments and the sequence signatures of mitochondrial genome rearrangements

BackgroundGenomic DNA frequently undergoes rearrangement of the gene order that can be localized by comparing the two DNA sequences. In mitochondrial genomes different mechanisms are likely at work, at least some of which involve the duplication of sequence around the location of the apparent breakpoints. We hypothesize that these different mechanisms of genome rearrangement leave distinctive sequence footprints. In order to study such effects it is important to locate the breakpoint positions with precision.ResultsWe define a partially local sequence alignment problem that assumes that following a rearrangement of a sequence F, two fragments L, and R are produced that may exactly fit together to match F, leave a gap of deleted DNA between L and R, or overlap with each other. We show that this alignment problem can be solved by dynamic programming in cubic space and time. We apply the new method to evaluate rearrangements of animal mitogenomes and find that a surprisingly large fraction of these events involved local sequence duplications.ConclusionsThe partially local sequence alignment method is an effective way to investigate the mechanism of genomic rearrangement events. While applied here only to mitogenomes there is no reason why the method could not be used to also consider rearrangements in nuclear genomes.

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome.

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena's germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.

Editorial: Marching Toward 100% Whole Genome Sequencing.

Editorial: Marching Toward 100% Whole Genome Sequencing.

Conformational polymorphysm of G-rich fragments of DNA ALU-repeats. I. Potential noncanonical structures

In this paper, we report results of systematic studies of conformational polymorphism of G-rich DNA fragments from Alu repeats. Alu retrotransposones are primate-specific short interspersed elements. Using the Alu sequence from the prooncogen bcl2 intron and the consensus AluSx sequence as representative examples, we determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites - PQSAlu), and demonstrated by bioinformatics methods that those sites are Alu-specific in the human genome. Genomic frequencies of PQSAlu were assessed (~1/10000 b.p.). The sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element from the human bcl2 gene (304 b.p.) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alubcl2 dsDNA and the absence of such structures in the case of Alu-PQS were shown using DMS-footprinting and AFM microscopy. Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions were obtained, and the effects of these insertions on the expression of the reporter gene in НЕК293 and HeLa cell lines were compared. Our findings on the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements which underlie many oncological and neurodegenerative diseases.

Genetics of Charcot-Marie-Tooth (CMT) Disease within the Frame of the Human Genome Project Success

Charcot-Marie-Tooth (CMT) neuropathies comprise a group of monogenic disorders affecting the peripheral nervous system. CMT is characterized by a clinically and genetically heterogeneous group of neuropathies, involving all types of Mendelian inheritance patterns. Over 1,000 different mutations have been discovered in 80 disease-associated genes. Genetic research of CMT has pioneered the discovery of genomic disorders and aided in understanding the effects of copy number variation and the mechanisms of genomic rearrangements. CMT genetic study also unraveled common pathomechanisms for peripheral nerve degeneration, elucidated gene networks, and initiated the development of therapeutic approaches. The reference genome, which became available thanks to the Human Genome Project, and the development of next generation sequencing tools, considerably accelerated gene and mutation discoveries. In fact, the first clinical whole genome sequence was reported in a patient with CMT. Here we review the history of CMT gene discoveries, starting with technologies from the early days in human genetics through the high-throughput application of modern DNA analyses. We highlight the most relevant examples of CMT genes and mutation mechanisms, some of which provide promising treatment strategies. Finally, we propose future initiatives to accelerate diagnosis of CMT patients through new ways of sharing large datasets and genetic variants, and at ever diminishing costs.

Abstract 2192: A novel mechanism of EML4-ALK rearrangement in a patient-derived cell line and its tumor growth inhibition by an ALK inhibitor CH5424802.

Abstract Purpose: EML4-ALK is a driver oncogene in non-small cell lung cancer (NSCLC) and has been developed into a promising molecular target for antitumor agents. Although EML4-ALK is reported to be formed by inversion of chromosome 2, other mechanisms of gene fusion remain unknown. Here we characterize the EML4-ALK fusion gene of a new cell line derived from a patient of NSCLC. In addition, the efficacy of ALK inhibitors against this new cell line was examined. Experimental Design: An EML4-ALK-positive cell line, termed LC649, was established from pleomorphic carcinoma, a rare subtype of NSCLC. We investigated the chromosomal aberrations using fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). CH5424802, a selective ALK inhibitor, was evaluated in the antitumor activity against LC649 in vitro and in vivo xenograft model. Results: Our initial characterization revealed that LC649 harbors variant 3 of EML4-ALK fusion with 2-fold copy number gain. Interestingly, CGH and metaphase-FISH analysis showed that LC649 contained, in addition to two normal chromosome 2, two aberrant chromosome 2 that were fragmented and scattered. These observations provided the first evidence that EML4-ALK fusion in LC649 cells was formed in chromosome 2 by a distinct mechanism of genomic rearrangement, termed “chromothripsis”. Additionally, CH5424802 inhibited phospho-ALK and suppressed tumor growth of the LC649 cells in vitro and in vivo in a xenograft model. Conclusions: Our results suggested that chromothripsis may be a mechanism of oncogenic rearrangement of EML4-ALK. CH5424802 was effective against EML4-ALK positive tumors with ALK copy number gain mediated by chromothripsis. Citation Format: Tatsushi Kodama, Noriko Motoi, Hironori Ninomiya, Hiroshi Sakamoto, Kunio Kitada, Toshiyuki Tsukaguchi, Yasuko Sato, Nobuya Ishii, Yasuhito Terui, Kiyohiko Hatake, Yuichi Ishikawa. A novel mechanism of EML4-ALK rearrangement in a patient-derived cell line and its tumor growth inhibition by an ALK inhibitor CH5424802. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2192. doi:10.1158/1538-7445.AM2013-2192

Improved systematic tRNA gene annotation allows new insights into the evolution of mitochondrial tRNA structures and into the mechanisms of mitochondrial genome rearrangements

Transfer RNAs (tRNAs) are present in all types of cells as well as in organelles. tRNAs of animal mitochondria show a low level of primary sequence conservation and exhibit ‘bizarre’ secondary structures, lacking complete domains of the common cloverleaf. Such sequences are hard to detect and hence frequently missed in computational analyses and mitochondrial genome annotation. Here, we introduce an automatic annotation procedure for mitochondrial tRNA genes in Metazoa based on sequence and structural information in manually curated covariance models. The method, applied to re-annotate 1876 available metazoan mitochondrial RefSeq genomes, allows to distinguish between remaining functional genes and degrading ‘pseudogenes’, even at early stages of divergence. The subsequent analysis of a comprehensive set of mitochondrial tRNA genes gives new insights into the evolution of structures of mitochondrial tRNA sequences as well as into the mechanisms of genome rearrangements. We find frequent losses of tRNA genes concentrated in basal Metazoa, frequent independent losses of individual parts of tRNA genes, particularly in Arthropoda, and wide-spread conserved overlaps of tRNAs in opposite reading direction. Direct evidence for several recent Tandem Duplication-Random Loss events is gained, demonstrating that this mechanism has an impact on the appearance of new mitochondrial gene orders.

Genome-Wide Analysis of Copy Number Variations in Normal Population Identified by SNP Arrays

Gene copy number change is an essential characteristic of many types of cancer. However, it is important to distinguish copy number variation (CNV) in the human genome of normal individuals from bona fide abnormal copy number changes of genes specific to cancers. Based on Affymetrix 50K single nucleotide polymorphism (SNP) array data, we identified genome-wide copy number variations among 104 normal subjects from three ethnic groups that were used in the HapMap project. Our analysis revealed 155 CNV regions, of which 37% were gains and 63% were losses. About 21% (30) of the CNV regions are concordant with earlier reports. These 155 CNV regions are located on more than 100 cytobands across all 23 chromosomes. The CNVs range from 68bp to 18 Mb in length, with a median length of 86 Kb. Eight CNV regions were selected for validation by quantitative PCR. Analysis of genomic sequences within and adjacent to CNVs suggests that repetitive sequences such as long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs) may play a role in the origin of CNVs by facilitating non-allelic homologous recombination. Thirty-two percent of the CNVs identified in this study are associated with segmental duplications. CNVs were not preferentially enriched in gene-encoding regions. Among the 364 genes that are completely encompassed by these 155 CNVs, genes related to olfactory sensory, chemical stimulus, and other physiological responses are significantly enriched. A statistical analysis of CNVs by ethnic group revealed distinct patterns regarding the CNV location and gain-to-loss ratio. The CNVs reported here will help build a more comprehensive map of genomic variations in the human genome and facilitate the differentiation between copy number variation and somatic changes in cancers. The potential roles of certain repeat elements in CNV formation, as corroborated by other studies, shed light on the origin of CNVs and will improve our understanding of the mechanisms of genomic rearrangements in the human genome.

Mechanisms of genomic rearrangements and gene expression changes in plant polyploids

Polyploidy is produced by multiplication of a single genome (autopolyploid) or combination of two or more divergent genomes (allopolyploid). The available data obtained from the study of synthetic (newly created or human-made) plant allopolyploids have documented dynamic and stochastic changes in genomic organization and gene expression, including sequence elimination, inter-chromosomal exchanges, cytosine methylation, gene repression, novel activation, genetic dominance, subfunctionalization and transposon activation. The underlying mechanisms for these alterations are poorly understood. To promote a better understanding of genomic and gene expression changes in polyploidy, we briefly review origins and forms of polyploidy and summarize what has been learned from genome-wide gene expression analyses in newly synthesized auto-and allopolyploids. We show transcriptome divergence between the progenitors and in the newly formed allopolyploids. We propose models for transcriptional regulation, chromatin modification and RNA-mediated pathways in establishing locus-specific expression of orthologous and homoeologous genes during allopolyploid formation and evolution.

Multiple variants of the archaeal DNA rudivirus SIRV1 in a single host and a novel mechanism of genomic variation

The DNA rudivirus SIRV1 of the hyperthermophilic archaeon Sulfolobus shows exceptional properties. Viral isolates invariably contain a population of variants with different but closely related genomes. Upon propagation in a given host strain, one or more genomes dominate in the viral population. However, upon passage into a new host strain the viral population undergoes changes and other dominant variants are selected. Sequencing and analysis of the variant genomes revealed that major differences occur in gene order, gene size and gene content at localized genomic sites. A previously unknown mechanism of genomic rearrangement involving putative 12 bp archaeal introns appears to facilitate alteration of the variant genomes. Inter-genomic recombination between the different variants also occurs. The variant genomes exhibit signature tetranucleotide sequences near their putative sites for replication initiation.

Genome plasticity in Enterobacteriaceae

The comparative analysis of multiple representatives of the genomes of particular species are leading us away from a view of bacterial genomes as static, monolithic structures towards the view that they are relatively variable, fluid structures. This plasticity is mainly the result of the rearrangement of genes within the genome and the acquisition of novel genes by horizontal transfer systems, e. g. plasmids, bacteriophages, transposons or gene cassettes. These mechanisms often act in concert thus generating a complex genetic structure. Genomic variations are not a phenomenon at the DNA level alone; they influence the phenotype of a bacterium as well and can render a formerly harmless organism into a hazardous pathogen. This review deals not only with the mechanisms of genome rearrangements and the horizontal transfer of genes in Enterobacteriaceae but also points out that mobile genetic elements themselves are subjected to variation.

Mitomycin C induces genomic rearrangements involving transposable elements in Drosophila melanognster

Mitomycin C was injected into the abdomen of male flies of the y2 sc1 waG strain of Drosophila melanogaster. They were mated with females bearing attached-X chromosomes, and the male offspring (F1) were analysed for the appearance of mutations in the X chromosome. We observed y+ and sc+ reversions induced either by excision of mdg4 (gypsy) with retention of one long terminal repeat (LTR) or by insertion of a foreign sequence into mdg4, partial reversion of the waG mutation, waG----waGd, and unstable f mutations. The overall mutation frequency was considerably higher than in control flies of the y2 sc1 waG strain. Possible mechanisms of genomic rearrangements induced by Mitomycin C, in particular the role of homologous recombination, are discussed.