Intrachromosomal Gene Amplification Triggered by Hairpin-Capped Breaks Requires Homologous Recombination and is Independent of Nonhomologous End-Joining

Gene amplification is one of the major mechanisms of acquisition of drug resistance and activation of oncogenes in tumors. In mammalian cells, amplified chromosomal regions are manifested cytogenetically as extrachromosomal double minutes (DMs) and chromosomal homogeneously staining regions (HSRs). We recently demonstrated using yeast model system that hairpin-capped double strand breaks (DSBs) generated at the location of human Alu-quasipalindromes can trigger both types of gene amplification. Specifically, the dicentric chromosomes arising from replication of hairpin-capped molecules can be precursors for intrachromosomal amplicons. The formation of HSRs can be accounted for either by breakage-fusion-bridge (BFB) cycle which necessitates nonhomologous end-joining pathway (NHEJ) or by the repair event involving homologous recombination (HR). In this study, we report that intrachromosomal gene amplification mediated by hairpin-capped DSBs is independent of NHEJ machinery, however requires the functions of Rad52 and Rad51 proteins. Based on our observations, we propose a HR-dependent mechanism to explain how the breakage of dicentric chromosomes can lead to the formation of HSRs.     

Intrastrand annealing leads to the formation of a large DNA palindrome and determines the boundaries of genomic amplification in human cancer

Amplification of large chromosomal regions (gene amplification) is a common somatic alteration in human cancer cells and often is associated with advanced disease. A critical event initiating gene amplification is a DNA double-strand break (DSB), which is immediately followed by the formation of a large DNA palindrome. Large DNA palindromes are frequent and nonrandomly distributed in the genomes of cancer cells and facilitate a further increase in copy number. Although the importance of the formation of large DNA palindromes as a very early event in gene amplification is widely recognized, it is not known how a DSB is resolved to form a large DNA palindrome and whether any local DNA structure determines the location of large DNA palindromes. We show here that intrastrand annealing following a DNA double-strand break leads to the formation of large DNA palindromes and that DNA inverted repeats in the genome determine the efficiency of this event. Furthermore, in human Colo320DM cancer cells, a DNA inverted repeat in the genome marks the border between amplified and nonamplified DNA. Therefore, an early step of gene amplification is a regulated process that is facilitated by DNA inverted repeats in the genome.     

Large DNA palindromes as a common form of structural chromosome aberrations in human cancers

Breakage-fusion-bridge cycles contribute to chromosome aberrations and generate large DNA palindromes that facilitate oncogene amplification in cancer cells. At the molecular level, large DNA palindrome formation is initiated by chromosome breaks, and genomic architecture such as short inverted repeat sequences facilitates this process in mammalian cells. However, the prevalence of DNA palindromes in cancer cells is currently unknown. To determine the prevalence of DNA palindromes in human cancer cells, we have developed a new microarray-based approach called Genome-wide Analysis of Palindrome Formation (GAPF, Tanaka et al., Nat Genet 2005; 37: 320-7). This approach is based on a relatively simple and efficient method to purify "snap-back DNA" from large DNA palindromes by intramolecular base-pairing, followed by elimination of single-stranded DNA by nuclease S1. Comparison of Genome-wide Analysis of Palindrome Formation profiles between cancer and normal cells using microarray can identify genome-wide distributions of somatic palindromes. Using a human cDNA microarray, we have shown that DNA palindromes occur frequently in human cancer cell lines and primary medulloblastomas. Significant overlap of the loci containing DNA palindromes between Colo320DM and MCF7 cancer cell lines suggests regions in the genome susceptible to chromosome breaks and palindrome formation. A subset of loci containing palindromes is associated with gene amplification in Colo320DM, indicating that the location of palindromes in the cancer genome serves as a structural platform that supports subsequent gene amplification.     

Integrated cytogenetic and high-resolution array CGH analysis of genomic alterations associated with MYCN amplification

Amplification of oncogenes and closely linked flanking genes is common in some types of cancer and can be associated with complex chromosome rearrangements and/or co-amplification of non-syntenic chromosomal regions. To better understand the etiology and structural complexity of focal MYCN amplicons in human neuronal cancer, we investigated the precise chromosomal locations of high copy number genomic regions in MYCN amplified cell lines. An integrated cytogenetic map of the MYCN amplicon was created using high-resolution array CGH, spectral karyotyping (SKY), multi-color banding (mBAND), and fluorescence in situ hybridization (FISH) in 4 human neuronal tumor cell lines. The evidence of complex intra- and inter-chromosomal events, providing clues concerning the nature of the genomic mechanisms that contributed to the process of MYCN amplification, was observed. The presence of multiple co-amplified syntenic or non-syntenic sequences in the MYCN amplicon is quite intriguing. MYCN is usually centrally located in the amplicon; however, the structure and complexity of the amplicons were highly variable. It is noteworthy that clusters of unstable repetitive regions characterized by CNV sequences were present throughout the regions encompassed by MYCN gene amplification, and these sequences could provide a mechanism to destabilize this region of the genome. Complex structural rearrangements involving genomic losses and gains in the 2p24 region lead to MYCN amplification and that these rearrangements can trigger amplification events.     

Linear amplicons as precursors of amplified circles in methotrexate-resistant Leishmania tarentolae.

Gene amplification is frequently observed in Leishmania cells selected for drug resistance. By gene targeting we have tagged both alleles of the H locus of Leishmania tarentolae with the neomycin and hygromycin phosphotransferase genes ( neo and hyg ). Selection of these recombinant parasites for low level methotrexate resistance led to amplification of the H locus as part of linear amplicons. The availability of tags has permitted us to determine that both alleles can be amplified in the same cell and that chromosomal deletions are frequent. When methotrexate concentration was increased in subsequent selection steps, circles were observed in several mutants. We have introduced a hyg marker into linear amplicons to test whether the circles originated from linear amplicons. After selection with a high methotrexate concentration, circles with the hyg marker were observed, showing that circles can indeed be formed from linear amplicons. The tagging of H locus alleles permits appreciation of the extent of genetic rearrangements leading to amplicon formation in Leishmania cells selected for drug resistance.     

Myc oncogene-induced genomic instability: DNA palindromes in bursal lymphomagenesis

Genetic instability plays a key role in the formation of naturally occurring cancer. The formation of long DNA palindromes is a rate-limiting step in gene amplification, a common form of tumor-associated genetic instability. Genome-wide analysis of palindrome formation (GAPF) has detected both extensive palindrome formation and gene amplification, beginning early in tumorigenesis, in an experimental Myc-induced model tumor system in the chicken bursa of Fabricius. We determined that GAPF-detected palindromes are abundant and distributed nonrandomly throughout the genome of bursal lymphoma cells, frequently at preexisting short inverted repeats. By combining GAPF with chromatin immunoprecipitation (ChIP), we found a significant association between occupancy of gene-proximal Myc binding sites and the formation of palindromes. Numbers of palindromic loci correlate with increases in both levels of Myc over-expression and ChIP-detected occupancy of Myc binding sites in bursal cells. However, clonal analysis of chick DF-1 fibroblasts suggests that palindrome formation is a stochastic process occurring in individual cells at a small number of loci relative to much larger numbers of susceptible loci in the cell population and that the induction of palindromes is not involved in Myc-induced acute fibroblast transformation. GAPF-detected palindromes at the highly oncogenic bic/miR-155 locus in all of our preneoplastic and neoplastic bursal samples, but not in DNA from normal and other transformed cell types. This finding indicates very strong selection during bursal lymphomagenesis. Therefore, in addition to providing a platform for gene copy number change, palindromes may alter microRNA genes in a fashion that can contribute to cancer development.     

How a Replication Origin and Matrix Attachment Region Accelerate Gene Amplification under Replication Stress in Mammalian Cells

The gene amplification plays a critical role in the malignant transformation of mammalian cells. The most widespread method for amplifying a target gene in cell culture is the use of methotrexate (Mtx) treatment to amplify dihydrofolate reductase (Dhfr). Whereas, we found that a plasmid bearing both a mammalian origin of replication (initiation region; IR) and a matrix attachment region (MAR) was spontaneously amplified in mammalian cells. In this study, we attempted to uncover the underlying mechanism by which the IR/MAR sequence might accelerate Mtx induced Dhfr amplification. The plasmid containing the IR/MAR was extrachromosomally amplified, and then integrated at multiple chromosomal locations within individual cells, increasing the likelihood that the plasmid might be inserted into a chromosomal environment that permits high expression and further amplification. Efficient amplification of this plasmid alleviated the genotoxicity of Mtx. Clone-based cytogenetic and sequence analysis revealed that the plasmid was amplified in a chromosomal context by breakage-fusion-bridge cycles operating either at the plasmid repeat or at the flanking fragile site activated by Mtx. This mechanism explains how a circular molecule bearing IR/MAR sequences of chromosomal origin might be amplified under replication stress, and also provides insight into gene amplification in human cancer.     

Amplification of the multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen as multiple independent events.

The multidrug resistance (MDR) phenotype in mammalian tumor cells can involve amplification of mdr genes that results in overexpression of the protein product termed P-glycoprotein. Chloroquine resistance (CQR) in Plasmodium falciparum has similarities with the MDR phenotype in tumor cells, and some isolates of P. falciparum have amplified levels of the pfmdr1 gene. To investigate the nature and origin of pfmdr1 amplicons, we have cloned large regions of a 110-kb amplicon from the CQR cloned isolate B8 by using the yeast artificial chromosome system. We have identified and sequenced the breakpoints of the amplicon by a novel method employing inverted polymerase chain reaction that is applicable to analysis of any large-scale repeat. We show that the five copies of the amplicon in this isolate are in a head to tail configuration. A string of 30 A's flank the breakpoints on each side of the amplified segment, suggesting a mechanism for the origin of the tandem amplification. Polymerase chain reaction analysis with oligonucleotides that cross the B8 breakpoint has shown in 26 independent CQR isolates, 16 of which contain amplified copies of pfmdr1, that amplification of the pfmdr1 gene in P. falciparum has arisen as multiple independent events. These results suggest that this region of the genome is under strong selective pressure.     

A Mechanism of Gene Amplification Driven by Small DNA Fragments

DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s). Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA) occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB) external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation in nature.     

回文序列诱导突变的机制及人类相关疾病

在原核和真核生物基因组中, 含有回文序列的区域高度可变且稳定性差, 主要原因是回文序列能形成发卡或十字形二级结构, 然后通过滑动错配、单链复性以及非同源末端连接(Non-homologous end joining, NHEJ)等机制导致缺失突变或染色体易位的发生。在人类基因组中, 回文序列较普遍存在于基因表达调控的重要作用元件中, 它诱导的缺失和易位突变还与男性不育、地中海贫血等多种疾病的发生、发展密切相关。文章综合近几年国内外相关文献, 初步阐释回文序列诱导突变的类型和可能机制, 及其与人类疾病的关系, 为进一步探讨回文序列在基因表达调控、基因突变及人类疾病中的作用及功能等相关研究提供参考。     

Regularities of karyotypic evolution during stepwise amplification of genes determining drug resistance.

Analysis of chromosomal alterations during stepwise development of mdr1, dhfr, or CAD gene amplifications in a large number of independently selected Djungarian hamster DM-15 and murine P388 sublines revealed typical patterns of karyotypic evolution, specific for multiplication of each of these genes in each cell type. Some principal similarities of karyotypic evolution were noted in at least two different systems. They include: (i) appearance at the first selection step of a new chromosomal arm bearing the resident gene copy followed at the next selection steps by the formation in these specific chromosomal arms of amplified DNA tandem arrays; (ii) translocations of amplified DNA from its initial site to other, also non-random, chromosomal sites; and (iii) emergence in the cell variants with high degrees of gene amplification of multiple extra-chromosomal elements. The most prominent distinctions among the systems were as follows: (i) different structures, evidently containing amplified DNAs, appeared at the initial steps of amplification of different genes--additional heterogeneously staining regions in specific chromosomal segments in the case of amplification of dhfr or CAD genes in DM-15 cells, and mini-chromosomes in the case of mdr1 gene amplification in both DM-15 and P380 cells; (ii) distinct patterns of location of the amplified mdr1 gene copies are characteristic of Djungarian hamster DM-15 and murine P388 cell derivatives after subsequent steps of selection--at the site of resident gene localization or in some other, also non-random, chromosomal sites in DM-15 sublines, and predominantly extra-chromosomal in P388 sublines. We propose that different mechanisms are responsible for the initial steps of amplification of dhfr and CAD genes on the one hand and the mdr1 gene on the other: non-equal sister-chromatid exchanges and autonomous replication of the extra-chromosomal elements. It seems, however, that both mechanisms may be involved in further rounds of amplification of each of these three genes.     

High-resolution in situ hybridization technique using biotinylated NMYC oncogene probe reveals periodic structure of HSRs in human neuroblastoma

Gene amplification in mammalian cells commonly manifests itself as homogeneously staining chromosomal regions (HSRs). These are frequently seen in neuroblastoma and have been shown to be the site of amplification of the NMYC oncogene. We have used a nonisotopic, high-resolution in situ hybridization technique to reveal a hitherto unrecognized periodic microstructure within HSRs of a human neuroblastoma cell line.     

Genome-wide amplifications caused by chromosomal rearrangements play a major role in the adaptive evolution of natural yeast

The relative importance of gross chromosomal rearrangements to adaptive evolution has not been precisely defined. The Saccharomyces cerevisiae flor yeast strains offer significant advantages for the study of molecular evolution since they have recently evolved to a high degree of specialization in a very restrictive environment. Using DNA microarray technology, we have compared the genomes of two prominent variants of S. cerevisiae flor yeast strains. The strains differ from one another in the DNA copy number of 116 genomic regions that comprise 38% of the genome. In most cases, these regions are amplicons flanked by repeated sequences or other recombination hotspots previously described as regions where double-strand breaks occur. The presence of genes that confer specific characteristics to the flor yeast within the amplicons supports the role of chromosomal rearrangements as a major mechanism of adaptive evolution in S. cerevisiae. We propose that nonallelic interactions are enhanced by ethanol- and acetaldehyde-induced double-strand breaks in the chromosomal DNA, which are repaired by pathways that yield gross chromosomal rearrangements. This mechanism of chromosomal evolution could also account for the sexual isolation shown among the flor yeast.     

Yeast clathrin has a distinctive light chain that is important for cell growth

The structure and physiologic role of clathrin light chain has been explored by purification of the protein from Saccharomyces cerevisiae, molecular cloning of the gene, and disruption of the chromosomal locus. The single light chain protein from yeast shares many physical properties with the mammalian light chains, in spite of considerable sequence divergence. Within the limited amino acid sequence identity between yeast and mammalian light chains (18% overall), three regions are notable. The carboxy termini of yeast light chain and mammalian light chain LCb are 39% homologous. Yeast light chain contains an amino-terminal region 45% homologous to a domain that is completely conserved among mammalian light chains. Lastly, a possible homolog of the tissue-specific insert of LCb is detected in the yeast gene. Disruption of the yeast gene (CLC1) leads to a slow-growth phenotype similar to that seen in strains that lack clathrin heavy chain. However, light chain gene deletion is not lethal to a strain that cannot sustain a heavy chain gene disruption. Light chain-deficient strains frequently give rise to variants that grow more rapidly but do not express an immunologically related light chain species. These properties suggest that clathrin light chain serves an important role in cell growth that can be compensated in light chain deficient cells.     

Genome-wide gene amplification during differentiation of neural progenitor cells in vitro

DNA sequence amplification is a phenomenon that occurs predictably at defined stages during normal development in some organisms. Developmental gene amplification was first described in amphibians during gametogenesis and has not yet been described in humans. To date gene amplification in humans is a hallmark of many tumors. We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells. Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells. We identified several amplified genes in neural progenitor cells that are known to be amplified in malignant tumors. There is also a striking overlap of amplified chromosomal regions between differentiating neural progenitor cells and malignant tumor cells derived from astrocytes. Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.