Interconversion of intra‐ and extra‐chromosomal sites of gene amplification by modulation of gene expression and DNA methylation

Shimizu, Noriaki; Hanada, Naoyuki; Utani, Koichi; Sekiguchi, Naoki

doi:10.1002/jcb.21313

Cited by 23 publications

(41 citation statements)

References 39 publications

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“…Gene expression in supercoil-derived transformants decreased significantly during cellular passage, which suggests the progression of epigenetic gene silencing in HSR, as we previously showed (10,11). In contrast, we found that expression in hairpin molecule-derived transformants did not significantly decrease (Fig.…”

Section: A Hairpin-capped Linear Ir/mar Plasmid Persists As Etes Insupporting

confidence: 74%

“…Now, we analyzed expression from ETEs. Because the plasmid p⌬BM-d2EGFP contains the d2EGFP expression cassette, which produces an enhanced GFP with a very short cellular half-life, we measured green fluorescence by flow cytometry as previously described (10). We found that d2EGFP expression was lower in cells with the linear plasmid than in cells with the supercoiled plasmid (Fig.…”

Section: A Hairpin-capped Linear Ir/mar Plasmid Persists As Etes Inmentioning

confidence: 86%

“…2H) cells. In our previous papers (5,6,10,14,15,22,23,26), we had detected DMs in COLO 320DM cells by FISH using DM-painting probe or c-myc cosmid probe. However, the chromatin of DMs in these cells is large enough; thus we could easily detect them by DNA staining (yellow arrows in Fig.…”

Section: A Hairpin-capped Linear Ir/mar Plasmid Persists As Etes Inmentioning

confidence: 99%

“…If the large circle is integrated into the chromosome, it efficiently initiates a breakage-fusion-bridge cycle, which generates an HSR. Plasmid-borne genes are expressed more actively when they are amplified in DMs than in HSRs (10). Indeed, most of an HSR is heterochromatic, and transcription is detected only at few discrete spots inside the HSR (11).…”

mentioning

confidence: 99%

“…If an inducible promoter in the HSR is activated, the entire HSR is loosened, and active transcription is detected throughout this region (11). Such transcriptional activation, in collaboration with DNA demethylation induced by 5-azacytidine treatment, results in fragmentation of the HSR and the generation of extrachromosomal DMs (10). In addition, gene amplification generated by the IR/MAR plasmid has been used for chromosome studies (12)(13)(14)(15)(16), because it creates large HSRs or numerous DMs composed of a defined plasmid sequence.…”

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confidence: 99%

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Episomal High Copy Number Maintenance of Hairpin-capped DNA Bearing a Replication Initiation Region in Human Cells

Harada

Uchida

Shimizu

2009

Journal of Biological Chemistry

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We previously found that a plasmid bearing a replication initiation region efficiently initiates gene amplification in mammalian cells and that it generates extrachromosomal double minutes and/or chromosomal homogeneously staining regions. During analysis of the underlying mechanism, we serendipitously found that hairpin-capped linear DNA was stably maintained as numerous extrachromosomal tiny episomes for more than a few months in a human cancer cell line. Generation of such episomes depended on the presence of the replication initiation region in the original plasmid. Despite extrachromosomal maintenance, episomal gene expression was epigenetically suppressed. The Southern blot analysis of the DNA of cloned cells revealed that the region around the hairpin end was diversified between the clones. Furthermore, the bisulfite-modified PCR and the sequencing analyses revealed that the palindrome sequence that derived from the original hairpin end or its end-resected structure were well preserved during clonal long term growth. From these data, we propose a model that explains the formation and maintenance of these episomes, in which replication of the hairpin-capped DNA and cruciform formation and its resolution play central roles. Our findings may be relevant for the dissection of mammalian replicator sequences.Gene amplification plays a pivotal role in mammalian malignant transformation through acquisition of growth advantages or drug resistances (for recent reviews, see Refs. 1-4). Amplified genes are cytogenetically detected at extrachromosomal double minutes (DMs) 2 or in chromosomal homogeneously staining region (HSRs). DMs are acentric, atelomeric chromatin bodies composed of amplified DNA of genomic origin. We previously found that a plasmid with a mammalian replication initiation region (IR) and a matrix attachment region (MAR) is efficiently amplified to high copy number in mammalian cells and that it generates DMs and/or HSRs composed of plasmid sequences (5, 6). Because IRs and MARs are scattered throughout the mammalian genome in a frequency of once per several tens of kilobase pairs and because it was proposed that circular molecules excised from the genome may mediate gene amplification in cancer cells (7, 8), we considered the plasmid system as a novel one that efficiently reproduces gene amplification in cultured cells. Thus, we examined the mechanism by which the IR/MAR plasmid mimics gene amplification (5, 9). We suggested that the IR/MAR plasmid undergoes multiplication to produce a large extrachromosomal circle consisting of tandem repeats of plasmid sequences. If multiplication is extensive, the circle forms DMs. If the large circle is integrated into the chromosome, it efficiently initiates a breakage-fusion-bridge cycle, which generates an HSR. Plasmid-borne genes are expressed more actively when they are amplified in DMs than in HSRs (10). Indeed, most of an HSR is heterochromatic, and transcription is detected only at few discrete spots inside the HSR (11). If an inducible promoter in the ...

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Section: A Hairpin-capped Linear Ir/mar Plasmid Persists As Etes Insupporting

confidence: 74%

Section: A Hairpin-capped Linear Ir/mar Plasmid Persists As Etes Inmentioning

confidence: 86%

Section: A Hairpin-capped Linear Ir/mar Plasmid Persists As Etes Inmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Episomal High Copy Number Maintenance of Hairpin-capped DNA Bearing a Replication Initiation Region in Human Cells

Harada

Uchida

Shimizu

2009

Journal of Biological Chemistry

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Double‐strand breakage in the extrachromosomal double minutes triggers their aggregation in the nucleus, micronucleation, and morphological transformation

Oobatake

Shimizu

2019

Genes Chromosomes & Cancer

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Gene amplification plays a pivotal role in malignant transformation. Amplified genes often reside on extrachromosomal double minutes (DMs). Low‐dose hydroxyurea induces DM aggregation in the nucleus which, in turn, generates micronuclei composed of DMs. Low‐dose hydroxyurea also induces random double‐strand breakage throughout the nucleus. In the present study, we found that double‐strand breakage in DMs is sufficient for induction of DM aggregation. Here, we used CRISPR/Cas9 to introduce specific breakages in both natural and artificially tagged DMs of human colorectal carcinoma COLO 320DM cells. Aggregation occurred in the S phase but not in the G1 phase within 4 hours after breakage, which suggested the possible involvement of homologous recombination in the aggregation of numerous DMs. Simultaneous detection of DMs and the phosphorylated histone H2AX revealed that the aggregation persisted after breakage repair. Thus, the aggregate generated cytoplasmic micronuclei at the next interphase. Our data also suggested that micronuclear entrapment eliminated the DMs or morphologically transformed them into giant DMs or homogeneously staining regions (HSRs). In this study, we obtained a model explaining the consequences of DMs after double‐strand breakage in cancer cells. Because double‐strand breakage is frequently involved in cancer therapy, the model suggests how it affects gene amplification.

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Advancements in Brain Tumors Classification

Noorani,

Di Ieva

2024

Advanced Imaging and Therapy in Neuro-Oncology

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Interconversion of intra‐ and extra‐chromosomal sites of gene amplification by modulation of gene expression and DNA methylation

Cited by 23 publications

References 39 publications

Episomal High Copy Number Maintenance of Hairpin-capped DNA Bearing a Replication Initiation Region in Human Cells

Episomal High Copy Number Maintenance of Hairpin-capped DNA Bearing a Replication Initiation Region in Human Cells

Double‐strand breakage in the extrachromosomal double minutes triggers their aggregation in the nucleus, micronucleation, and morphological transformation

Advancements in Brain Tumors Classification

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