Abstract
A plasmid bearing both a replication initiation region and a matrix attachment region is spontaneously amplified in transfected mammalian cells and generates plasmid repeats in the extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region (HSR). Generally, the repeat sequences are subject to repeat-induced gene silencing, the mechanism of which remains to be elucidated. Previous research showed that gene expression from the same plasmid repeat was higher from repeats located at DMs than at the HSR, which may reflect the extrachromosomal environment of the DMs. In the current study, plasmid repeats in both DMs and HSR were associated with repressive histone modifications (H3K9me3, H3K9me2), and the levels of repressive chromatin markers were higher in HSR than in DMs. Inactive chromatin is known to spread to neighboring regions in chromosome arm. Here, we found that such spreading also occurs in extrachromosomal DMs. Higher levels of active histone modifications (H3K9Ac, H3K4me3, and H3K79me2) were detected at plasmid repeats in DMs than in HSR. The level of DNA CpG methylation was generally low in both DMs and HSR; however, there were some hypermethylated copies within the population of repeated sequences, and the frequency of such copies was higher in DMs than in HSR. Together, these data suggest a “DNA methylation-core and chromatin-spread” model for repeat-induced gene silencing. The unique histone modifications at the extrachromosomal context are discussed with regard to the model.
Highlights
Gene amplification of oncogenes or drug-resistance genes can play a pivotal role in mammalian carcinogenesis
If the plasmid repeat integrated into the chromosome arm, a breakage-fusion-bridge cycle (BFB) was initiated, and a large homogeneously staining region (HSR) composed solely of plasmid repeats was formed in human COLO 320 cells [5,6,7] or a fine ladder-type HSR was formed in hamster CHO DG44 cells [8,9]
Repressive histone modifications accumulate at higher levels in HSR plasmid repeats than in double minutes (DMs) plasmid repeats
Summary
Gene amplification of oncogenes or drug-resistance genes can play a pivotal role in mammalian carcinogenesis. We found that a plasmid with a mammalian replication initiation region (IR) and a matrix attachment region (MAR) efficiently initiated gene amplification in transfected. Repeat-Induced Gene Silencing in the Chromosomal and Extrachromosomal Context cells, and that DMs and HSR were generated in the transfected cells [4,5]. The IR/MAR plasmid was initially maintained extrachromosomally in the transfected cells, and multimerized to form a large circular molecule in which the plasmid was arranged as a direct repeat [5]. The large circular molecules appeared as separate cytogenetically detectable DMs. If cells contained pre-existing DMs, the plasmid repeat efficiently recombined with these DMs [6]. If the plasmid repeat integrated into the chromosome arm, a breakage-fusion-bridge cycle (BFB) was initiated, and a large HSR composed solely of plasmid repeats was formed in human COLO 320 cells [5,6,7] or a fine ladder-type HSR was formed in hamster CHO DG44 cells [8,9]
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