Repeat induces not only gene silencing, but also gene activation in mammalian cells.

Yusuke Ogaki,Noriaki Shimizu,Miki Fukuma,Arthur J Lustig

doi:10.1371/journal.pone.0235127

Abstract

Repeat-induced gene silencing (RIGS) establishes the centromere structure, prevents the spread of transposons and silences transgenes, thereby limiting recombinant protein production. We previously isolated a sequence (B-3-31) that alleviates RIGS from the human genome. Here, we developed an assay system for evaluating the influence of repeat sequences on gene expression, based on in vitro ligation followed by our original gene amplification technology in animal cells. Using this assay, we found that the repeat of B-3-31, three core sequences of replication initiation regions (G5, C12, and D8) and two matrix attachment regions (AR1 and 32-3), activated the co-amplified plasmid-encoded d2EGFP gene in both human and hamster cell lines. This upregulation effect persisted for up to 82 days, which was confirmed to be repeat-induced, and was thus designated as a repeat-induced gene activation (RIGA). In clear contrast, the repeat of three bacterial sequences (lambda-phage, Amp, and ColE1) and three human retroposon sequences (Alu, 5'-untranslated region, and ORF1 of a long interspersed nuclear element) suppressed gene expression, thus reflecting RIGS. RIGS was CpG-independent. We suggest that RIGA might be associated with replication initiation. The discovery of RIGS and RIGA has implications for the repeat in mammalian genome, as well as practical value in recombinant production.

Highlights

Amplification of oncogenes or drug-resistant genes plays a pivotal role in malignant transformation of human cells
Addition of the B-3-31 or G5 repeat sequence consistently increased the expression of d2EGFP from all three vector constructs with or without initiation region (IR) and/or matrix attachment region (MAR) in both the hamster CHO DG44 and human COLO 320DM cells with or without butyrate addition (Fig 2A and 2B)
We examined the influence of plasmid-derived Colicin E1 (ColE1), an ampicillin-resistant gene, and three different transposon sequences that are most abundant in the human genome (50 untranslated region (UTR) of long interspersed nuclear element (LINE), ORF1 of LINE, and Alu)

Summary

Introduction

Amplification of oncogenes or drug-resistant genes plays a pivotal role in malignant transformation of human cells. We have previously reported that a plasmid bearing both a replication initiation region (IR) and a nuclear matrix attachment region (MAR) efficiently mimics gene amplification, and spontaneously generates DMs and HSR in transfected cells [1, 2]. Since this discovery, we have been using this IR/MAR gene amplification technology to uncover the underlying mechanism of gene amplification [3,4,5], investigate basic cell biology (see [6] for an early review), and efficiently produce recombinant proteins [7,8,9]. This was caused by IR/MAR plasmid amplification into the tandem repeat

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