Transcription-dependent silencing of inducible convergent transgenes in transgenic mice

Andrew G Bert,Fernando J Calero-Nieto,Peter N Cockerill

doi:10.1186/1756-8935-3-3

Abstract

BackgroundSilencing of transgenes in mice is a common phenomenon typically associated with short multi-copy transgenes. We have investigated the regulation of the highly inducible human granulocyte-macrophage colony-stimulating-factor gene (Csf2) in transgenic mice.ResultsIn the absence of any previous history of transcriptional activation, this transgene was expressed in T lineage cells at the correct inducible level in all lines of mice tested. In contrast, the transgene was silenced in a specific subset of lines in T cells that had encountered a previous episode of activation. Transgene silencing appeared to be both transcription-dependent and mediated by epigenetic mechanisms. Silencing was accompanied by loss of DNase I hypersensitive sites and inability to recruit RNA polymerase II upon stimulation. This pattern of silencing was reflected by increased methylation and decreased acetylation of histone H3 K9 in the transgene. We found that silenced lines were specifically associated with a single pair of tail-to-tail inverted repeated copies of the transgene embedded within a multi-copy array.ConclusionsOur study suggests that epigenetic transgene silencing can result from convergent transcription of inverted repeats which can lead to silencing of an entire multi-copy transgene array. This mechanism may account for a significant proportion of the reported cases of transgene inactivation in mice.

Highlights

Silencing of transgenes in mice is a common phenomenon typically associated with short multi-copy transgenes
We demonstrated that this transgene is expressed in activated spleen cells in an inducible copy numberdependent fashion at levels equivalent to the endogenous mouse granulocyte- macrophage colony-stimulating-factor (GM-CSF) gene in 10 out of 11 independent lines of transgenic mice
We suggest that the mere act of convergent transcription is unlikely to be sufficient since we have clearly demonstrated that the GM-CSF locus generates both 3’ sense and 5’ anti-sense transcripts and so there must logically be a point in our head-to-tail multi-copy transgenic lines where these opposing transcripts converge without this process leading to silencing of these loci

Summary

Introduction

Silencing of transgenes in mice is a common phenomenon typically associated with short multi-copy transgenes. The mechanism of transgene silencing in mammalian cells is not fully understood, in plants there is evidence that silencing occurs predominantly at sites where transgene arrays include inverted repeated DNA sequences [4,8,9,10,11,12]. In such instances it has been suggested that transgene silencing occurs via convergent transcription and the synthesis of palindromic RNAs and RNAi. In such instances it has been suggested that transgene silencing occurs via convergent transcription and the synthesis of palindromic RNAs and RNAi Until recently, such a mechanism was not believed to exist in mammalian cells. There is substantial evidence that targeting of siRNAs to active genes can direct epigenetic silencing in mammalian cells via histone deacetylation and methylation [13,14,15,16,17,18]

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