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Molecular and Cellular Biology, June 2006, p. 4028-4040, Vol. 26, No. 11
0270-7306/06/$08.00+0     doi:10.1128/MCB.02189-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Assembly and Maintenance of Heterochromatin Initiated by Transgene Repeats Are Independent of the RNA Interference Pathway in Mammalian Cells{dagger}

Fangwei Wang,1,2 Naoki Koyama,1 Hiroko Nishida,1 Tokuko Haraguchi,3 Walter Reith,4 and Toshiro Tsukamoto1*

Genomics Research Institute, Utsunomiya University, 350 Mine-machi, Utsunomiya, Tochigi 321-8505, Japan,1 College of Animal Sciences, Zhejiang University, Hangzhou 310029, People’s Republic of China,2 Cell Biology Group, Kansai Advanced Research Center, National Institute of Information and Communications Technology, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan,3 Department of Pathology and Immunology, University of Geneva Medical School, Centre Medical Universitaire, 1 rue Michel-Servet, CH-1211 Geneva, Switzerland4

Received 11 November 2005/ Returned for modification 3 January 2006/ Accepted 8 March 2006

A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.

* Corresponding author. Mailing address: Genomics Research Institute, Utsunomiya University, 350 Mine-machi, Utsunomiya, Tochigi 321-8505, Japan. Phone: 81-28-649-5527. Fax: 81-28-649-8651. E-mail: tsukamot{at}cc.utsunomiya-u.ac.jp.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, June 2006, p. 4028-4040, Vol. 26, No. 11
0270-7306/06/$08.00+0     doi:10.1128/MCB.02189-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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