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Journal of Virology, August 2003, p. 9000-9007, Vol. 77, No. 16
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.16.9000-9007.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification of an Insulator in AAVS1, a Preferred Region for Integration of Adeno-Associated Virus DNA

Toshihiko Ogata,1 Takuyo Kozuka,2 and Tadahito Kanda1*

Division of Molecular Genetics, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo 162-8640,1 Department of Radiotherapy, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Kamiikebukuro, Toshima-ku, Tokyo 170-8455, Japan2

Received 17 March 2003/ Accepted 13 May 2003

In latent adeno-associated virus (AAV) infection, the viral genome is integrated preferentially into the human chromosome 19 q arm at a specific region designated AAVS1, which has an open chromatin conformation as indicated by the presence of a DNase I-hypersensitive site (DHS-S1). We examined whether an insulator, which defines the domain of gene expression by directionally blocking the action of enhancers and by preventing the spread of heterochomatin, is present near the DHS-S1 in the middle of a 2.6-kbp AAVS1-related DNA fragment used in this study. The fragment, cloned into an Epstein-Barr virus (EBV)-based eukaryotic episomal plasmid, was introduced into HEK293 cells. The DHS-S1 on the plasmid replicating in the nuclei was hypersensitive to DNase I digestion, and thus, the EBV plasmid system was used in an enhancer-blocking assay with the 2.6-kbp DNA and two shortened DNAs, of 1.6 kbp and 336 bp, containing DHS-S1. The three DNA fragments, when inserted in the proper direction between the cytomegalovirus immediate-early enhancer and minimal promoter, repressed the expression of a reporter gene. Thus, the enhancer-blocking activity was located within the 336-bp DNA containing the entire region (300 bp) of DHS-S1. To investigate the prevention of repression caused by heterochromatin, a transgene-expressing cassette flanked by the two 336-bp DNAs placed in the enhancer-blocking direction was introduced into HEK293 and HeLa cells. All the cell clones examined with the cassette integrated into cell DNA continued to express the transgene, which indicates that the pair of 336-bp DNA apparently prevented the spread of heterochromatin. The results show that an insulator lies between nucleotides 17 and 354 near the DHS-S1 in AAVS1. In a gel shift test, the 336-bp DNA did not bind an in vitro-prepared CCCTC-binding factor that binds to the chicken ß-globin insulator, suggesting that the AAVS1 insulator requires an as yet unidentified binding protein. The newly identified AAVS1 insulator is likely to contribute to the maintenance of an open chromatin conformation that affects the life cycle of AAV.

* Corresponding author. Mailing address: Division of Molecular Genetics, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Phone: (81) 3-5285-1111. Fax: (81) 3-5285-1166. E-mail: kanda{at}nih.go.jp.

Journal of Virology, August 2003, p. 9000-9007, Vol. 77, No. 16
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.16.9000-9007.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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