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Molecular and Cellular Biology, January 2001, p. 298-309, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.298-309.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Position Effects Are Influenced by the Orientation of a Transgene with Respect to Flanking Chromatin

Yong-Qing Feng,1 Matthew C. Lorincz,2 Steve Fiering,3 John M. Greally,4 and Eric E. Bouhassira1,*

Division of Hematology, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York1; Fred Hutchinson Cancer Research Center, Seattle, Washington2; Microbiology Department, Dartmouth Medical School, Hanover, New Hampshire3; and Department of Genetics, Yale University School of Medicine, New Haven, Connecticut4

Received 30 June 2000/Returned for modification 10 August 2000/Accepted 28 September 2000

We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

* Corresponding author. Mailing address: Division of Hematology, Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430 2188. Fax: (718) 824 3153. E-mail: Bouhassi{at}aecom.yu.edu.

Molecular and Cellular Biology, January 2001, p. 298-309, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.298-309.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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