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Molecular and Cellular Biology, May 1999, p. 3714-3726, Vol. 19, No. 5
Fred Hutchinson Cancer Research
Center1 and University of Washington
School of Medicine,2 Seattle, Washington;
Microbiology Department, Dartmouth Medical Center, Lebanon, New
Hampshire3; Albert Einstein College
of Medicine, Bronx, New York4; and
Department of Biochemistry, University of Sydney, Sydney,
New South Wales, Australia5
Received 22 September 1998/Returned for modification 11 November
1998/Accepted 16 February 1999
A constitutive DNase I-hypersensitive site 5' of the chicken
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Chicken
-Globin 5'HS4 Boundary Element
Blocks Enhancer-Mediated Suppression of Silencing
-globin locus, termed 5'HS4 or cHS4, has been shown to insulate a
promoter from the effect of an upstream enhancer and to reduce position
effects on mini-white expression in Drosophila
cells; on the basis of these findings, it has been designated a
chromatin insulator. We have examined the effect of the cHS4 insulator
in a system that assays both the level of gene expression and the rate
of transcriptional silencing. Because transgenes flanked by insulator
elements are shielded from position effects in Drosophila cells, we tested the ability of cHS4 to protect transgenes from position effects in mammalian cells. Flanking of an expression vector
with the cHS4 insulator in a colony assay did not increase the number
of G418-resistant colonies. Using lox/cre-based recombinase-mediated cassette exchange to control integration position, we studied the
effect of cHS4 on the silencing of an integrated -geo reporter at
three genomic sites in K562 erythroleukemia cells. In this assay,
enhancers act to suppress silencing but do not increase expression
levels. While cHS4 blocked enhancement at each integration site, the
strength of the effect varied from site to site. Furthermore, at some
sites, cHS4 inhibited the enhancer effect either when placed between
the enhancer and the promoter or when placed upstream of the enhancer.
These results suggest that the activity of cHS4 is not dominant in all
contexts and is unlikely to prevent silencing at all genomic
integration sites.
*
Corresponding author. Present address: The Victor Chang
Cardiac Research Institute, 384 Victoria St., Darlinghurst, Sydney, NSW
2010, Australia. Phone: 61-2-9295-8500. Fax: 61-2-9295-8501. E-mail:
dmartin{at}fhcrc.org.
Molecular and Cellular Biology, May 1999, p. 3714-3726, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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