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Vol. 12, No. 18, pp. 2852-2862, September 15, 1998
Laboratory of Molecular Biology, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, Maryland 20892 USA
The constitutive DNase I hypersensitive site at the 5' end of
the chicken 
-globin locus marks the boundary of the active chromatin
domain in erythroid cells. The DNA sequence containing this site has
the properties of an insulator, as shown by its ability in stable
transformation experiments to block enhancer-promoter interaction when
it lies between the two, but not when it lies outside, and to protect
against position effects in Drosophila. We now show that the
chicken insulator can protect a stably integrated gene, which is
otherwise subject to great variability of expression, from
chromatin-mediated repression in cell culture. When the integrated reporter gene is surrounded by insulator elements, stably transformed cell lines display consistent enhancer-dependent expression levels, in
accord with the strength of the enhancer. In the absence of insulators,
long-term nonselective propagation of cells carrying the integrated
reporter gene results in gradual extinction of the reporter's
expression, with expression patterns from tandemly repeated inserted
genes suggesting that the extinction of adjacent genes is coupled. We
show that the uninsulated reporter genes, in addition to becoming
transcriptionally inactive, lose several epigenetic hallmarks of active
chromatin, including nuclease accessibility, DNA hypomethylation, and
histone hyperacetylation during time in culture. Treatment with
inhibitors of histone deacetylase or DNA methylation reverses the
extinction of the uninsulated genes. Extinction is completely prevented
by flanking the reporter construct with insulators. Furthermore, in
contrast to the uninsulated reporter genes, chromatin over the
insulated genes retains nuclease accessibility and histone
hyperacetylation. However, there is no clear correlation between the
presence of the insulators and the level of DNA methylation. This leads
us to propose a model for the insulator's ability to protect against
extinction in the transformed cell lines and to function as a chromatin
boundary for the chicken -globin locus in normal erythroid cells.
[Key Words: Insulator; transcription; DNA methylation; histone acetylation; silencing]
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