Nucleic Acids Research, Vol 24, Issue 24 4902-4909, Copyright © 1996 by Oxford University Press
D Garrick, H Sutherland, G Robertson and E Whitelaw
There are now many mammalian examples in which single cell assays of
transgene activity have revealed variegated patterns of expression. We have
previously reported that transgenes in which globin regulatory elements
drive the lacZ reporter gene exhibit variegated expression patterns in
mouse erythrocytes, with transgene activity detectable in only a
sub-population of circulating erythroid cells. In order to elucidate the
molecular mechanism responsible for variegated expression in this system,
we have compared the chromatin structure and methylation status of the
transgene locus in expressing and non- expressing populations of
erythrocytes. We find that there is a difference in the chromatin
conformation of the transgene locus between the two states. Relative to
active transgenes, transgene loci which have been silenced exhibit a
reduced sensitivity to general digestion by DNase I, as well as a failure
to establish a transgene-specific DNase I hypersensitive site, suggesting
that silenced transgenes are situated within less accessible chromatin
structures. Surprisingly, the restrictive chromatin structure observed at
silenced transgene loci did not correlate with increased methylation, with
transgenes from both active and inactive loci appearing largely
unmethylated following analysis with methylation-sensitive restriction
enzymes and by sequencing PCR products derived from bisulphite-converted
genomic DNA.
ARTICLES
Variegated expression of a globin transgene correlates with chromatin accessibility but not methylation status
Department of Biochemistry, University of Sydney, Australia.
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