Abstract
The 5'-flank of the H19 gene harbors a differentially methylated imprinting control region that represses the maternally derived Igf2 and paternally derived H19 alleles. Here we show that the H19 imprinting control region (ICR) is a potent silencer when positioned in a promoter-proximal position. The silencing effect is not alleviated by trichostatin A treatment, suggesting that it does not involve histone deacetylase functions. When the H19 ICR is separated from the promoter by more than 1.2 +/- 0.3 kb, however, trichostatin A stimulates promoter activity 10-fold. Deletion analyses revealed that the silencing feature extended throughout the ICR segment. Finally, chromatin immunopurification analyses revealed that the H19 ICR prevented trichostatin A-dependent reacetylation of histones in the promoter region in a proximal but not in a distal position. We argue that these features are likely to be side effects of the H19 ICR, rather than explaining the mechanism of silencing of the paternal H19 allele. We issue a cautionary note, therefore, that the interpretation of insulator/silencer data could be erroneous should the distance issue not be taken into consideration.
Highlights
From the ‡Department of Development & Genetics, Evolution Biology Centre, Uppsala University, Norbyvagen 18A, S-752 36 Uppsala, Sweden and the ¶Laboratory of Immunopathology, NIAID, National Institutes of Health, Bethesda, Maryland 20892
We show that the H19 imprinting control region (ICR) is a potent silencer when positioned in a promoter-proximal position
We focused on a 1.2-kb segment of the H19 ICR that encompasses a major portion of the differentially methylated domain (Fig. 1A) and displays strong insulator activity (4)
Summary
Received for publication, September 26, 2001, and in revised form, December 27, 2001. Chromatin immunopurification analyses revealed that the H19 ICR prevented trichostatin A-dependent reacetylation of histones in the promoter region in a proximal but not in a distal position We argue that these features are likely to be side effects of the H19 ICR, rather than explaining the mechanism of silencing of the paternal H19 allele. The notion that the methylated status of the H19 ICR involves recruitment of repressive factors that propagate an inactive chromatin toward the H19 promoter (3) is supported by the observation that MeCP2 and MBD 2/3 are associated preferentially with the paternal H19 ICR allele in chromatin immunopurification assays.[2] because the unmethylated H19 ICR performed as an efficient silencer in transgenic Drosophila assays, it has been claimed that the H19 ICR contains silencing features by default (9). We set out to examine if the unmethylated H19 ICR can act as a silencer under certain circumstances
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