Multiple cis elements within the Igf2/H19 insulator domain organize a distance-dependent silencer. A cautionary note.
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.
Ginjala
Vasudeva
V
Department of Development & Genetics, Evolution Biology Centre, Uppsala University, Norbyvägen 18A, S-752 36 Uppsala, Sweden.
Holmgren
Claes
C
Ullerås
Erik
E
Kanduri
Chandrasekhar
C
Pant
Vinod
V
Lobanenkov
Victor
V
Franklin
Gary
G
Ohlsson
Rolf
R
D000935:Antifungal Agents / Q000494:pharmacology; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002822:Choriocarcinoma; D019175:DNA Methylation; D017931:DNA Primers; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing; D017930:Genes, Reporter; D018392:Genomic Imprinting; D006657:Histones / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000187:drug effects; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D014594:Uterine Neoplasms
D000935:Antifungal Agents; D017931:DNA Primers; C094335:H19 long non-coding RNA; D006657:Histones; D006877:Hydroxamic Acids; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; C012589:trichostatin A; D015500:Chloramphenicol O-Acetyltransferase