title: Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9.
abstract: Transgenes stably integrated into cells or animals in many cases are silenced rapidly, probably under the influence of surrounding endogenous condensed chromatin. This gene silencing correlates with repressed chromatin structure marked by histone hypoacetylation, loss of methylation at H3 lysine 4, increase of histone H3 lysine 9 methylation as well as CpG DNA methylation at the promoter. However, the order and the timing of these modifications and their impact on transcription inactivation are less well understood. To determine the temporal order of these events, we examined a model system consisting of a transgenic cassette stably integrated in chicken erythroid cells. We found that histone H3 and H4 hypoacetylation and loss of methylation at H3 lysine 4 all occurred during the same window of time as transgene inactivation in both multicopy and low-copy-number lines. These results indicate that these histone modifications were the primary events in gene silencing. We show that the kinetics of silencing exclude histone H3 K9 and promoter DNA methylation as the primary causative events in our transgene system.
authors.affiliation: Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Vesco
authors.initials: V
authors.lastname: Mutskov
authors.forename: Gary
authors.initials: G
authors.lastname: Felsenfeld
mesh_terms: D000107:Acetylation; D001355:Alpharetrovirus / Q000235:genetics; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D002460:Cell Line; D002472:Cell Transformation, Viral; D002645:Chickens; D002843:Chromatin / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation / Q000187:drug effects; D041905:Erythroid Cells; D005434:Flow Cytometry; D018628:Gene Dosage; D020868:Gene Silencing / Q000187:drug effects*; D017930:Genes, Reporter; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008745:Methylation; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D019076:Transgenes
chemical_list: D002843:Chromatin; D006657:Histones; D012333:RNA, Messenger; D008239:Lysine; D001374:Azacitidine