title: cHS4 insulator-mediated alleviation of promoter interference during cell-based expression of tandemly associated transgenes.
abstract: Expression of multiple transgenes in cells or whole organisms is a powerful tool for basic research of various biological functions and potentially for clinical applications such as gene therapy. As a model system for this purpose, multi-cDNA expression clones were constructed harboring two tandemly situated fluorescent protein cDNAs as reporter genes on a single plasmid. When 293 cells were transfected transiently, the downstream gene displayed significantly lower expression when compared with the upstream cDNA. Such transcriptional interference was markedly alleviated by inserting an insulator cassette of cHS4 elements derived from the chicken beta-globin locus at a site between two neighboring cDNAs. The introduction of cHS4 resulted in a drastic increase of the expression level of the downstream cDNA, ensuring comparable expression levels of the tandem transgenes. Using a chromatin immunoprecipitation assay, we demonstrated that CTCF and USF1 that recruit histone-modifying complexes are bound to the cHS4 region. Depletion of CTCF or USF1 by siRNA resulted in relief of the diminished effect. Our data thus indicate that CTCF and histone modifiers recruited by USF1 cooperatively mediate the suppression of transcriptional interference between apposed genes, presumably by facilitating active chromatin conformation over the transgenes.
authors.affiliation: Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Yahata
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Maeshima
authors.forename: Takefumi
authors.initials: T
authors.lastname: Sone
authors.forename: Taichi
authors.initials: T
authors.lastname: Ando
authors.forename: Masaru
authors.initials: M
authors.lastname: Okabe
authors.forename: Naoko
authors.initials: N
authors.lastname: Imamoto
authors.forename: Fumio
authors.initials: F
authors.lastname: Imamoto
mesh_terms: D000818:Animals; D002460:Cell Line; D002645:Chickens; D047369:Chromatin Immunoprecipitation; D018076:DNA, Complementary; D020868:Gene Silencing; D006657:Histones / Q000378:metabolism*; D006801:Humans; D011401:Promoter Regions, Genetic; D019076:Transgenes
chemical_list: D018076:DNA, Complementary; D006657:Histones