title: Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects.
abstract: The prototypic chromatin insulator cHS4 has proven effective at reducing repressive chromosomal position effects on retroviral vector expression. We report here studies designed to identify the minimal chicken hypersensitive site-4 (cHS4) sequences necessary for this activity. Using a gammaretroviral reporter vector and expression analysis in cell lines and primary mouse hematopoietic progenitor colonies, we found that a 250-bp core fragment reported to contain most of the cHS4 insulating activity failed to prevent silencing when used alone, although some barrier activity was observed when this fragment was combined with a 790-bp, but not 596-bp, spacer. Similar studies showed that four copies of a 90-bp fragment containing the cHS4 enhancer-blocking activity actually repressed vector green fluorescent protein (GFP) expression. In contrast, a 400-bp fragment containing the 250-bp core plus 3' flanking sequences protected vector expression to the same degree as the full-length 1.2-kb fragment. The 400-bp fragment activity was confirmed in a lentiviral vector expressing human beta-globin in murine erythroid leukemia (MEL) cells. Taken together, these studies indicate that the insulating activity of the 250-bp cHS4 core can be influenced by distance, and identify an extended core element that confers full barrier activity in the setting of two different classes of retroviral vectors.
authors.affiliation: Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA.
authors.forename: Mari
authors.initials: M
authors.lastname: Aker
authors.forename: Julie
authors.initials: J
authors.lastname: Tubb
authors.forename: Amy C
authors.initials: AC
authors.lastname: Groth
authors.forename: Anatoly A
authors.initials: AA
authors.lastname: Bukovsky
authors.forename: Adam C
authors.initials: AC
authors.lastname: Bell
authors.forename: Gary
authors.initials: G
authors.lastname: Felsenfeld
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Kiem
authors.forename: George
authors.initials: G
authors.lastname: Stamatoyannopoulos
authors.forename: David W
authors.initials: DW
authors.lastname: Emery
mesh_terms: D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics*; D005914:Globins / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D038101:Insulator Elements / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D041681:NIH 3T3 Cells; D017422:Sequence Analysis, DNA; D014161:Transduction, Genetic
chemical_list: D049452:Green Fluorescent Proteins; D005914:Globins; D004247:DNA