The sea urchin sns insulator blocks CMV enhancer following integration in human cells.
Insulators are a new class of genetic elements that attenuate enhancer function directionally. Previously, we characterized in sea urchin a 265-bp-long insulator, termed sns. To test insulator activity following stable integration in human cells, we placed sns between the CMV enhancer and a tk promoter upstream of a GFP transgene of plasmid or retroviral vectors. In contrast to controls, cells transfected or transduced with insulated constructs displayed a barely detectable fluorescence. Southern blot and PCR ruled out vector rearrangement following integration into host DNA; RNase protection confirmed the enhancer blocking activity. Finally, we demonstrate that two cis-acting sequences, previously characterized in sea urchin, are also specific binding sites for human proteins. We conclude that sns interferes with enhancer promoter interaction also in a human chromatin context. The relatively small size, evolutionary conservation and apparent lack of enhancer specificity might result useful in gene transfer experiments in human cells.
Di Simone
P
P
Department of Cell and Developmental Biology, "A.Monroy" University of Palermo, viale delle Scienze, Palermo, 90128, Italy.
Di Leonardo
A
A
Costanzo
G
G
Melfi
R
R
Spinelli
G
G
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003587:Cytomegalovirus / Q000235:genetics*; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D062559:NAV1.8 Voltage-Gated Sodium Channel; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D012617:Sea Urchins; D013539:Simian virus 40 / Q000235:genetics; D015222:Sodium Channels / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D016662:Virus Integration
D004279:DNA, Viral; D062559:NAV1.8 Voltage-Gated Sodium Channel; D011994:Recombinant Proteins; C568317:SCN10A protein, human; D015222:Sodium Channels