Inhibition of interleukin-4- and CD40-induced IgE germline gene promoter activity by 2'-aminoethoxy-modified triplex-forming oligonucleotides.
Elevated levels of IgE are intimately associated with a number of allergic diseases, such as allergic rhinitis or asthma. Therefore, prevention of IgE production in human B-cells represents an attractive therapeutic target. IL-4-induced IgE germline gene transcription represents a crucial early step during IgE isotype switch differentiation. Gene induction is orchestrated by the coordinated action of the transcription factors STAT6 (signal transducer and activator of transcription), NF-kappaB, PU.1, and C/EBP. This study shows that 2'-aminoethoxy-modified oligonucleotides, which partially overlap with the STAT6 and the adjacent PU.1/NF-kappaB binding site, inhibit DNA binding of all three proteins with high affinity in a dose- and time-dependent fashion in vitro. Loss of protein binding correlated strongly with increasing DNA triplex formation. Importantly, the oligomers also effectively displaced pre-bound recombinant NF-kappaB p50 from double-stranded DNA in vitro. Functionally, the oligonucleotides led to a selective inhibition of IL-4-induced reporter gene activity from a construct driven by the IgE germline gene promoter in human B-cells. These data confirm the critical role of this cytokine-responsive regulatory region in IgE germline gene induction and further support the concept of specific modulation of gene expression by DNA triplex formation induced with chemically modified oligonucleotides.
Department of Allergic Diseases, Novartis Research Institute, Brunnerstrasse 59, A-1230 Vienna, Austria.
A M
AM
Stütz
J
J
Hoeck
F
F
Natt
B
B
Cuenoud
M
M
Woisetschläger
D001483:Base Sequence; D019013:CD40 Antigens / Q000187:drug effects* / Q000494:pharmacology; D002460:Cell Line; D004247:DNA; D005786:Gene Expression Regulation / Q000187:drug effects; D005854:Germ Cells; D006801:Humans; D007073:Immunoglobulin E / Q000235:genetics*; D015847:Interleukin-4 / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D009841:Oligonucleotides / Q000737:chemistry / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation
D019013:CD40 Antigens; D009841:Oligonucleotides; D015847:Interleukin-4; D007073:Immunoglobulin E; D004247:DNA