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.
Stütz
A M
AM
Department of Allergic Diseases, Novartis Research Institute, Brunnerstrasse 59, A-1230 Vienna, Austria.
Hoeck
J
J
Natt
F
F
Cuenoud
B
B
Woisetschläger
M
M
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