Oligonucleotide inhibition of IL2R alpha mRNA transcription by promoter region collinear triplex formation in lymphocytes.
The promoter region of the IL2R alpha gene 5' flanking sequence contains enhancer elements crucial for binding nuclear factors which upregulate transcription following T lymphocyte activation. A 3' exonuclease resistant oligonucleotide (3'A-IL28p, terminated by a free amine group at its 3' end) was designed to bind to the IL2R alpha promoter region from -273 to -246, forming a collinear triplex spanning the kappa B enhancer (-266 to -256) as well as most of the serum response element (CArG box, -251 to -244). The binding site specificity of this oligonucleotide was demonstrated in electrophoretic mobility shift assays and by inhibition of restriction endonuclease (HinfI) cleavage within the segment of the target DNA predicted to form a triplex with the oligonucleotide. Intact normal lymphocytes, preincubated for 2h with 3'A-IL28p, accumulated less IL2Ralpha mRNA relative to other mRNAs (c-myc, beta-actin, IL2R beta, IL-6) for up to 12h after PHA stimulation, than did lymphocytes treated with a control oligomer of similar composition but different sequence. Nuclear run-on studies demonstrated that the rate of IL2R alpha mRNA synthesis relative to c-myc and beta-actin was also selectively diminished by treatment with 3'A-IL28p. These experiments suggest that transcription of individual genes can be selectively modulated in living cells by sequence specific collinear triplex formation in regulatory enhancer sequences.
Orson
F M
FM
Veterans Affairs Medical Center, Houston, TX 77030.
Thomas
D W
DW
McShan
W M
WM
Kessler
D J
DJ
Hogan
M E
ME
D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D006801:Humans; D007700:Kinetics; D008213:Lymphocyte Activation; D008214:Lymphocytes; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects*
D009838:Oligodeoxyribonucleotides; D015375:Receptors, Interleukin-2