Class switch recombination signals induce lymphocyte-derived Spo11 expression and Spo11 antisense oligonucleotide inhibits class switching.
Recently, we showed that mouse Spo11 is induced in normal mu(+) B cells by class switch recombination (CSR) stimuli, by RT-PCR using primers based on the reported cDNA sequence of testis-derived Spo11 (test-Spo11) cDNA. In the present study, we first determined the cDNA sequence of lymphocyte-derived Spo11 (lym-Spo11). The 5' upstream portion had an as yet unreported sequence but the remaining part from exons 2 to 12 and the subsequent 3'UTR was completely identical to that of test-Spo11. RT-PCR analysis indicated that lymphocytes express lym-Spo11 but not test-Spo11. Second, we showed that lym-Spo11 is strongly induced (above eightfold) in the IgA CSR system of LPS-stimulated mu(+)B cells in the presence of all-trans retinoic acid and IL-4. Finally, we examined whether lym-Spo11 antisense S-oligonucleotide (AS) can inhibit CSR reactions in three in vitro CSR systems, IgA,IgG1, and IgE. Lym-Spo11 AS or the sense oligonucleotide was added to the cultures at the start, and total RNA was extracted after 4 days. IgA, IgG1, and IgE mRNAs (J(H)C(H)) and mature germline C(H) transcripts (I(H)C(H)) were quantitatively assayed by RT-PCR. AS inhibited J(H)C(H) expression dose-dependently. In all three systems, the maximum inhibition by 20 microM AS was in the range of 60 to 90%. Interestingly, I(H)C(H) was also inhibited by AS to a similar extent as J(H)C(H). These results suggested that lym-Spo11 plays an important role in the initiation step of CSR.
Tokuyama
H
H
Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa-ken, 920-0934, Japan.
Tokuyama
Y
Y
D000818:Animals; D001483:Base Sequence; D004706:Endodeoxyribonucleases; D004950:Esterases / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D007070:Immunoglobulin A / Q000276:immunology; D017578:Immunoglobulin Class Switching / Q000235:genetics*; D015847:Interleukin-4 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008214:Lymphocytes; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D020319:Oligodeoxyribonucleotides, Antisense; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000235:genetics*; D013873:Thionucleotides; D014212:Tretinoin / Q000494:pharmacology; D000099257:Meiotic Recombination Protein SPO11
D004706:Endodeoxyribonucleases; D004950:Esterases; D007070:Immunoglobulin A; D015847:Interleukin-4; D008070:Lipopolysaccharides; D020319:Oligodeoxyribonucleotides, Antisense; D013873:Thionucleotides; D014212:Tretinoin; D000099257:Meiotic Recombination Protein SPO11