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 th e present study, we first determined the cDNA sequence of lymphocyte-derive d Spo11 (lym-Spo11). The 5' upstream portion had an as yet unreported seque nce 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 ly m-Spo11 is strongly induced (above eightfold) in the IgA CSR system of LPS- stimulated mu(+)B cells in the presence of all-traps retinoic acid and IL-4 . Finally, we examined whether lym-Spo11 antisense S-oligonucleotide (AS) c an 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 (INCH) were quantitatively as sayed by RT-PCR. AS inhibited JHCH expression dose-dependently. In all thre e systems, the maximum inhibition by 20 muM AS was in the range of 60 to 90 %. Interestingly, IHCH was also inhibited by AS to a similar extent as JHCH . These results suggested that lym-Spo11 plays an important role in the ini tiation step of CSR. (C) 2001 Academic Press.