STAT1 binds to Prdm1 promoter and regulates Prdm1 expression and IgM production in MZ B cells in response to TLR stimulation. (A) CH12F3 cells were subjected to lentivirus-based knockdown of luciferase or Stat1, and then stimulated with or without LPS for 4 d. IgM in the culture supernatant was measured by ELISA (n = 3). Results represent three experiments. (B) Same as in A, except total RNA from the cells treated for 2 d were subjected to RT-QPCR using primers to Prdm1 (n = 6). Results represent three experiments. (C) ChIP-seq data (Robertson et al., 2007) were analyzed for STAT1 binding regions in human Prdm1 loci. The corresponding exons and introns of Prdm1 are shown. Comparison of conserved GAS-like 1 (GASL1) and GAS-like 2 (GASL2) in human and mouse Prdm1 promoters are shown. (D) CH12F3 cells were stimulated with 5 µg/ml LPS for 6 h, and ChIP was performed using anti-STAT1 antibody. Relative abundance of GASL1 (A), GASL2 (B), and exon 7 are shown (n = 6). Results represent three experiments. (E) Reporter plasmid containing the promoter region of Prdm1 (–2,052 to +207 bp) was cotransfected with pCMV-DsRed, an internal control plasmid, into shLacZ- or shStat1-treated CH12F3 cells for 24 h, followed by stimulation with 5 µg/ml LPS for the indicated times before being harvested and subjected to reporter activity assay. Reporter activity was normalized to the percentage of DsRed-positive cells (n = 3). Results represent three experiments. (F) MZ B cells from WT or Stat1^−/− mice were stimulated with 2 µg/ml LPS for 24 h and transduced with recombinant retrovirus pGC-YFP or pGC-Blimp-1-YFP for 2 d. The YFP⁺ cells were sorted out and incubated for another 24 h before measuring IgM production by ELISA (n = 3). Results represent three experiments. All values are shown as the means ± SD *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test.