American Society of Hematology

Figure 2

$Down-regulation of AID mediated by E2A, Pax5, E2f7, and E2f8 is responsible for CSR impairment by IM. (A) Ectopic expression of AID completely rescued reduction of IgG1 expression caused by IM. Spleen cells were cultured in IL-4 and LPS conditioning medium with or without IM. After 24 hours of prestimulation culture, cells were transduced with retrovirus encoding AID-eGFP or retrovirus encoding eGFP only (control). After a further 48 hours, IgG1 expression was analyzed (bottom panel). A mononuclear cell fraction based on forward scatter/side scatter profiles was gated and sequentially subdivided into an eGFP-positive fraction. This eGFP-positive fraction was analyzed. Ectopic AID expression with 10μM IM increased IgG1 expression from 13.0% to 25.1% versus 20.8% without IM. The BrdU assay revealed that DNA synthesis decreased in the 10μM IM culturing condition. Although the BrdU assay was similar with or without ectopic expression of AID, IgG1 expression was completely rescued by ectopic expression of AID. (B) Average of the IgG1 expression rescue rate among 4 rescue experiments. IgG1 expression of AID(+) IM at 10μM was completely rescued by ectopic expression of AID. (C) Schema illustrating transcriptional binding sites in the Aicda gene promoter region, focusing particularly on region 2 in the first intron. PAX5 and E2A activate the Aicda promoter, whereas E2f7 and E2f8 have silencing effects. (D) The expression levels of 4 transcriptional factors (PAX5, E2A, E2f7, and E2f8) in spleen cells cultured in conditioning medium containing IL-4 and LPS for 72 hours were determined by real-time RT-PCR. All were reduced by IM. E2A expression was most markedly reduced.*P < .05. The y-axis represents mRNA levels of the PAX5, E2A, E2f7, and E2f8 relative to the no-IM control. Levels of each transcriptional factor mRNA were calculated relative to the internal control (GAPDH); n = 2. (E) Protein expression and DNA-binding activity of E2A in spleen cells cultured in conditioning medium containing IL-4 and LPS for 72 hours. The E2A gene encodes 2 transcription factors: E12 and E47. Western blot analysis revealed that expression of E47 in splenocytes cultured in conditioning medium containing LPS and IL-4 was down-regulated by IM to a barely detectable level. DNA affinity precipitation analysis of the same cell extracts using biotinylated E-box probe and its mutant revealed that E-box binding activity of E47 in the extracts was similarly reduced by IM.$

Down-regulation of AID mediated by E2A, Pax5, E2f7, and E2f8 is responsible for CSR impairment by IM. (A) Ectopic expression of AID completely rescued reduction of IgG1 expression caused by IM. Spleen cells were cultured in IL-4 and LPS conditioning medium with or without IM. After 24 hours of prestimulation culture, cells were transduced with retrovirus encoding AID-eGFP or retrovirus encoding eGFP only (control). After a further 48 hours, IgG1 expression was analyzed (bottom panel). A mononuclear cell fraction based on forward scatter/side scatter profiles was gated and sequentially subdivided into an eGFP-positive fraction. This eGFP-positive fraction was analyzed. Ectopic AID expression with 10μM IM increased IgG1 expression from 13.0% to 25.1% versus 20.8% without IM. The BrdU assay revealed that DNA synthesis decreased in the 10μM IM culturing condition. Although the BrdU assay was similar with or without ectopic expression of AID, IgG1 expression was completely rescued by ectopic expression of AID. (B) Average of the IgG1 expression rescue rate among 4 rescue experiments. IgG1 expression of AID(+) IM at 10μM was completely rescued by ectopic expression of AID. (C) Schema illustrating transcriptional binding sites in the Aicda gene promoter region, focusing particularly on region 2 in the first intron. PAX5 and E2A activate the Aicda promoter, whereas E2f7 and E2f8 have silencing effects. (D) The expression levels of 4 transcriptional factors (PAX5, E2A, E2f7, and E2f8) in spleen cells cultured in conditioning medium containing IL-4 and LPS for 72 hours were determined by real-time RT-PCR. All were reduced by IM. E2A expression was most markedly reduced.*P < .05. The y-axis represents mRNA levels of the PAX5, E2A, E2f7, and E2f8 relative to the no-IM control. Levels of each transcriptional factor mRNA were calculated relative to the internal control (GAPDH); n = 2. (E) Protein expression and DNA-binding activity of E2A in spleen cells cultured in conditioning medium containing IL-4 and LPS for 72 hours. The E2A gene encodes 2 transcription factors: E12 and E47. Western blot analysis revealed that expression of E47 in splenocytes cultured in conditioning medium containing LPS and IL-4 was down-regulated by IM to a barely detectable level. DNA affinity precipitation analysis of the same cell extracts using biotinylated E-box probe and its mutant revealed that E-box binding activity of E47 in the extracts was similarly reduced by IM.

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