Engagement of membrane immunoglobulin enhances Id3 promoter activity in WEHI‐231 B lymphoma cells

Aim : We have recently shown that engagement of membrane immunoglobulin (mIg) induced upregulation of inhibitor of differentiation 3 (Id3) mRNA, resulting in growth arrest at G1 phase in WEHI‐231 cells. In the present study, we examined whether engagement of mIg will affect promoter activity of the Id3 gene in WEHI‐23 1 cells. Methods : DNA fragments corresponding to the 5′‐flanking region of mId3 gene were amplified by polymerase chain reaction (PCR) using genomic DNA as the template. Three DNA fragments upstream of the transcription start site (+1) of the mId3 gene were subcloned into the luciferase reporter vector PGV‐B2. The recombinant constructs were transiently transfected into WEHI‐231 cells by an electroporation method. After incubation for 24 h, WEHI‐231 cells were stimulated with 10 mg/L anti‐IgM or irradiated CD40L‐expressing NIH3T3 cells or control NIH3T3 cells for further 24 h, followed by assay for luciferase activity. Results : The luciferase analysis demonstrated that basal promoter activity of the Id3 gene was found in the region between ‐200 and +54. The Id3 promoter activity was increased 2‐fold following stimulation with anti‐IgM, but not CD40L, compared with medium alone. Conclusion : The mIg‐mediated upregulation of Id3 expression is controlled, at least in part, through transcriptional regulation, as assessed by luciferase assay.