Induction of germline (GL) ε transcripts, an essential step preceding immunoglobulin (Ig) isotype switching to IgE, requires activation of transcription factors by IL-4 and a B cell activator, e.g. CD40 ligand (CD40L). AP-1 (Fos and Jun), induced transiently by CD40L, binds a DNA element in the mouse GL ε promoter. AP-1 synergizes with Stat6 to activate both the intact GL ε promoter and a minimal heterologous promoter driven by the AP-1 and Stat6 sites of the mouse GL ε promoter. By contrast, C/EBPβ, which transactivates the human GL ε promoter, inhibits IL-4 induction of the mouse promoter, probably by attenuating the synergistic interaction between AP-1 and Stat6. Furthermore, AP-1 does not transactivate the human GL ε promoter. Thus, due to selective binding of either AP-1 or C/EBP proteins, induction of GL ε transcripts in mouse and human may be regulated differently. In addition to AP-1, NF-кB activity is also induced by CD40L stimulation in normal B cells. Using GST pulldown assays and coimmunoprecipitation techniques I show that NF-кB and tyrosine-phosphorylated Stat6 can directly bind each other in vitro and in vivo. When Stat6 and NF-кB proteins are co-expressed in human embryonic kidney 293 (HEK 293) cells, an IL-4-inducible reporter gene containing both cognate binding sites in the promoter is synergistically activated in the presence of IL-4. Furthermore, the same IL-4-inducible reporter gene is also synergistically activated by the endogenous Stat6 and NF-кB proteins in IL-4-stimulated B lymphoma cells I.29μ. Consistently, by using nuclear extracts from transfected HEK 293 cells and from I.29μ B cells in electrophoretic mobility shift assays (EMSAs), I show that Stat6 and NF-кB bind cooperatively to a DNA probe containing both sites, and the presence of a complex formed by their cooperative binding correlates with the synergistic activation of the promoter by Stat6 and NF-кB. I conclude that the direct interaction between Stat6 and NF-кB may provide the basis for synergistic activation of the GL ε promoter. Finally, although mouse GL ε transcripts have a half-life of approximately 100 min, the RNA level continues to increase for up to 24 h and the promoter appears to be active for at least 2 days after B cell activation. These data suggest that induction of AP-1 and NF-кB activities by CD40L, although transient, is required for activation of the mouse GL ε promoter by IL-4-induced Stat6.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-1317 |
| Date | 01 July 2000 |
| Creators | Shen, Ching-Hung |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved. |
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