title: Efficiency of Iε promoter-directed switch recombination in GFP expression-based switch constructs works synergistically with other promoter and/or enhancer elements but is not tightly linked to the strength of transcription.
abstract: One key unresolved issue in immunoglobulin class switch recombination (CSR) is how the accessibility of the switch region for CSR is controlled. To better understand the nature of accessibility control for human Ig CSR, we developed a novel inducible switch recombination assay based on expression of green fluorescence protein (GFP) from switch constructs undergoing substrate switch recombination (SSR). Efficient SSR depends on the cytokine-inducible Iepsilon promoter and co-stimulation with IL-4+anti-CD40. Characterization of SSR reveals that both S-S deletional recombination and S-S inversion occur. We show that the IL-4-inducible Iepsilon promoter (pIepsilon) selectively determines the efficiency of the accessibility for SSR. However, the pIepsilon-induced transcription, by itself,is not sufficient to direct efficient SSR. For efficient SSR, both pIepsilon-driven transcriptional activity and an additional promoter/enhancer-derived activity are required. The efficiency of SSR is not tightly correlated with the strength of the combined transcriptional activity. Our results suggest that the mechanism(s) underlying the transcriptional activity, e.g. DNA modification is important for controlling the accessibility for efficient switch recombination.
authors.affiliation: Division of Clinical Immunology, Department of Medicine, UCLA School of Medicine, Los Angeles, USA.
authors.forename: Ke
authors.initials: K
authors.lastname: Zhang
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.forename: Takechiyo
authors.initials: T
authors.lastname: Yamada
authors.forename: Michael
authors.initials: M
authors.lastname: Vu
authors.forename: Anna
authors.initials: A
authors.lastname: Lee
authors.forename: Andrew
authors.initials: A
authors.lastname: Saxon
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001483:Base Sequence; D019013:CD40 Antigens / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D017578:Immunoglobulin Class Switching; D015847:Interleukin-4 / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014158:Transcription, Genetic
chemical_list: D000911:Antibodies, Monoclonal; D019013:CD40 Antigens; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D015847:Interleukin-4; D004247:DNA