S region transcription per se promotes basal IgE class switch recombination but additional factors regulate the efficiency of the process.
Stimulation of B lymphocytes with a combination of lipopolysaccharide (LPS) and interleukin-4 (IL-4) induces germline transcription of and subsequent switching to the epsilon heavy chain constant region (C epsilon) gene. Mature germline C epsilon transcripts contain a non-coding exon (I epsilon exon) spliced to the C epsilon exons. To distinguish between the potential roles of germline transcription and those of germline transcripts in regulating the class switch process, we replaced the LPS- and IL-4-inducible I epsilon promoter and exon in ES cells with an LPS-inducible E mu enhancer/VH promoter expression cassette. Wildtype, heterozygous or homozygous mutant ES cells were injected into RAG-2 deficient blastocysts to generate somatic chimeras in which all B cells derived from ES cells. In contrast to normal B cells, heterozygous and homozygous mutant B cells had substantial transcription through the epsilon switch recombination region (S epsilon) following treatment with LPS alone and, under these conditions, both underwent low level switching (10- to 100-fold less than wildtype cells stimulated with LPS + IL-4) to IgE production. Heterozygous mutant cells underwent switching to IgE at essentially wildtype levels when stimulated with LPS and IL-4. However, homozygous mutant cells still showed extremely low levels of switching to IgE upon LPS and IL-4 stimulation. Analyses of hybridomas from heterozygous mutants indicated that the mutation is cis-acting and normal switching to other isotypes indicated that it is specific for IgE. Thus transcription per se generates low levels of class switch recombination in the absence of I region sequences. However, we demonstrate for the first time that, for optimal efficiency, the process requires the presence of the intact I region and/or I region promoter in cis, implicating factors beyond transcription through the S region in the regulation of class switching.
Bottaro
A
A
Howard Hughes Medical Institute, Children's Hospital, Boston, MA.
Lansford
R
R
Xu
L
L
Zhang
J
J
Rothman
P
P
Alt
F W
FW
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D005091:Exons; D005786:Gene Expression Regulation; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D005812:Genes, Switch; D006579:Heterozygote; D006720:Homozygote; D007073:Immunoglobulin E / Q000235:genetics*; D015847:Interleukin-4 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D015183:Restriction Mapping; D013234:Stem Cells; D014158:Transcription, Genetic
D008070:Lipopolysaccharides; D015847:Interleukin-4; D007073:Immunoglobulin E; D004247:DNA