Treatment of splenic B lymphocytes and certain B-lineage cell lines with the mitogen lipopolysaccharide (LPS) and the lymphokine interleukin-4 (IL-4) induces expression of germ line immunoglobulin Ce transcripts and class switching to the CE gene. We show that LPS-plus-IL-4 induction of germ induce germ line E transcripts in freshly isolated B cells (13,14). The correlation between class switching and induction of germ line transcripts has raised the question of whether germ line transcription is an essential component of the switch process or a by-product of changes in chromatin structure prior to recombination. The former model implies the presence of inducible cis-acting elements that control germ line CH transcription. These elements would allow agents such as IL-4 to direct class switching by modulating germ line transcription at different CH loci. This model provides a mechanism by which different agents, through interacting effects on transcriptional activity, could combine to regulate class switching. If transcription played a mechanistic role in targeting recombination, one would also expect induction of germ line transcripts to occur at the transcriptional level. In this regard, IL-4 has been shown to induce a DNase I-hypersensitive site upstream of the yl switch region (5, 41). However, until now, no cis element controlling inducible germ line CH transcription been described. In this report, we demonstrate that LPS-plus-IL-4 (LPS/IL-4) treatment of a pre-B-cell line induces transcription of germ line E sequences. In addition, we identify a cis-acting element located near the transcriptional initiation site of germ line E transcripts that can contribute to this induction and describe several nuclear binding proteins that bind to this region. MATERIALS AND METHODSCell culture. The 18-81A20 Abelson murine leukemia virus (A-MuLV)-transformed cell line and M12.4.1 B-cell lymphoma cell line have been described previously (1, 15).

To further analyze the VDJ recombination defect in lymphoid pre-B cells from mice with severe combined immune deficiency (scid mice), we have assayed the ability of Abelson murine leukemia virus (A-MuLV) transformed pre-B cells from scid mice to rearrange a recombination substrate in which inverted VH to DJH joins activate a selectable (gpt) gene. In unselected populations, substrate rearrangements occurred frequently, but were aberrant and probably analogous to the aberrant rearrangements observed at endogenous scid Ig gene loci. In contrast, populations of scid pre-B lines selected for gpt activity within the substrate contained mostly "normal" VH to DJH joins within the introduced substrate. These findings demonstrate that scid pre-B cells can make normal joins at low efficiency and are discussed with respect to the potential mechanism of the scid defect and the occurrence of Igs in leaky scid mice.

We describe two retroviral vector-based recombination substrate systems designed to assay for lymphoid VDJ recombinase activity in cultured cells. Both substrates incorporate a constitutive dominant marker gene (the simian virus promoter-driven neo gene) to allow selection of cells that stably integrate the substrate. Both substrates also include a second marker gene that becomes transcriptionaHly active only when inverted by a site-specific recombination event between flanking immunoglobulin variable-region gene segments. The first vector, similar in structure to previous retrovirus-based recombination substrates, utilizes the bacterial guanine-xanthine phosphoribosyltransferase gene (gpt) as its activatable marker; detection of inversion (VDJ recombinase activity) involves drug selection and Southern blotting analyses. We have used this vector to make a more extensive and quantitative survey of VDJ recombinase activity in B-lineage cell lines than has previously been performed with stable substrates, and we have compared our results with those of other studies that use transient recombination substrates. In the second vector, the activatable gene is the bacterial 0-galactosidase gene (lacZ). Detection for inversional activation of this gene is achieved by a fluorogenic assay, termed FACS-Gal, that detects ,-galactosidase activity in viable cells. The latter assay has the unique advantage of rapidly detecting cells that undergo recombination and also allows viable sorting of cells on the basis of the presence or absence of VDJ recombinase activity. We have used the lacZ vector to rapidly quantitate VDJ recombinase activity in B-lineage cell lines and compared the results with those obtained with the gpt vector. We have also used the lacZ vector to isolate variant pre-B-cell lines with low and high levels of VDJ recombinase activity.

Treatment of splenic B lymphocytes and certain B-lineage cell lines with the mitogen lipopolysaccharide (LPS) and the lymphokine interleukin-4 (IL-4) induces expression of germ line immunoglobulin C epsilon transcripts and class switching to the C epsilon gene. We show that LPS-plus-IL-4 induction of germ line epsilon transcripts (termed I epsilon transcripts) occurs at the transcriptional level in an Abelson murine leukemia virus-transformed pre-B-cell line. A 1.1-kb region of DNA surrounding the I epsilon promoter endows inducible transcription to a heterologous reporter gene stably transfected into these cells; such inducible expression depends on combined treatment with LPS and IL-4. Analyses of constructs transiently introduced into a B-cell lymphoma line demonstrated that LPS-plus-IL-4-inducible expression can be conferred by a 179-bp segment of DNA spanning the I epsilon transcriptional initiation site. Mutational analyses demonstrated that this expression depended on DNA sequences within a conserved region directly upstream from the I epsilon transcriptional initiation region. One nuclear protein that is constitutively expressed in normal B cells binds to the downstream end of the conserved sequence; its binding specificity correlates with the functional effect of several mutations. Two additional proteins, which are induced by IL-4 treatment of splenic B cells, bind to the transcription initiation sites of I epsilon. These proteins are indistinguishable in binding assays from proteins previously shown to bind an enhancer region of the class II major histocompatibility complex gene A alpha.