Normal Isotype Switching in B Cells Lacking the Iμ Exon Splice Donor Site: Evidence for Multiple Iμ-Like Germline Transcripts

Abstract

Ig class switch recombination (CSR) in activated B cells is preceded by the generation of “switch” transcripts from the heavy chain constant region (CH) genes targeted for rearrangement. Switch transcripts include a sterile “I” exon spliced onto the first CH exon. Targeted mutations disrupting the expression or splicing of I exons severely hamper CSR to all tested CH loci, except μ. However, all μ switch transcript mutations tested so far have left the Iμ exon splice donor site intact. To test the possibility that the residual CSR activity in Iμ mutants could be due to splicing of a truncated Iμ exon, we generated new mutants specifically lacking the Iμ splice donor site. Surprisingly, normal CSR was observed in the Iμ splice donor mutants even in the absence of detectable spliced Iμ transcripts. In a search for potential alternative sources of switch-like transcripts in the μ locus, we identified two novel exons which map just upstream of the Sμ region and splice onto the Cμ1 exon. Their expression is detectable from early B cell developmental stages, and, at least in hybridomas, it does not require the Eμ enhancer. These studies highlight a unique structure for the μ locus I exon region, with multiple nested switch transcript-like exons mapping upstream of Sμ. We propose that all of these transcripts directly contribute to μ class switching activity.

Cyclin D1 activation in B-cell malignancy: association with changes in histone acetylation, DNA methylation, and RNA polymerase II binding to both promoter and distal sequences

“…These data are consistent with a transcriptional mechanism of cyclin D1 gene activation in B-cell malignancies, in contrast to the recently reported LCR-mediated regulation of mouse ␤-globin gene expression by transcriptional elongation. 36 Pol II was also found in vivo at the IgH regulatory regions, including the E intronic enhancer, 3Ј C␣ LCR HS3 and HS4. Sterile germline transcripts originating from the E enhancer have been described, 37 so the presence of Pol II at the E enhancer in B cells was not surprising.…”

Regulation of Immunoglobulin Class-Switch Recombination

“…This notion was further validated through mutational analyses. Deletion of I-exon promoters or regulatory regions (3′-RR) at the 3′-end of Igh abolished or severely reduced CSR, while replacing the inducible I-exon promoters with heterologous constitutive promoters drove CSR in a cytokine-independent fashion (Bottaro et al, 1994; Cogne et al, 1994; Jung et al, 1993; Kuzin et al, 2000; Lorenz, Jung, & Radbruch, et al, 1995; Manis, van der Stoep, et al, 1998; Michaelson, Giannini, & Birshtein, et al, 1995; Pinaud et al, 2001; Qiu, Harriman, & Stavnezer, et al, 1999; Seidl et al, 1998; Vincent-Fabert et al, 2010; Zhang et al, 1993). These studies thus provided experimental evidence for the notion that germline transcription of particular C h genes renders them “accessible” for CSR (Stavnezer-Nordgren & Sirlin, 1986; Yancopoulos et al, 1986).…”

AID and Somatic Hypermutation

“…In a critical experiment, Radbruch and colleagues (Hein et al ., 1998) reported that switching was abrogated when the splice donor site for Iγ1 was deleted, even though Iγ1 transcripts were made. Another study showed that mice lacking the splice donor site for the Iμ exon had switching, but splicing still occurred in transcripts using pseudo-splice donor sites (Kuzin et al ., 2000). Thus, AID could be brought to the Ig loci through interaction with cis -acting elements (potentially using E2A family members (Michael et al ., 2003; Schoetz et al ., 2006; Tanaka et al ., 2010)), bind to the RNA polymerase II spliceosome complex through CTNNBL1, and load onto DNA at donor splice sites to interact with RPA and deaminate cytosine residues.…”