Regulation and targeting of recombination in extrachromosomal substrates carrying immunoglobulin switch region sequences.

Abstract

We have used extrachromosomal substrates carrying immunoglobulin heavy-chain S,u and Sy3 switch region sequences to study activation and targeting of recombination by a transcriptional enhancer element. Substrates are transiently introduced into activated primary murine B cells, in which recombination involving S-region sequences deletes a conditionally lethal marker, and recombination is measured by transformation of Escherichia coli in the second step of the assay. Previously we found that as many as 25% of replicated substrates recombined during 40-h transfection of lipopolysaccharide (LPS)-stimulated primary cells and that efficient recombination was dependent on the presence of S-region sequences as well as a transcriptional activator region in the constructs (H. Leung and N. Maizels, Proc. Natl. Acad. Sci. USA 89:4154-4158, 1992). Here we show that recombination of the switch substrates is threefold more efficient in LPS-cultured primary B cells than in the T-cell line EL4; the activities responsible for switch substrate recombination thus appear to be more abundant or more active in cells which can carry out chromosomal switch recombination. We test the role of the transcriptional activator region and show that the immunoglobulin heavy-chain intron enhancer (E,u) alone stimulates recombination as well as E,L combined with a heavy-chain promoter and that mutations that diminish enhancer-dependent transcription 500-fold diminish recombinational activation less than 2-fold. These observations suggest that the enhancer stimulates recombination by a mechanism that does not depend on transcript production or that is insensitive to the level of transcript production over a very broad range.Furthermore, we find that E,u stimulates recombination when located either upstream or downstream of SFL but that the position of the recombinational activator does affect the targeting of recombination junctions, suggesting that the relatively imprecise targeting of switch junctions in vivo may reflect the availability of many potential activator sites within each switch region.Immunoglobulin isotype switch recombination is a regulated recombination event which joins an expressed heavychain variable [V(D)J] region to a new downstream constant (C) region, deleting the DNA between (for reviews, see references 5, 6, and 8). Isotype switching occurs in B cells which have accomplished V(D)J joining and have been activated by antigen. Recombination involves G-rich switch or S regions, from 1 to 8 kb in length, which are found in the intron upstream of each C region that undergoes switching-C,u, Cy, Ca, and Ce; expression of Ca is regulated by alternative splicing, not recombination, and there is no G-rich S region upstream of Ca. In contrast to V(D)J joining, in which heptamer-nonamer recognition elements precisely target recombination (13), switch recombination is notably imprecise. No conserved sequences comparable to the heptamer-nonamer elements are apparent within S regions or at switch recombination sites, and chromosomal swi...

Sequence Dependence of Chromosomal R-Loops at the Immunoglobulin Heavy-Chain Sμ Class Switch Region

“…Several groups, including ours, have demonstrated R-loop formation in switch regions in vitro when these regions are transcribed by prokaryotic polymerases (4,13,27,31). In 2003, we demonstrated kilobase-length chromosomal R-loops in the murine S␥3 and S␥2b switch regions in vivo (35), and this finding has been confirmed (28).…”

Germline Transcription and Switch Recombination of a Transgene Containing the Entire H Chain Constant Region Locus: Effect of a Mutation in a STAT6 Binding Site in the γ1 Promoter

“…S recombination to ␥1 is also not as sensitive as other isotypes to both cis and trans mutations (12, 60 -64). In contrast, we note that recombination of some S substrates with different S regions (S␥3 or S␣) does not require abundant transcription (22,23).…”

Efficient AID targeting of switch regions is not sufficient for optimal class switch recombination

“…3b), and features the entire repetitive region, previously described 36 . Equivalent S regions were previously shown to efficiently support CSR in several studies [37][38][39] . From 5 0 to 3 0 , the following parts were then added: the murine core Sm, a 1.5-kb genomic fragment containing human Eik and Ck, a 0.3-kb fragment containing Vk4-63 promoter and the following leader exon (in which the last codon was replaced by a stop codon to mimic a non-translatable I-exon) and the murine core Sg3.…”