Comparison of Mouse and Rabbit Eiκ Enhancers Indicates That Different Elements Within the Enhancer May Mediate Activation of Transcription and Recombination

Abstract

The intronic Ig κ-light chain enhancer (Eiκ) has been implicated in regulation of transcription and Vκ-Jκ recombination at the κ locus. To identify sequences within the Eiκ enhancer which are involved in control of recombination, we have made use of the finding that the Eiκ element from the rabbit b9 κ locus is capable of inducing rearrangement, but not transcription of κ genes in mouse lymphoid cells. We have therefore compared the binding of murine nuclear proteins to the mouse and rabbit Eiκ elements. DNase I footprinting and gel mobility shift assays indicate that only the κB, κE1, and κE2 sites of the rabbit enhancer are able to interact with murine trans-acting factors. Moreover, although the rabbit κB site binds murine NF-κB p50/p50 and p50/p65 complexes with high affinity, this site is not capable of mediating transcriptional activation of transient transfection reporter constructs in mouse B lineage cells. These results therefore suggest that, in contrast to the maintenance of κ enhancer transcription which requires all of the Eiκ sites, only the κB, κE1, and κE2 sites may be necessary for the recombinational activity of the enhancer. Furthermore, NF-κB-mediated effects on transcription and recombination appear to involve separate downstream activation pathways.

Important Roles for E Protein Binding Sites within the Immunoglobulin κ Chain Intronic Enhancer in Activating Vκ Jκ Rearrangement

“…By analyzing the functional motifs within iE κ in vivo, we demonstrated that the κB site mutation had no apparent impact on κ rearrangement. The κB site is bound by NF-κB family members and was found to play an important role in activating κ rearrangement of recombination substrates in cell line transfection and transgenic studies ( 16 , 25 ). However, the findings that the impacts of κE1 and κE2 double mutation on κ rearrangement are very similar to the deletion of the entire iE κ rule out any important roles of other protein-binding motifs within iE κ , such as the NF-κB binding site, in activating κ rearrangement.…”

Chromatin Remodeling at the Ig Loci Prior to V(D)J Recombination

“…Nuclear extracts of lymphoid cell lines and gel retardation experiments were performed as described previously (30). The mouse NF-B oligonucleotide used was 5Ј-GACAGAGGGGACTTTCCGAGAG-3Ј.…”

Activation of V(D)J Recombination at the IgH Chain JH Locus Occurs within a 6-Kilobase Chromatin Domain and Is Associated with Nucleosomal Remodeling

“…Cell nuclei were prepared from RAG2 Ϫ/Ϫ CD19 ϩ pro-B cell precursors and S17 stromal cells as described previously (25). For restriction enzyme digest, 5 ϫ 10 6 nuclei were resuspended in 300 l of appropriate digestion buffer (New England Biolabs) and incubated with 50 U of restriction enzyme at 37°C.…”