The Proximal J Kappa Germline-Transcript Promoter Facilitates Receptor Editing through Control of Ordered Recombination

<div><p>V(D)J recombination creates antibody light chain diversity by joining a Vκ gene segment with one of four Jκ segments. Two Jκ germline-transcript (GT) promoters control Vκ-Jκ joining, but the mechanisms that govern Jκ choice are unclear. Here, we show in gene-targeted mice that the proximal GT promoter helps targeting rearrangements to Jκ1 by preventing premature DNA breaks at Jκ2. Consequently, cells lacking the proximal GT promoter show a biased utilization of downstream Jκ segments, resulting in a diminished potential for receptor editing. Surprisingly, the proximal—in contrast to the distal—GT promoter is transcriptionally inactive prior to Igκ recombination, indicating that its role in Jκ choice is independent of classical promoter function. Removal of the proximal GT promoter increases H3K4me3 levels at Jκ segments, suggesting that this promoter could act as a suppressor of recombination by limiting chromatin accessibility to RAG. Our findings identify the first <i>cis</i>-element critical for Jκ choice and demonstrate that ordered Igκ recombination facilitates receptor editing.</p></div

The proximal Jκ GT promoter is inactive prior to Igκ recombination.

<p><b>A)</b> Flow cytometry detects expression of κGFP (proximal GT promoter reporter; top) and κhCD4 (distal GT promoter reporter; bottom) in RAG-deficient developing B cells carrying either no transgene, a B1-8^wt HC transgene, or a B1-8^wt HC transgene plus a κHEL transgene. Pro-B and pre-B cells are gated B220⁺ IgM⁻, immature (imm) B cells are gated B220⁺ IgM⁺ IgD⁻, transitional (trans) B cells are gated B220⁺ IgM⁺ IgD^low, and mature (mat) B cells are gated B220⁺ IgM⁺ IgD^high. Grey shaded histograms show cells from a C57Bl/6 control mouse. Results are representative of at least two independent experiments. <b>B)</b> Flow cytometry detects expression of κGFP (top) and κhCD4 (bottom) in non-editing (B1-8^wtHC/κHEL/RAG−/−) or receptor-editing (B1-8^lowHC/κHEL/RAG−/−) B cells (gated B220+ IgM−). Grey shaded histograms show cells from a C57Bl/6 control mouse. Results are representative of at least two independent experiments. <b>C)</b> Northern blotting of Jκ GTs in an Abelson virus-transformed pre-B cell line treated with either STI-571 (STI, which mimics pre-BCR signaling) or the TLR4 ligand LPS. mRNA was hybridized with a Cκ-specific probe (top) that recognizes mature Igκ transcripts, distal GTs, and proximal GTs, the latter of which can be identified by their smaller size. Additionally, the blot was hybridized with a probe specific for distal GTs (middle). Beta-tubulin transcripts (bottom) served as a loading control. Results are representative of two independent experiments. </p

The proximal Jκ GT promoter controls Jκ choice.

<p><b>A)</b> LM-PCR detects total DNA breaks at Jκ gene segments in pre-B cells from wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Linker ligated genomic DNA was first amplified with several Jκ-specific forward primers (FP) and a linker-specific reverse primer (LP) and then hybridized with Jκ RSS probes. Results are representative of at least two independent experiments. <b>B)</b> LM-PCR detects premature DNA breaks at Jκ2, Jκ4, and Jκ5 in pre-B cells from wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Linker ligated genomic DNA was first amplified with several Jκ-specific forward primers (FP) and a linker-specific reverse primer (LP) and then hybridized with Jκ RSS probes. Results are representative of at least two independent experiments. <b>C)</b> VJ coding joint PCR detects individual Jκ segments in completed VκJκ joints in B cells from bone marrow or spleen of wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Genomic DNA was first amplified with a degenerate Vκ-specific forward primer and a reverse primer (MAR35) that binds downstream of Jκ5 and then hybridized with a probe (5’MAR35) that binds downstream of Jκ5 but upstream of the reverse primer. Results are representative of at least two independent experiments. </p

Removal of the proximal Jκ GT promoter increases H3K4me3 levels in the Jκ region and upregulates distal GT promoter activity.

<p><b>A)</b> ChIP analysis of H3K4me3 levels in pre-B cells from wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Immunoprecipitated genomic DNA was analyzed by qPCR. Specific enrichment was calculated with the formula 2^{Ct(Input)-Ct(IP)}. Results are representative of two independent experiments. Error bars represent standard deviations of triplicate qPCR assays. <b>B)</b> RT-qPCR analysis of distal GT promoter activity in pre-B cells from wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Jκ GT specific amplification was normalized to HPRT. Locations of forward (FP) and reverse (RP) primers are indicated above the diagram (D, distal GT promoter). Results are representative of two independent experiments. Error bars represent standard deviations of triplicate qPCR assays. </p