Individual C_H genes are organized into germline transcription units which consist, from 5’ to 3’, of a non-coding “I” exon, a switch (S) region and the C_H exons⁶. CSR to a particular C_H gene requires introduction of AID-initiated DSBs into the donor S region upstream of Cμ (Sμ) and into a downstream acceptor S region⁶. The I exon is preceded by a germline promoter that is up-regulated by particular activation treatments, with transcription targeting AID to specific S regions¹⁷. The IgH3’RR contains multiple enhancer elements^18-20 and controls germline transcription of certain C_H promoters over distances of 100kb or more⁸. However, the IgH3’RR is not required for V(D)J recombination, expression of rearranged IgH genes, or transcription through Sμ or Sγ1¹⁰. To test potential roles of the IgH3’RR in B cell lymphomagenesis, we bred an IgH3’RR inactivating mutation, which deletes the key HS3b and HS4 enhancers¹⁰ (Supp. Fig. 1), into non-homologous end-joining (NHEJ) and p53 tumor suppressor deficient backgrounds that predispose to either pro-B or peripheral B cell lymphoma.

XRCC4 and DNA ligase 4 (Lig4) form a NHEJ ligation complex required for V(D)J recombination²¹. Mice with germline inactivation of either Lig4 or Xrcc4 and the p53 tumor suppressor develop pro-B cell lymphomas with complex translocations (“complicons”) involving IgH on chromosome 12 (chr12) and a region downstream of c-myc on chromosome 15 (chr15)²². These translocations arise from joining RAG1/2-induced DSBs in the IgH J_H region to DSBs downstream of c-myc, leading to dicentric chr12;15 translocations and c-myc amplification via breakage-fusion-bridge cycles²²,²³. Analysis of Lig4/p53 double-deficient mice (“LP” mice) that harbored IgH3’RR inactivating mutations on either one or both IgH alleles (referred to as LPR^+/- or LPR^-/- respectively) revealed that both genotypes succumbed to pro-B cell lymphomas with kinetics similar to those of LP mice²² (Fig. 1a; Supp. Table 1). Likewise, all analyzed LPR^+/- and LPR^-/- tumors had characteristic chr12 to15 translocations (T(12;15)) and 15;12 complicons (C(15;12)) harboring amplified c-myc (Fig. 1b; Supp. Fig. 2; Supp. Table 1). In addition, Southern blotting with a probe that distinguishes IgH3’RR-deleted from wild-type (wt) IgH alleles (Fig. 1c, top) revealed that IgH/c-myc translocations/amplifications involved the wt allele in some LPR^+/- tumors and the IgH3’RR-deleted allele in others (Fig. 1c, bottom). Thus, the IgH3’RR is dispensable for LP pro-B cell lymphomas with IgH/c-myc complicons.

Specific inactivation of Xrcc4 by a loxP/Cre approach in peripheral B cells of p53-deficient mice (referred to as “CXP” mice) leads to surface Ig-negative peripheral B cell lymphomas²⁴,²⁵. CXP B cell lymphomas arise from progenitors that delete or aberrantly rearrange their Igκ and Igλ light chain loci and routinely harbor a T(12;15) that fuses IgH S regions to sequences just upstream of c-myc, leading to high level c-myc expression from a translocated single copy c-myc gene²⁵,²⁶. Such T(12;15)s lack iEμ as they occur downstream of this element²⁴. To test potential roles of the IgH3’RR in CXP tumorigenesis, we followed tumor development in cohorts of CXPR^+/- and CXPR^-/- mice. CXPR^+/- mice succumbed to the same tumor spectrum as CXP mice²⁵, with 40% developing surface Ig-negative B cell lymphomas that appeared identical to CXP B cell lymphomas (Fig. 2a and Supp. Table 2). The remaining CXPR^+/- mice succumbed to thymic lymphomas or other tumors associated with germline p53 deficiency. In contrast, none of 17 analyzed CXPR^-/- mice developed B cell lymphoma; instead, most died from thymic lymphoma (Fig. 2a and Supp. Table 2). Thus, homozygous IgH3’RR inactivation abrogates CXP B cell lymphomas.

All analyzed CXPR^+/- B cell tumors had a clonal T(12;15), based on spectral karyotyping (Fig. 3c, Supp. Fig. 3). As in CXP B cell lymphomas²⁵, most T(12;15) from CXPR^+/- B cell lymphomas split c-myc, as evidenced by Southern blotting with 5’ and 3’ c-myc locus probes (Fig. 2b, top panels). However, two tumors (393 and 959) had c-myc amplification without detectable c-myc rearrangements (Fig. 2b), and tumor 796, while harboring a clonal T(12;15) that split c-myc, also contained metaphases with a translocation that fused this T(12;15) to chromosome 16, resulting in low level IgH/c-myc amplification (Supp. Fig.3; data not shown). Northern blotting revealed elevated c-myc expression in all CXPR^+/- B cell tumors, with tumors 393, 796 and 959 showing highest levels (Supp. Fig. 4), suggesting that amplification may sometimes be selected secondary to ectopic activation to achieve maximal expression, which may be relevant to certain human lymphomas that acquire c-myc amplification during tumor progression¹².

To determine which IgH allele was involved in CXPR^+/- lymphoma T(12;15)s, we analyzed tumor metaphases by fluorescence in situ hybridization (FISH) with a probe specific for the deleted portion of the IgH3’RR (3’RR wt probe, green) and a chr15 paint (red). In this assay, the translocated portion of chr15 (red paint) co-localizes with a green signal if the wt IgH allele is translocated, but not if the IgH3’RR-deleted allele is translocated (Fig. 3a, left panel). Sequential re-probing of these metaphases with a green chr12 paint (FISH 2, green) plus a 3’ IgH BAC probe (FISH 2, red) (Fig. 3a, right panel) revealed that all CXPR^+/- tumor IgH/c-myc translocations involved the wt IgH allele (Fig. 3b, c and Supp. Fig. 5). These results, coupled with the absence of B cell tumors in CXPR^-/- mice, demonstrate that the IgH3’RR is required for peripheral CXP B cell lymphomas via a role in oncogenic IgH/c-myc translocations.

The IgH3’RR might influence appearance of oncogenic IgH/c-myc translocations by mechanistically promoting them through induction of CSR at certain S regions and/or by long-range activation of translocated c-myc expression. To distinguish between these potential mechanisms, we asked whether the IgH3’RR-deleted allele is a substrate for AID-induced DSBs. As one measure, we activated CXPR^+/- and CXPR^-/- B cells for 4 days with αCD40/IL4 and found that both genotypes switched to IgG1, as expected by the fact that the 3’IgHRR is not required for CSR to this IgH isotype⁸,¹⁰ (Supp. Fig. 6a). We also analyzed metaphases from αCD40/IL4-stimulated CXPR^+/-and CXPR^-/- B cells via two-color FISH²⁷ and found that the increased level of IgH breaks in the absence of XRCC4²⁴ was not markedly affected by the IgH3’RR mutation (Fig. 4a). CSR to IgG3 is severely impaired in mice homozygous for IgH3’RR inactivating mutations due to inhibition of Iγ3 transcription⁸,¹⁰. However, LPS/αIgD-dextran stimulation to induce IgG3 CSR led to similarly increased IgH breaks in CXPR^+/- and CXPR^-/- B cells (Supp. Fig. 6b; Fig. 4a), likely reflecting unimpeded AID activity on Sμ, which is transcribed independently of the IgH3’RR¹⁰. Southern blotting further revealed that most CXPR^+/- B cell tumors had Sμ rearrangements or deletions on both alleles, again indicative of substantial AID activity on IgH3’RR-deleted alleles (Fig. 4b). We conclude that introduction of AID-induced IgH lesions is not markedly impaired by the IgH3’RR deletion.

While AID-induced IgH breaks occur at high frequency on IgH3’RR-deleted alleles, IgH/c-myc translocations still might be inhibited, for example by a different distribution of IgH DSBs or by effects on proximity of the two loci²⁶. Therefore, we employed a PCR assay²⁸ to directly evaluate potential effects of the IgH3’RR deletion on IgH/c-myc translocation frequency and found IgH/c-myc translocations, indeed, occurred at similar frequencies in αCD40/IL4-stimulated CXPR^+/- and CXPR^-/- B cells (Fig. 4c). Moreover, one CXPR^+/- B cell tumor had a T(12;11) involving the IgH3’RR-deleted allele in addition to its T(12;15), again indicating the IgH3’RR-deleted allele is a translocation target (Fig. 3c, Supp. Fig. 3). We conclude that the IgH3’RR is dispensable for generation of IgH/c-myc translocations in XRCC4-deficient B cells.

The IgH locus has long been speculated to have cis-acting elements that activate c-myc or other oncogenes in the context of translocations. We now demonstrate that the IgH3’RR is required for oncogenicity of IgH/c-myc translocations that ectopically activate c-myc in mouse CXP B cell lymphomas. As a substantial proportion of CXPR^+/- (Supp. Fig. 7) and CXP lymphomas²⁵ have translocations that fuse c-myc to Sμ, oncogenic IgH3’RR activity extends at least 200kb upstream. Thus, our findings define a major oncogenic role for the IgH3’RR in activating c-myc subsequent to IgH/c-myc translocations; although we do not rule out an additional role in promoting translocations by enhancing AID-mediated lesions in certain S regions regulated by this element. Although high-copy iEμ transgenes predispose to pro-B cell lymphoma in mice¹², the role of iEμ in IgH/oncogene translocations remains to be determined. In this context, knock-in of c-myc into the IgH J_H region led to peripheral B cell lymphomas, as opposed to pro-B cell lymphomas¹², suggesting iEμ alone may not always be sufficient to activate c-myc in the endogenous setting. In this regard, our finding that the IgH3’RR is dispensable for LP pro-B cell lymphomas may explain why c-myc is amplified in these tumors and ectopically activated in peripheral CXP B cell tumors. Specifically, the IgH3’RR is not active in pro-B cells⁹ and would not activate IgH translocations upstream of a single-copy c-myc, favoring selection for translocations downstream of c-myc that promote gene amplification. Given the similar organization of mouse and human IgH⁹, our findings suggest that the IgH3’RR also supports activated oncogene expression in human B cell tumors with IgH S region translocations that eliminate iEμ (e.g. sporadic BL²⁹) and, potentially, even in some with IgH J_H region translocations that leave iEμ intact (e.g. endemic BL²⁹) In this regard, targeted inhibition of the B cell-specific IgH3’RR could theoretically provide a therapeutic strategy for such human B lymphomas.