Long-range oncogenic activation of Igh-c-myc translocations by the Igh 3' regulatory region

Abstract

B-cell malignancies, such as human Burkitt's lymphoma, often contain translocations that link c-myc or other proto-oncogenes to the immunoglobulin heavy chain locus (IgH, encoded by Igh). The nature of elements that activate oncogenes within such translocations has been a long-standing question. Translocations within Igh involve DNA double-strand breaks initiated either by the RAG1/2 endonuclease during variable, diversity and joining gene segment (V(D)J) recombination, or by activation-induced cytidine deaminase (AID, also known as AICDA) during class switch recombination (CSR). V(D)J recombination in progenitor B (pro-B) cells assembles Igh variable region exons upstream of mu constant region (Cmu) exons, which are the first of several sets of C(H) exons ('C(H) genes') within a C(H) locus that span several hundred kilobases (kb). In mature B cells, CSR deletes Cmu and replaces it with a downstream C(H) gene. An intronic enhancer (iEmu) between the variable region exons and Cmu promotes V(D)J recombination in developing B cells. Furthermore, the Igh 3' regulatory region (Igh3'RR) lies downstream of the C(H) locus and modulates CSR by long-range transcriptional enhancement of C(H) genes. Transgenic mice bearing iEmu or Igh3'RR sequences fused to c-myc are predisposed to B lymphomas, demonstrating that such elements can confer oncogenic c-myc expression. However, in many B-cell lymphomas, Igh-c-myc translocations delete iEmu and place c-myc up to 200 kb upstream of the Igh3'RR. Here we address the oncogenic role of the Igh3'RR by inactivating it in two distinct mouse models for B-cell lymphoma with Igh-c-myc translocations. We show that the Igh3'RR is dispensable for pro-B-cell lymphomas with V(D)J recombination-initiated translocations, but is required for peripheral B-cell lymphomas with CSR-associated translocations. As the Igh3'RR is not required for CSR-associated Igh breaks or Igh-c-myc translocations in peripheral B-cell lymphoma progenitors, we conclude that this regulatory region confers oncogenic activity by long-range and developmental stage-specific activation of translocated c-myc genes.

Figure 1

Deletion of the Igh3’RR does not affect development of pro-B-cell lymphomas. a, Left, Kaplan–Meier curve of the LPR1+/− (n=12) and LPR−/− (n=5) cohorts. Curves represent total survival. Right, the percentage of mice in the LPR+/− and LPR−/− cohorts succumbing to B-cell lymphomas, thymic lymphomas or other causes of death. b, Examples of cytological aberrations in representative LPR+/− tumours with translocations to the wild-type (mouse number 434) or the 3’RR-deleted (mouse number 421) Igh alleles. In each set of panels: left, paints specific for chr12 (red) and chr15 (green); middle, FISH analysis on separated metaphases with 3’Igh (green) and c-myc (red) probes; right, graphic representation. Only chromosomes involved in translocations are shown. Whole metaphases are presented in Supplementary Fig. 2. c, Southern blot analysis of LPR+/− (left) and LPR−/− (right) tumour DNA with a probe downstream of hs4, which distinguishes the wild-type (WT) and 3’RR-deleted (del) Igh alleles. A schematic of the wild-type and del Igh locus, with the position of the probe, is on the top. Numbers refer to individual mice in the cohort (see Supplementary Table 1). C, control, total spleen DNA from wild-type mouse; RI, EcoRI.

Figure 2. Deletion of the IgH3’RR abrogates development of peripheral B cell lymphomas

a. Left: Kaplan-Meier curve of the CXPR^+/- (n=22) and CXPR^-/- (n=17) cohorts. Curves represent total survival. Right: Percentage of mice in the CXPR^+/- and CXPR^-/- cohorts succumbing to B cell lymphomas, thymic lymphomas or to other cause of death. b. Southern blot analysis of CXPR^+/- tumor DNA with probes indicated on the right of each panel. Numbers refer to individual mice in the cohort (see Supp. Table 2). K, kidney, used as control; Tu, tumor. A schematic of the c-myc locus, indicating the 5’ and 3’ probes used to detect c-myc rearrangements is on the top.

Figure 3. T(12;15) in CXPR^+/- B cell tumors always involves the wt IgH allele

a. Example of FISH and chromosome paints on CXPR^+/- tumor metaphases. The same metaphase was sequentially analyzed with the different set of probes indicated at the top of each panel. A schematic of the different chromosomal species detected is shown at the bottom. b. Summary of FISH and chromosome paint analyses on all analyzed CXPR^+/- tumors. Numbers refer to individual mice in the cohort. Sequential hybridization with the set of probes indicated on the left was performed. Only chromosomes involved in translocations are shown, along with a graphic representation. The whole metaphases are presented in Supp. Fig. 4. c. Table summarizing SKY and FISH data for CXPR^+/- B cell tumors.

Figure 4. Deletion of the IgH3’RR does not affect IgH locus breaks and IgH/c-myc translocations

a. Left, top: Diagram showing location of 3’ IgH (red) and 5’ IgH (green) BAC probes used for FISH. An intact IgH locus on chr12 shows colocalized red and green signals, a broken locus shows split red and green signals. Left, bottom: Examples of metaphases from CXPR^+/-and CXPR^-/- αCD40/IL4-stimulated splenic B cells showing IgH breaks. Right: Quantification of IgH locus breaks in day 4 αCD40/IL4-activated or day 5 LPS/αIgD-dextran-activated B cells from control, CXPR^+/-, and CXPR^-/- mice. At least three mice per each genotype and at least 60 metaphases per mouse were analyzed; data are presented as average ± sd. b. Southern blot analysis of CXPR^+/- tumor DNA with a Cμ probe that detects Sμ rearrangements. A schematic of the IgH locus, with position of the probe is on the top. Position of germline bands in C57/B6 and 129Sv backgrounds is indicated on the left; the 3’RR-deleted allele is from 129Sv background. Numbers refer to individual mice in the cohort. K, kidney, used as control; Tu, tumor. Note that in some cases kidneys contained infiltration of tumor cells, as judged by tumor-specific rearranged J_H and c-myc bands (Fig. 2b). c. Frequency of IgH/c-myc translocations was measured by PCR assays using Sμ and c-myc primers. DNA samples were isolated from day4 αCD40/IL4-activated wt, CX, CXPR^+/- and CXPR^-/- splenic B cells.