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

Gostissa M, Yan CT, Bianco JM, Cogné M, Pinaud E, Alt FW - Nature (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

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.

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

Mentions: 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.


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

Gostissa M, Yan CT, Bianco JM, Cogné M, Pinaud E, Alt FW - Nature (2009)

Deletion of the IgH3’RR does not affect IgH locus breaks and IgH/c-myc translocationsa. 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 JH 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.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Deletion of the IgH3’RR does not affect IgH locus breaks and IgH/c-myc translocationsa. 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 JH 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.
Mentions: 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.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

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.

Show MeSH
Related in: MedlinePlus