The IgH 3' regulatory region influences lymphomagenesis in Igλ-Myc mice.

The IgH 3'regulatory region (3'RR), encompassing the four transcriptional enhancers hs3a-hs1,2-hs3b-hs4, has a key role on class switch recombination, somatic hypermutation, IgH transcription and B-cell fate. In plasma cells, transcribed IgH and IgL loci often colocalized in transcription factories and an IgL transcription defect might translate into lowered IgH transcription. We explored whether the 3'RR would affect lymphomagenesis in Igλ-Myc transgenic mice prone to lymphoproliferations. Breeding Igλ-Myc transgenics in a background deficient for the 3'RR influences lymphomagenesis toward less mature lymphomas (16% vs 54%, p = 0.01, Z test for two population proportions). In a 3'RR-deficient background mature tumors less often expressed the CD43 antigen (54% vs 0%, p = 0.02), a membrane glycoprotein expressed on activated mature B-cells. In contrast, in a 3'RR-deficient background tumors more often expressed the CD5 antigen (32% vs 12%, p = 0.05) that may serve to control autoimmunity and that is suspected to play a role in leukemic transformation. Lymphoma myc transcript levels, the Ki67 index of proliferation, the clonality, the usage of V(D)J segments, and their somatic hypermutation status were not affected in the 3'RR-deficient background. In conclusion, most probably through its action during the maturation process, the 3'RR can influence lymphomagenesis even when not linked with an oncogene.


INTRODUCTION
The immunoglobulin heavy chain (IgH) locus undergoes multiple changes along B-cell differentiation, affecting transcription and accessibility to V(D) J recombination, somatic hypermutation (SHM) and class switch recombination (CSR) [1,2]. Since Ig gene remodelling events require transcription, cis-regulatory regions and especially transcriptional enhancers are major locus regulators. The IgH 3' regulatory region (3'RR) enhancers (hs3a, hs1,2, hs3b and hs4) promote IgH transcription [3], SHM [4] and CSR [5,6] but not V(D)J recombination [7]. Ongoing recombination and mutation all along B-cell development make the IgH and Ig light (IgL) chain locus hotspots for translocations [8,9]. Numerous lymphomas are thus marked by proto-oncogene translocation into the IgH locus such as cyclin D1, Bcl-2 and c-myc for mantle cell lymphoma, follicular lymphoma and Burkitt lymphoma, respectively. Convincing demonstration of the key contribution of the IgH and IgL enhancers in mature B-cell lymphomagenesis has been done by transgenic animal models. Thus, c-myc-3'RR and Igλ-Myc transgenics developed Burkitt lymphomalike proliferation [10][11][12][13][14][15]. IgH and IgL enhancers may thus be potent activators of IgH/IgL-translocated oncogene transcription, even when breakpoints lie several hundred kb away from the enhancers. Thus, long-range interactions between the two regions of chromatin, through formation of a loop structure constitute an important mechanism of normal and abnormal gene transcription regulation by the 3'RR [2,16]. Although IgL and IgH chains are encoded by loci located in different chromosome, nuclear positions of Ig alleles are coordinated according to ordered V(D) J recombination [17]. During B-cell activation one IgH allele more often associates with the nuclear periphery [18], while the chromatin context of both alleles appears similar in 4C experiments [19]. Observation of frequent inter-allelic (trans-) CSR in mammals also implies inter-Oncotarget 20303 www.impactjournals.com/oncotarget allelic proximity favoring recombination and synapsis between alleles [20,21]. In plasma cells, transcribed IgH and IgL loci often colocalized in transcription factories and an IgL transcription defect might translate into lowered IgH transcription [22]. Until now, the role of the 3'RR in the development of B-cell lymphomas due to a IgL enhancer-mediated oncogene deregulation is unknown. We have explored whether the 3'RR would affect lymphomagenesis in Igλ-Myc transgenic mice prone to lymphoproliferations. The Igλ-Myc transgene induces the B-cell specific overexpression of the human c-myc oncogene leading to the progressive development of B-cell lymphomas [14].

Generation of 3'RR-deficient lymphoma mice
3'RR-deficient mice were crossed with Igλ-Myc mice (considered as wt in this study) to derive heterozygous 3'RR-deficient/Igλ-Myc and homozygous 3'RR-deficient/Igλ-Myc mice. The presence of the Igλ-Myc transgene ( Figure 1A) and of the 3'RR-deleted allele ( Figure 1B) was followed by using specific PCR.
Oncotarget 20305 www.impactjournals.com/oncotarget of flow cytometry labelling were also similar ( Figure 2) showing that lymphoma cells in lymph nodes, spleen and blood were identical. Lymphoma cells were not associated with liver and gut-associated lymphoid tissues (data not shown). All data reported below were obtained with lymph node lymphomas.
Lymphomas from 79 animals were investigated (24 wt mice, 30 heterozygous mice and 25 homozygous mice). During precursor B-cell differentiation, genes encoding H and L chains of an Ig molecule are somatically assembled from germline DNA. This process occurs in the bone marrow prior to antigenic challenge and leads to the successive formation of pro-B, pre-B and immature B-cells. B-cells mature in germinal centers and once activated differentiate into Ig-secreting plasma cells (schematized in Figure 1D). Whether the membrane B220 marker is present all along B-cell maturation, stage specific markers characterise these different stages: expression of the membrane CD117/CD43/CD138 antigens on pro-B/ pre-B-cells, expression of membrane IgM on immature B-cells, expression of membrane IgD on mature B-cells, re-expression of membrane CD138 on plasma cells and re-expression of membrane CD43 on activated mature B-cells (schematized in Figure 1D) [23]. Flow cytometry was used to monitor the immunophenotypic profile of lymphomas (typical profiles are reported in Figure  3). A B220 + population was characterized in all cases (100%, 79/79) while staining for T lineage (CD4 and with flow cytometry. Lymphoma analysis revealed four major subtypes. A. pro-B/pre-B lymphomas (B220 + CD117 + IgM -); B. immature B-cell lymphoma (B220 + CD117 -IgM + IgD -); C. mature B-cell lymphoma (B220 + IgM + IgD + with or without CD43); D. plasmablastic B-cell lymphoma (B220 + CD138 + ). E. Some lymphomas were CD5 + . Representative B220/CD117, B220/IgM, B220/IgD, B220/CD43, B220/ CD138 and B220/CD5 labelling were shown. Anti-B220 was conjugated with PC5. Anti-IgM, anti-IgD, anti-CD43 and anti-CD5 were conjugated with FITC. Anti-CD117 and anti-CD138 were conjugated with PE.
All along B-lymphocyte development, cell survival is dependent from BCR expression and signalling. IgH transcription is markedly reduced in 3'RR-deficient mice even if BCR expression, signalling and B-cell compartments (except for marginal zone B-cell one) are roughly normal [3]. The lower percentage of total mature B-cell lymphomas in the 3'RR-deficient background might be linked to a lowered maturation speed unmasked by the c-myc-induced lymphoproliferation context. The lower percentage of CD43 activated mature B-cell lymphomas in the 3'RR-deficient background is coherent with this hypothesis. CD5 + B-cells have distinct functional properties compared with B-cells lacking CD5. In B-cells, CD5 associates with the BCR and is suspected to maintain anergy in mouse B-cells. CD5 also promotes multiple intracellular signalling pathways in B-lymphocytes [26] and CD5 + B-cells are more resistant to apoptose than CD5 -B-cells [25]. The elevated CD5 + B-cell lymphomas in 3'RR-deficient Igλ-Myc mice might unmask a role of the CD5 antigen in leukemic transformation as previously suggested [26]. The role of the 3'RR deletion on the CD5 + B-cell fate deserves now further investigations. Finally, taken altogether these results reinforce a recent study reporting that the class-specific BCR tonic signal modulates lymphomagenesis in a c-myc deregulation transgenic model [13].

Clonal origin of lymphomas
Southern analysis of V(D)J recombinations showed that all B-cell lymphomas had undergone clonotypic Ig rearrangements. Thus, the use of a J H4 probe revealed rearranged bands in addition to the germline band indicating the clonal origin of lymphomas from Igλ-Myc mice with or without the 3'RR ( Figure 4E).

Ki67 expression of lymphomas
The proliferative activity of tumor cells was investigated with the nuclear protein Ki67 found during G 1 , S, G 2 and M phase of the cell cycle. The percentage of Ki67 + cells was high and similar in Igλ-Myc mice (91%, 12 mice), heterozygous 3'RR-deficient/Igλ-Myc mice (87%, 21 mice) and homozygous 3'RR-deficient/Igλ-Myc mice (89%, 21 mice) showing that the intensive lymphoma proliferation was not affected by deletion of the 3'RR. These results are in agreement with the lack of effect of the 3'RR deletion on the growth of normal B-cells [5].

Mouse and human c-myc transcripts in lymphomas
Igλ-Myc mice carried an Igλ-Myc transgene containing a translocated MYC gene from the human Burkitt lymphoma cell line IARC-BL60 under the transcriptional control of the IgL λ chain regulatory sequences [14]. Thus the dual analysis of both human transgenic myc transcripts and endogenous mouse myc transcripts can be investigated in Igλ-Myc mice. We analyzed mouse splenic B-cells in 8 week old homozygous 3'RR-deficient/Igλ-Myc mice and Igλ-Myc mice before any manifestation of disease. Mouse and human myc transcripts levels in splenic B-cells were not affected by the 3'RR-deficient background ( Figure 5A and 5B). Similarly, the shift between mouse and human myc transcript levels in B-cell lymphomas of Igλ-Myc mice was not affected. For these experiments purified splenic B-cells of 8-weeks old mice prior any manifestation of disease were used as controls. This was done because lymphomas arose from axillary, parotid, and submandibular lymph nodes ( Figure  1C) and that in steady state conditions it is impossible to obtain sufficient amounts of purified lymph node B-cells from 8-weeks old mice. Finally, human myc transcript levels were similar in immature and mature B-cell lymphomas ( Figure 5B). Taken together these results suggest that the tumoral process is not affected by the 3'RR-deficient background, reinforcing the hypothesis that the lowered lymphoma maturity in 3'RR-deficient/ Igλ-Myc mice is related to a difference on cell maturity of the cell of origin of the B-cell lymphoma.

Analysis of Ig VH mutations in lymphomas
We then analysed the mutation status of V H rearranged genes in B-cell lymphomas. This study was made possible because of their clonal nature ( Figure  4E). The 150 bp located downstream the J H4 exon were investigated as an hot spot of somatic hypermutation [4]. All lymphomas revealed unmutated sequences and thus featured a pre-germinal centre origin (results from four wt lymphomas, three heterozygous 3'RR lymphomas, three homozygous 3'RR lymphomas, ten sequences for each lymphoma) (data not shown). The lack of somatic hypermutations in lymphomas from Igλ-Myc mice is in agreement with their absence in lymphomas from iMyc Eµ mice [27] and reinforced the hypothesis that most mice lymphomas arise from naïve B-cells. It is interesting to note that most human B-cell lymphomas are mutated suggesting that the cell of origin of human and mouse lymphomas differs suggesting multifactorial route to tumor development and that correlations between human lymphomas and mice models should be made cautiously.

Analysis of V(D)J recombination in lymphomas
The mouse IgH locus contains about 200 variable (V H ) genes subdivided into domain-organized gene families, including the distal V H (the largest family) and the proximal V H family. V H genes are followed by a dozen of diversity (D) segments and four junction (J) segments. We then analysed if 3'RR deficiency affected the type of V, D and J used to express the B-cell receptor at the membrane of B-cell lymphomas. This study was made possible because of their clonal nature. As shown in Figure  6, lack of the 3'RR did not affect the use of either V, D and J segments. This study confirms that the 3'RR is not implicated in V(D)J recombination confirming previous studies using non lymphoma B-cells [7,28].

CONCLUDING REMARKS
Results of the present study indicate that the IgH 3'RR influences lymphomagenesis in Igλ-Myc mice. The 3'RR did not directly act on the lymphomagenetic process but on the maturity of the cell of origin of the lymphoma. The 3'RR is a major lymphoma oncogene deregulator [8,[10][11][12][13]. Targeted inhibition of the 3'RR could provides a therapeutic strategy for the treatment of a wide range of mature B-cell lymphomas, specifically those with oncogene translocation into the IgH locus [29,30].

Generation of transgenic mice
3'RR-deficient mice [5] were crossed with Igλ-Myc mice [14] to derive heterozygous 3'RR-deficient/ Igλ-Myc mice and homozygous 3'RR-deficient/Igλ-Myc mice. Mice exhibiting obvious tumors or presenting signs of illness were immediately sacrificed. Our research has been approved by our local ethics committee review board (Comité Régional d'Ethique sur l'Expérimentation Animale du Limousin, Limoges, France) and carried according the European guidelines for animal experimentation.

Clonality assay
Genomic DNA prepared from lymph node lymphomas was digested with EcoRI and analysed by Southern blot with a 32 P-labeled J H probe [10].

mRNA expression
Total RNA was extracted from lymph node lymphomas. RNA was reverse-transcribed into cDNA by addition of reverse transcriptase to 2 µg total RNA in a final volume of 20 µl. Real time PCR was performed in duplicate by using TaqMan assay reagents and analysed on an ABI Prism 7000 system (Applied Biosystems Foster City, CA) [32]. Product reference: mouse c-myc, Mm00487803-m1; human c-myc, Hs00153408-m1. Mouse actin (Mn00607939-s1) was used for normalization of gene expression levels (Applied Biosystems). In another set of experiments, RNA was extracted from splenic B-cells (purified by CD43 magnetic cell sorting) of 8 week old Igλ-Myc/3'RR-deficient mice and Igλ-Myc mice (i.e., before any manifestation of disease).