Activated B-cell factor 1 (ABF-1) is a member of the bHLH transcription factor family. Its expression was detected in lymph nodes, appendix and other tissues, but not in thymus and peripheral blood lymphocytes (PBL). It contains a transrepression domain and was shown to inhibit transactivation of the related E2A transcription factors.1 E2A proteins are necessary for B-cell survival and proliferation.2 They are highly expressed in many types of B-cell non-Hodgkin's lymphomas (B-NHL), that is, follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) and Burkitt's lymphoma (BL).3, 4 In contrast, expression of the E2A antagonist ABF-1 is low or absent in these types of tumor. Interestingly, an inverse situation is observed in classical Hodgkin's lymphoma (cHL) where the levels of E2A are relatively low and ABF-1 expression is high.3 Indeed, ABF-1 expression was found to be limited to lymphoblastoid cell lines (LCL),5 cHL3 and primary effusion lymphoma.6 We therefore hypothesized that stable downregulation ABF-1 expression in FL, DLBCL and BL might endow a selective advantage to these tumors. Along with genomic mutations robust and stable gene inactivation has been shown to be achieved by epigenetic gene silencing.7 We therefore determined whether this mechanism contributes to the regulation of ABF-1 expression in distinct types of B-NHLs.
In agreement with earlier work, we found high-level ABF-1 mRNA and protein expression in cHL cell lines KM-H2, L1236 and L428, but not in BL cell lines Namalwa, BJA-B, Raji and Ramos (Figures 1a and b). To evaluate a possible contribution of epigenetic silencing to this lack of expression, BL cell lines were treated with the DNA methyltransferase I inhibitor 5-aza-dC. ABF-1 expression was restored in all BL cell lines at the mRNA level (Figure 1b). The ABF-1 reactivation was efficient enough to be detected at the protein level indicating that methylation is the main mechanism of ABF-1 repression at least in some of the BL cases (Figure 1c). The ABF-1 promoter region contains a cytosine-phosphate-guanine (CpG) island, a region with a high density of CpG dinucleotides (Figure 2a). Methylation of promoter CpG islands generally results in gene silencing.7 We investigated the methylation status of the ABF-1 promoter CpG island using two commonly accepted methods: bisulfite sequencing and pyrosequencing. We first compared ABF-1 promoter methylation in BL and cHL cell lines (Figure 2b). Although ABF-1 was hypomethylated in all cHL cell lines and the control LCL UM-1, virtually complete methylation of all CpG residues was seen in the BL cell lines. Thus, promoter methylation and ABF-1 expression levels show the expected inverse correlation.
We then extended our study using bisulfite sequencing toward primary normal or hyperplastic lymphoid tissues as well as samples of primary B-NHLs. The methylation status of the ABF-1 promoter was determined in hyperplastic tonsil samples, obtained from four patients. In addition, we analyzed PBL samples from three healthy donors (Figure 3a). Although methylated CpGs were occasionally present in both tonsil and in PBL samples, we did not find complete promoter methylation as in BL cell lines (Figures 2b and 3a). In contrast, hypermethylation of the ABF-1 promoter was observed in primary tumor samples. In FL, DLBCL and BL we found high levels of promoter methylation in 3 of 5, 6 of 6 and 3 of 3 cases, respectively (Figure 3a). The primary tumor samples show less complete and somewhat variegated hypermethylation pattern than the established BL cell lines (Figures 2b). The heterogeneity of the methylation patterns observed in primary tumors can be explained by the fact that in contrast to genetic mutations occurring in one step, promoter methylation increases gradually after initiation. Hence, complete uniform ABF-1 methylation pattern in BL cell lines may reflect continuously progressing methylation in vitro of partially methylated alleles in the tumors of origin. Alternatively, hypomethylated ABF-1 alleles in the B-NHL samples may be derived from contaminating reactive cells, which were below 20% in each tumor tissue examined. To further corroborate ABF-1 hypermethylation in B-NHLs we analyzed additional cases of B-NHLs as well as seven samples of CD19+ cells obtained from the blood of healthy donors using pyrosequencing (Figure 3b). Again we found ABF-1 promoter methylation in most cases of B-NHLs (Figure 3b). The combination of the data obtained by bisulfite sequencing and pyrosequencing showed that in 12 out of 16 cases of DLBL, 9 out of 15 cases of FL and 7 out of 8 cases of BL, the methylation of ABF-1 promoter was substantially higher than in normal tissues.
Our results suggest that epigenetic silencing of ABF-1 is commonly observed in diverse types of B-NHL, but not in cHL. In the past, primary mediastinal B-cell lymphoma (PMBL) was shown to share several features, particularly constitutive nuclear factor-κB activation, with cHL.8 Additionally, gene expression array data hint at the presence of ABF-1 in PMBL.8 Indeed, the PMBL cell line MedB-1 shows high level ABF-1 protein expression similar to the LCL UM-1 (Figure 4a). Furthermore, analysis of ABF-1 promoter methylation in MedB-1 cells demonstrated that the promoter is unmethylated (Figure 4b). In addition, we analyzed the methylation status of seven primary cases of PMBL. Only a single PMBL sample showed increased levels of ABF-1 promoter methylation, whereas the other six primary cases were completely unmethylated (Figure 4b). Finally, we analyzed whether the methylation status in these primary tumor samples correlated with ABF-1 expression level. Q-PCR analyses were therefore performed on several tissues and tumor cases. Very low levels of ABF-1 expression were seen in all FL, DLBCL and most BL cases. In contrast, PMBL cases showed significantly higher ABF-1 expression levels in most cases (Figure 4c). In summary, this analysis revealed a striking inverse correlation between methylation status and expression level. Interestingly, low levels of ABF-1 expression were also found in tonsils indicating that the ABF-1 gene is not expressed in these cells, although the promoter is accessible. This suggests that tumor cells employ an additional level of gene regulation, namely epigenetic silencing to permanently repress genes inappropriate with proliferation and/or survival.
Massari ME, Rivera RR, Voland JR, Quong MW, Breit TM, van Dongen JJ et al Characterization of ABF-1, a novel basic helix-loop-helix transcription factor expressed in activated B lymphocytes. Mol Cell Biol 1998; 18: 3130–3139.
Zhao F, Vilardi A, Neely RJ, Choi JK . Promotion of cell cycle progression by basic helix-loop-helix E2A. Mol Cell Biol 2001; 21: 6346–6357.
Mathas S, Janz M, Hummel F, Hummel M, Wollert-Wulf B, Lusatis S et al. Intrinsic inhibition of transcription factor E2A by HLH proteins ABF-1 and Id2 mediates reprogramming of neoplastic B cells in Hodgkin lymphoma. Nat Immunol 2006; 7: 207–215.
Renne C, Martin-Subero JI, Eickernjager M, Hansmann ML, Kuppers R, Siebert R et al. Aberrant expression of ID2, a suppressor of B-cell-specific gene expression, in Hodgkin's lymphoma. Am J Pathol 2006; 169: 655–664.
Kuppers R, Klein U, Schwering I, Distler V, Brauninger A, Cattoretti G et al. Identification of Hodgkin and Reed–Sternberg cell-specific genes by gene expression profiling. J Clin Invest 2003; 111: 529–537.
Lietz A, Janz M, Sigvardsson M, Jundt F, Dorken B, Mathas S . Loss of bHLH transcription factor E2A activity in primary effusion lymphoma confers resistance to apoptosis. Br J Haematol 2007; 137: 342–348.
Ushijima T, Okochi-Takada E . Aberrant methylations in cancer cells: where do they come from? Cancer Sci 2005; 96: 206–211.
Rosenwald A, Wright G, Leroy K, Yu X, Gaulard P, Gascoyne RD et al. Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma. J Exp Med 2003; 198: 851–862.
This study was supported in part by grant 107547 from Deutsche Krebshilfe eV to TW and AU.
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Ushmorov, A., Leithäuser, F., Ritz, O. et al. ABF-1 is frequently silenced by promoter methylation in follicular lymphoma, diffuse large B-cell lymphoma and Burkitt's lymphoma. Leukemia 22, 1942–1944 (2008) doi:10.1038/leu.2008.70
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