ABF-1 is frequently silenced by promoter methylation in follicular lymphoma, diffuse large B-cell lymphoma and Burkitt's lymphoma

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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.

Figure 1
figure1

Activated B-cell factor 1 (ABF-1) expression can be reactivated by 5-aza-dC in follicular lymphoma (FL) and Burkitt's lymphoma (BL) cell lines. (a) ABF-1 is expressed in classical Hodgkin's lymphoma (cHL) but not in BL cell lines. Expression of the ABF-1 protein in cHL and BL cell lines was assessed by western blot using the specific goat antibodies (sc-9556) and, as a control, rabbit antibodies against RelA/p65 (sc-372) (Santa Cruz Biotechnology, Heidelberg, Germany). (b) Reactivation of ABF-1 transcription in BL cell lines by 5-aza-dC. Cells (2.5 × 106) were seeded in 25 ml of the complete RPMI 1640 medium (Day 0). On the next day 5-aza-dC was added at a final concentration of 2 μM. Control wells were treated with the relevant amount of the vehicle (dimethyl sulfoxide (DMSO), 5 μl). After 24 h the cells were collected by centrifugation and resuspended in the fresh medium. On day 5, cells were harvested for RNA and protein purification. cDNA was amplified with ABF-1-specific primers (5′-CGGCGGCGCGAGGTGGTAG-3′ and 5′-TCTTCCCATCTCACGAGCTCTCCCTTCT-3′). HMBS (hydroxymethylbilane synthase) was amplified as loading control (primers: 5′-AGCTGCAGAGAAAGTTCCC-3′ and 5′-GTTACGAGCAGTGATGCC-3′). PCR products were resolved on 1.5% agarose gel and stained with ethidium bromide (EB). (c). Reactivation of ABF-1 can be observed on the protein level. ABF-1 expression in Raji cells treated or not treated with 5-aza-dC was analyzed by western blot. Experiments were repeated at least two times and a representative result is shown.

Figure 2
figure2

Activated B-cell factor 1 (ABF-1) promoter is hypermethylated in Burkitt's lymphoma (BL) cell lines. (a) Structure of the ABF-1 gene and location of the bisulfite sequencing primers. The cytosine-phosphate-guanine (CpG) island located in the promoter region of the ABF-1 gene was amplified with the help of bisulfite sequencing primers (designated as: F, forward primer and R, reverse primer). The obtained amplicon contained 13 CpG dinucleotides. The first and last CpGs are at positions −323 and −147 counting from the start site of transcription, respectively. The first exon is depicted as an open box. The CpG dinucleotides are represented as vertical bars. (b) Methylation status of the ABF-1 promoter in B-cell lymphomas was analyzed by pyrosequencing. gDNA (1 μg) was converted using the Methylamp DNA Modification Kit (Epigenetek, NY, USA). The converted gDNA was amplified with primers 5′-GGGTTTTAGAGTTTAAATTT-3′ and 5′-TATACTCCTTAAAACAAACCACC-3′ using the HotStarTaq Polymerase Master Kit (15 min at 95 °C followed by five cycles at 95 °C for 30 s, 52.6 °C for 1 min, 72 °C for 2 min and 45 cycles 95 °C for 30 s, 52.6 °C for 45 s, 72 °C for 1 min, followed by 7 min incubation at 72 °C). The PCR products were pyrosequenced with the sequencing primer 5′-GGGTTTTAGAGTTTAAAT-3′. Pyrosequencing was performed with a PSQ 96MA system using Pyro Gold Reagents. Data were analyzed with the Pyro Q-CpG software (all from Biotage, Uppsala, Sweden). The methylation density of the part of the ABF-1 promoter including CpGs 7–12 is shown as the percentage of methylated single CpGs using the color coding.

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.

Figure 3
figure3

Activated B-cell factor 1 (ABF-1) promoter hypermethylation is rare in normal tonsils and peripheral blood lymphocytes (PBL) but common in B-cell non-Hodgkin's lymphomas (B-NHLs). The methylation status of the ABF-1 promoter in samples of lymphoid organs and primary cases of B-NHLs was analyzed using bisulfite sequencing (a) or pyrosequencing (b). Primary cases of follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), primary mediastinal B-cell lymphoma (PMBL) as well as hyperplastic tonsillar tissues were from our bank of fresh tissues. The tumor material was pseudonymized to comply with the German law for correct usage of archival tissue for clinical research (Deutsches Ärzteblatt 2003; 100 A1632). Approval for this procedure was obtained from the local ethics committee. For gDNA methylation analyses, frozen tissue samples were cut in 25 μm thick slices. Sections (2 μm) were hematoxylin and eosin (H&E) stained and assessed microscopically. PBL were obtained from buffy coats by gradient separation using 1.077 g l−1 ficoll (PAN, Aidenbach, Germany). CD19+ cells were isolated from PBL using human CD19 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The purity of this CD19+ fraction was about 90% as assessed by anti-CD19-fluorescein isothiocyanate (FITC) staining and fluorescence-activated cell sorting (FACS) analysis. gDNAs from cells and tissue samples were purified using the DNease tissue kit, no. 69504 (Qiagen, Hilden, Germany). For bisulfite sequencing, 100 ng of the bisulfite-converted DNA was amplified with primers specific for the ABF-1 promoter cytosine-phosphate-guanine (CpG) island (5-TAAGATTATTTGGGGGTAGAAGTAT-3 and 5′-AAAAACCAAAAAATAAAACAAAAAC-3′). The annealing temperature was 55 °C. For the first 30 cycles of amplification we used the HotStarTaq Polymerase Master Kit (Qiagen). The ends of PCR products were blunted by 15 cycles with Pwo DNA polymerase (Roche, Mannheim, Germany). PCR products were then cloned into pCAPs vector using the PCR cloning kit (Roche). XL-1 Blue competent cells (Stratagene, Amsterdam, the Netherlands) were transformed with the vector. The cloned plasmids were purified with Quiaprep Spin Miniprep Kit (Qiagen) and sequenced using BigDye Terminator v1.1 Cycle Sequencing Kit and ABI Prism 310 Genetic Analyzer (both from Applied Biosystems, Warrington, UK). Clones (5–8) were sequenced from each sample. The methylation intensity was expressed as percent of methylated CpGs and shown by color coding. The pyrosequencing was done as described in legend of Figure 2.

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.

Figure 4
figure4

Activated B-cell factor 1 (ABF-1) promoter methylation is associated with downregulation of transcription. (a) ABF-1 is expressed in lymphoblastoid cell line (LCL) UM1 and primary mediastinal B-cell lymphoma (PMBL) cell line MedB-1. Western blots performed with the ABF-1-specific and, as a control, RelA/p65-specific antibodies are shown. (b) ABF-1 promoter methylation was determined by pyrosequencing in MedB-1 and primary PMBL cases as described above. Methylation levels were again color coded. (c) ABF-1 expression in samples of hyperplastic tonsils and B-cell non-Hodgkin's lymphomas (B-NHLs) was assessed by Q-PCR using QuantiTect SYBR Green PCR Kit (Qiagen). PMBL were diagnosed according to standard clinical, histomorphological and immunohistological criteria outlined by the World Health Organization Classification of Tumours (F Menestrina, JL Harris, P Möller. ‘Primary mediastinal large B-cell lymphoma’. In: Jaffe ES, Harris NL, Stein H, Vardiman JW (eds). World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart, IARC Press: Lyon, 2004 . The primers for the ABF-1 were forward 5′-GCTTCCAGTTACATCGCTCA-3′, reverse 5′-GAATGGCCATGTCAGGTTC-3′. RPL13a was used as internal control, forward: 5′-CGGACCGTGCGAGGTAT-3′, reverse: 5′-CACCATCCGCTTTTTCTTGTC-3′. The expression levels of ABF-1 were normalized to the sample TON1.

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Acknowledgements

This study was supported in part by grant 107547 from Deutsche Krebshilfe eV to TW and AU.

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Correspondence to T Wirth.

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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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