Hypermethylation of Bcl6 Is a Potential Cause of Development of Lymphoma with Tfh Features in Tet2 Knockdown Mice

TET2 is known as an enzyme which converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Loss-of-function mutations in TET2 are frequent in angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS). Normal counterpart of AITL is thought to be follicular helper T cells (Tfh). PTCL-NOS is likely to consist of heterogeneous groups. Some of the PTCL-NOS cases also have features of Tfh. In the last annual meeting, we reported that aged homozygous Tet2 gene-trap mice (Tet2^gt/gt), which showed 80% reduction in the Tet2 mRNA level in various hematopoietic cells, developed T-cell lymphoma. To further investigate the mechanism of T-lymphomagenesis, we analyzed methylome, hydroxymethylome, and transcriptome in the lymphoma cells.

Tet2^gt mice have a trapping vector inserted into the second intron of the Tet2 locus. In all the analyses, we used CD4+ T cells prepared from lymphoma cells developed in these mice, as well as CD4+ T cells prepared from spleen of wild-type mice as a control. To investigate genome-wide methylation and hydroxymethylation statuses, we performed MeDIP and hMeDIP sequencing, and bisulfite sequencing. To examine comprehensive gene expression, we performed microarray-based analysis, followed by Gene Set Enrichment Analysis (GSEA).

After observation over a year (median, 67 weeks), 5 out of 7 Tet2^gt/gt mice developed T-cell lymphomas with Tfh-like immunostaining pattern. No differences were found in the average levels of 5mC between lymphoma and control CD4+ cells throughout the regions around transcription start sites (TSS) +/- 5 kb. In contrast, when the same regions were analyzed for 5hmC levels, those in the lymphoma cells were significantly lower at the regions around TSS +/- 1 kb. When focused on regions having high 5mC contents (MACS score>5.0), lymphoma cells demonstrated a significant enrichment at regions around TSS +/- 1 kb, intragenic regions, and CpG islands (p=0.013, 0.006, 0.022 respectively). On the other hand, 5hmC was significantly decreased at regions around TSS +/- 1 kb in lymphoma cells than control cells (p=0.018). In a set of genes whose expression was higher in lymphoma cells than control cells, 5hmC levels were significantly lower in lymphoma cells. GSEA analysis revealed upregulation of Tfh-associated genes such as Bcl6 and cMaf (FDR q value=0.0004), key transcription factors for Tfh differentiation, in lymphoma cells compared with control cells. The expression of upregulated Tfh-associated genes was validated by real-time PCR. We focused on the epigenetic change of Bcl6 because it is among the most important transcription factors for Tfh development. It was reported that hypermethylation at intron 1 of Bcl6 upregulated its transcriptional activity in B cell lymphomas. Bisulfite sequencing revealed that the CpG sites in the intron 1 of Bcl6 were massively methylated/hydroxymathylated in lymphoma cells, whereas those in control cells were mostly at an unmodified status. MeDIP sequencing indicated that intron 1 of Bcl6 had more methylated status in lymphoma cells than control cells. We also found that CpG sites in the same region were densely methylated/hydroxymathylated in EL4, mouse T-cell lymphoma cell line, and that the decitabine treatment converted them into unmodified CpG along with the decrease in the Bcl6 expression levels.

Tet2^gt/gt mice developed T-cell lymphoma with both Tfh-like immunohistological character and gene expression pattern. We found distinct changes in methylome, hydroxymethylome, and transcriptome. We also found a tight linkage between the increased methylation of intron 1 of Bcl6 and increased expression of its mRNA level in lymphoma cells developed in Tet2 knockdown mice. The same scenario is indicated in a T-cell lymphoma cell line. These observations imply that, in normal CD4+ T cells, reduced Tet2 function might increase the methylation status of the CpG sites in intron 1 of Bcl6, which may result in upregulation of Bcl6 expression and deviated Tfh generation. These processes might be an initiating event for the development of T-cell lymphoma with the Tfh features. Because of the long latency before lymphoma development in Tet2^gt/gtmice, it is likely that additional hits are necessary.

No relevant conflicts of interest to declare.