BackgroundMYC is a key transcription factor involved in central cellular processes such as regulation of the cell cycle, histone acetylation and ribosomal biogenesis. It is overexpressed in the majority of human tumors including aggressive B-cell lymphoma. Especially Burkitt lymphoma (BL) is a highlight example for MYC overexpression due to a chromosomal translocation involving the c-MYC gene. However, no genome-wide analysis of MYC-binding sites by chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) has been conducted in BL so far.Methodology/Principal FindingsChIP-Seq was performed on 5 BL cell lines with a MYC-specific antibody giving rise to 7,054 MYC-binding sites after bioinformatics analysis of a total of approx. 19 million sequence reads. In line with previous findings, binding sites accumulate in gene sets known to be involved in the cell cycle, ribosomal biogenesis, histone acetyltransferase and methyltransferase complexes demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC-binding sites also accumulate in many B-cell relevant genes. To assess the functional consequences of MYC binding, the ChIP-Seq data were supplemented with siRNA- mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Interestingly, amongst others, genes involved in the B-cell function were up-regulated in response to MYC silencing.Conclusion/SignificanceThe 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in BL and shed further light on the enormous complexity of the MYC regulatory network. Especially our observations that (i) many B-cell relevant genes are targeted by MYC and (ii) that MYC down-regulation leads to an up-regulation of B-cell genes highlight an interesting aspect of BL biology.

A unique feature of the tumor cells (Hodgkin/Reed-Sternberg (HRS)) of classical Hodgkin lymphoma (cHL) is the loss of their B-cell phenotype despite their B-cell origin. Several lines of evidence suggest that epigenomic events, especially promoter DNA methylation, are involved in this silencing of many B-cellassociated genes. Here, we show that DNA demethylation alone or in conjunction with histone acetylation is not able to reconstitute the B-cell-gene expression program in cultured HRS cells. Instead, combined DNA demethylation and histone acetylation of B-cell lines induce an almost complete extinction of their B-cell-expression program and a tremendous upregulation of numerous Hodgkin-characteristic genes, including key players such as Id2 known to be involved in the suppression of the B-cell phenotype. Since the upregulation of Hodgkincharacteristic genes and the extinction of the B-cell-expression program occurred simultaneously, epigenetic changes may also be responsible for the malignant transformation of cHL. The epigenetic upregulation of Hodgkin-characteristic genes thus playsFin addition to promoter DNA hypermethylation of B-cellassociated genesFa pivotal role for the reprogramming of HRS cells and explains why DNA demethylation alone is unable to reconstitute the B-cell-expression program in HRS cells.

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The online version of this article has a Supplementary Appendix. BackgroundEpigenetic changes are involved in the extinction of the B-cell gene expression program of classical Hodgkin's lymphoma. However, little is known regarding epigenetic similarities between cells of classical Hodgkin's lymphoma and plasma cell myeloma, both of which share extinction of the gene expression program of mature B cells. Design and MethodsGlobal histone H3 acetylation patterns were determined in cell lines derived from classical Hodgkin's lymphoma, plasma cell myeloma and B-cell lymphoma by chromatin immunoprecipitation and subsequent hybridization onto promoter tiling arrays. H3K27 trimethylation was analyzed by chromatin immunoprecipitation and real-time DNA polymerase chain reaction for selected genes. Epigenetic modifications were compared to gene expression data. ResultsCharacteristic B-cell genes were hypoacetylated in classical Hodgkin's lymphoma and plasma cell myeloma cell lines as demonstrated by comparison of their histone H3 acetylation patterns to those of B-cell lines. However, the number of genes jointly hyperacetylated and expressed in classical Hodgkin' lymphoma and plasma cell myeloma cell lines, such as IRF4/MUM1 and RYBP, is limited. Moreover, H3K27 trimethylation for selected characteristic B-cell genes revealed that this additional epigenetic silencing is much more prevalent in classical Hodgkin's lymphoma than in plasma cell myeloma. ConclusionsOur epigenetic data support the view that classical Hodgkin's lymphoma is characterized by abortive plasma cell differentiation with a down-regulation of characteristic B-cell genes but without activation of most genes typical of plasma cells.

Enhancer of zeste homolog 2 (EZH2) tri-methylates histone 3 at position lysine 27 (H3K27me3). Overexpression and gain-of-function mutations in EZH2 are regarded as oncogenic drivers in lymphoma and other malignancies due to the silencing of tumor suppressors and differentiation genes. EZH2 inhibition is sought to represent a good strategy for tumor therapy. In this study, we treated Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) cell lines with 3-deazaneplanocin—A (DZNep), an indirect EZH2 inhibitor which possesses anticancer properties both in-vitro and in-vivo. We aimed to address the impact of the lymphoma type, EZH2 mutation status, as well as MYC, BCL2 and BCL6 translocations on the sensitivity of the lymphoma cell lines to DZNep-mediated apoptosis. We show that DZNep inhibits proliferation and induces apoptosis of these cell lines independent of the type of lymphoma, the EZH2 mutation status and the MYC, BCL2 and BCL6 rearrangement status. Furthermore, DZNep induced a much stronger apoptosis in majority of these cell lines at a lower concentration, and within a shorter period when compared with EPZ-6438, a direct EZH2 inhibitor currently in phase II clinical trials. Apoptosis induction by DZNep was both concentration-dependent and time-dependent, and was associated with the inhibition of EZH2 and subsequent downregulation of H3K27me3 in DZNep-sensitive cell lines. Although EZH2, MYC, BCL2 and BCL6 are important prognostic biomarkers for lymphomas, our study shows that they poorly influence the sensitivity of lymphoma cell lines to DZNep-mediated apoptosis.