Loss of Dnmt3a induces CLL and PTCL with distinct methylomes and transcriptomes in mice

www.nature.com/scientificreports OPEN received: 13 April 2016 accepted: 07 September 2016 Published: 28 September 2016 Loss of Dnmt3a induces CLL and PTCL with distinct methylomes and transcriptomes in mice Staci L. Haney1,*, Garland M. Upchurch2,*, Jana Opavska2, David Klinkebiel3, Adams Kusi Appiah4, Lynette M. Smith4, Tayla B. Heavican5, Javeed Iqbal5,6, Shantaram Joshi1,6 & Rene Opavsky1,2,6 Cytosine methylation of DNA is an epigenetic modification involved in the repression of genes that affect biological processes including hematopoiesis. It is catalyzed by DNA methyltransferases, one of which -DNMT3A- is frequently mutated in human hematologic malignancies. We have previously reported that Dnmt3a inactivation in hematopoietic stem cells results in chronic lymphocytic leukemia (CLL) and CD8-positive peripheral T cell lymphomas (PTCL) in EμSRα-tTA;Teto-Cre;Dnmt3afl/fl; Rosa26LOXPEGFP/EGFP (Dnmt3aΔ/Δ) mice. The extent to which molecular changes overlap between these diseases is not clear. Using high resolution global methylation and expression analysis we show that whereas patterns of methylation and transcription in normal B-1a cells and CD8-positive T cells are similar, methylomes and transcriptomes in malignant B-1a and CD8+ T cells are remarkably distinct, suggesting a cell-type specific function for Dnmt3a in cellular transformation. Promoter hypomethylation in tumors was 10 times more frequent than hypermethylation, three times more frequent in CLL than PTCL and correlated better with gene expression than hypermethylation. Crossspecies molecular comparison of mouse and human CLL and PTCL reveals significant overlaps and identifies putative oncogenic drivers of disease. Thus, Dnmt3aΔ/Δ mice can serve as a new mouse model to study CLL and PTCL in relevant physiological settings. Cytosine methylation of DNA is an epigenetic modification affecting gene transcription and the integrity of the mammalian genome. Basic methylation patterns are established and maintained by catalytic activity of three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Promoter methylation is associated with transcriptional repression and plays a role in a variety of normal physiologic processes, including X-chromosome inactivation, genomic imprinting, differentiation and hematopoiesis1,2. Non-Hodgkin’s lymphoma (NHL) is a heterogeneous group of lymphoid malignancies that arise from transformation of B, T, and NK cells. The majority of NHLs are B-cell lymphomas, the most common of which is chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL): an indolent low-grade lymphoproliferation of mature B-cells3. However, T cell lymphomas develop in ~10% of NHL patients4. Approximately 30% of these T cell malignancies will be diagnosed as peripheral T cell lymphoma-not otherwise specified (PTCL-NOS): a group of high-grade mature T cell neoplasms not classified by other WHO criteria5. Both CLL and PTCL are life-threatening conditions that present in late adulthood and despite recent advances in chemotherapy, these diseases remain refractory to cure. A better understanding of deregulated molecular landscapes and a contribution of individual changes to the development of these two NHLs is needed to generate new therapeutic approaches. Two types of molecular changes likely involved in the pathogenesis of CLL and PTCL are genetic alterations and epimutations such as de-regulated cytosine methylation. It was reported that an average of 45 somatic mutations are present in human CLL samples, with most genes mutated in less than 5% of cases6. About one 1 Department of Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska, 68198, USA. 2Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, 68198, USA. 3Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, 68198, USA. 4College of Public Health, UNMC, Omaha, Nebraska, 68198, USA. 5Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, 68198, USA. 6Center for Leukemia and Lymphoma Research, University of Nebraska Medical Center, Omaha, Nebraska, 68198, USA. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to R.O. (email: ) Scientific Reports | 6:34222 | DOI: 10.1038/srep34222 1 www.nature.com/scientificreports/ third of cases did not have recurrent mutations, suggesting a high degree of heterogeneity and no clear genetic drivers of CLL. Interestingly, global gene expression profiling identified two DNA methyltransferases, Dnmt3b and Dnmt3a, as the top 1% of underexpressed genes in human CLL7, suggesting that the DNA methylation landscape may be deregulated. Indeed, DNA methylation profiling revealed a substantial genome wide promoter and gene-body hypomethylation in tumors relative to normal B cells8. Consistently with possible roles of Dnmt3a and Dnmt3b in CLL development we have previously reported that conditional inactivation of Dnmt3a in hematopoietic stem cells and progenitors in EμSRα-tTA;Teto-Cre;Dnmt3afl/fl;Rosa26 LOXP EGFP/EGFP (Dnmt3aΔ/Δ) mice resulted in the development of chronic lymphocytic leukemia (CLL) around 1 year of age and is accelerated when Dnmt3b is deleted as well9. Such data strongly suggest that Dnmt3a represses genes in normal B cells likely through promoter methylation whose up-regulation upon hypomethylation may contribute to the development of CLL. The mutational landscape of T cell lymphoma (TCL) appears to be less diverse than in CLL. Interestingly, one of the most frequently mutated gene is DNMT3A, suggesting a possible involvement in disease development10. Although no comparable large scale profiling of the methylation landscape has been performed on TCL to date, our functional studies utilizing EμSRα-tTA;Teto-Cre;Dnmt3afl/fl;Rosa26 LOXPEGFP/EGFP mice demonstrated that Dnmt3a likely play a role in pathogenesis of PTCL. Although these mice primarily develop CLL, 30% of mice develop CD8-positive PTCL either in combination with CLL or by itself 9,11. However, the nature of deregulated events in both mouse diseases and how they relate to human diseases remains poorly understood. To better understand the molecular changes occurring in Dnmt3a-deficient mice we performed global methylation profiling using whole genome bisulfite sequencing (WGBS) and gene expression profiling using RNA-seq on CLL and PTCL tumors isolated from Dnmt3aΔ/Δ mice, as well as control B-1a and CD8+T cells. This analysis revealed that while normal B-1a and CD8+T cells had remarkably similar methylomes and transcriptomes, Dnmt3a loss induced unique changes in DNA methylation and gene transcription in CLL and PTCL. Importantly, analysis of available expression data from human CLL and PTCL samples, revealed a significant overlap between human and mou (...truncated)