Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia

Wernig‑Zorc et al. Epigenetics & Chromatin https://doi.org/10.1186/s13072-018-0252-7 (2019) 12:4 Epigenetics & Chromatin Open Access RESEARCH Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia Sara Wernig‑Zorc1, Mukesh Pratap Yadav2, Pradeep Kumar Kopparapu2, Mats Bemark3, Hallgerdur Lind Kristjansdottir4, Per‑Ola Andersson4,5, Chandrasekhar Kanduri1 and Meena Kanduri2* Abstract Background: Chronic lymphocytic leukemia (CLL) has been a good model system to understand the functional role of 5-methylcytosine (5-mC) in cancer progression. More recently, an oxidized form of 5-mC, 5-hydroxymethylcytosine (5-hmC) has gained lot of attention as a regulatory epigenetic modification with prognostic and diagnostic implica‑ tions for several cancers. However, there is no global study exploring the role of 5-hydroxymethylcytosine (5-hmC) levels in CLL. Herein, using mass spectrometry and hMeDIP-sequencing, we analysed the dynamics of 5-hmC during B cell maturation and CLL pathogenesis. Results: We show that naïve B-cells had higher levels of 5-hmC and 5-mC compared to non-class switched and classswitched memory B-cells. We found a significant decrease in global 5-mC levels in CLL patients (n = 15) compared to naïve and memory B cells, with no changes detected between the CLL prognostic groups. On the other hand, global 5-hmC levels of CLL patients were similar to memory B cells and reduced compared to naïve B cells. Interestingly, 5-hmC levels were increased at regulatory regions such as gene-body, CpG island shores and shelves and 5-hmC distribution over the gene-body positively correlated with degree of transcriptional activity. Importantly, CLL samples showed aberrant 5-hmC and 5-mC pattern over gene-body compared to well-defined patterns in normal B-cells. Inte‑ grated analysis of 5-hmC and RNA-sequencing from CLL datasets identified three novel oncogenic drivers that could have potential roles in CLL development and progression. Conclusions: Thus, our study suggests that the global loss of 5-hmC, accompanied by its significant increase at the gene regulatory regions, constitute a novel hallmark of CLL pathogenesis. Our combined analysis of 5-mC and 5-hmC sequencing provided insights into the potential role of 5-hmC in modulating gene expression changes during CLL pathogenesis. Keywords: Hydroxymethylation, CpG islands, Enhancers and chronic lymphocytic leukemia *Correspondence: 2 Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska University Hospital, 413 45 Gothenburg, Sweden Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wernig‑Zorc et al. Epigenetics & Chromatin (2019) 12:4 Introduction DNA methylation is a well-investigated, stable, heritable regulatory epigenetic modification in the mammalian genome and is established by interplay between maintenance DNA methyltransferase DNMT1 and de novo methyltransferases DNMT3A, and DNMT3B [1]. Evidence over the last two decades revealed that cancer DNA methylomes significantly differ from their normal tissue counter parts. Cancer genomes are characterized by global hypomethylation of the DNA, with frequent focal hypermethylation of tumor-suppressors [2, 3]. Chronic lymphocytic leukemia (CLL) has served as an ideal model system to understand the functional role of DNA methylation during cancer progression, evolution and risk stratification [4–7]. Previous studies have documented a progressive loss of methylation during B-cell development and CLL maturation [8, 9]. This progressive loss of methylation can be achieved in two alternate ways: (1) passive demethylation, by the failure of maintenance methylation following DNA replication, or (2) active demethylation, by replication-independent processes. Active demethylation is carried out by 5-methylcytosine hydroxylases TET1, TET2 and TET3 (TET refers to TenEleven-Translocation); which convert 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) via oxidation [10, 11]. Recent studies have shown that 5-hmC is not just an intermediate product during the DNA demethylation process, but rather a stable epigenetic mark, which regulates chromatin modifications and gene transcription during developmental stages and cellular differentiation [12]. Like DNA methylation, global loss of hydroxymethylation has also been observed in many different cancers [13–16]. Importantly, loss of hydroxymethylation serves as a prognostic marker in different cancers and solid tumors [17–20]. Reduction of 5-hmC levels was also observed in certain cell types that are highly proliferative in nature and contain stem cell character such as cryptic cells of small intestine [21] and proliferative mouse neural progenitor cells [22]. Important genes regulating DNA demethylation and methylation such as TET genes, IDH genes and DNMT3A are frequently mutated in myeloid malignancies [23, 24]. Moreover, 5-hmC levels positively correlate with better overall survival [25]. In CLL, the loss of global DNA methylation levels is well documented by many researchers, including our earlier studies. Interestingly, unlike other lymphomas, CLL do not show TET mutations [26]. Hence, it would be interesting to analyze the global levels of 5-hmC in CLL and compare them with global DNA methylation levels. Also, as promoter 5-hmC has been shown to positively correlate with gene expression, it would be of interest to know whether 5-hmC plays a functional role in CLL pathogenesis via Page 2 of 15 modulation of DNA methylation levels. Previous studies analysed DNA methylation changes during normal B-cell development (naïve B-cells to more differentiated memory B-cells) and CLL pathogenesis. They proposed that DNA methylation changes occurring during B-cell maturation are also recapitulated during CLL progression [27], indicating that different CLL prognostic groups derive from a continuum of maturation states reflected in normal B-cell development. Therefore, there is a need to distinguish between normal and disease-specific epigenetic events to explore the functionally important epigenetic changes that occur during disease progression. Most studies to date have not distinguished between 5-mC and 5-hmC levels in CLL or during normal B-cell maturation, due to limitations of available methods (for example Bisulphite sequencing). (...truncated)