Charting the dynamic epigenome during B-cell development

Accepted Manuscript Title: Charting the dynamic epigenome during B-cell development Authors: Jose I. Martin-Subero, Christopher C. Oakes PII: DOI: Reference:

S1044-579X(17)30177-3 http://dx.doi.org/10.1016/j.semcancer.2017.08.008 YSCBI 1373

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Seminars in Cancer Biology

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28-6-2017 21-8-2017 22-8-2017

Please cite this article as: Martin-Subero Jose I, Oakes Christopher C.Charting the dynamic epigenome during B-cell development.Seminars in Cancer Biology http://dx.doi.org/10.1016/j.semcancer.2017.08.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Invited review Seminars in Cancer Biology Special issue on "Epigenetics on hematological malignancies"

Charting the dynamic epigenome during B-cell development Jose I. Martin-Subero1 & Christopher C. Oakes2

1. Biomedical Epigenomics Group, Institut d'investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain. 2. Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA.

Contact details: Jose Ignacio Martin-Subero Centre Esther Koplowitz, Rossello 153, 2nd floor 08036 Barcelona Spain Email: [email protected]

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Abstract The epigenetic landscape undergoes a widespread modulation during embryonic development and cell differentiation. Within the hematopoietic system, B cells are perhaps the cell lineage with a more dynamic DNA methylome during their maturation process, which involves approximately one third of all the CpG sites of the genome. Although each B-cell maturation step displays its own DNA methylation fingerprint, the DNA methylome is more extensively modified in particular maturation transitions. These changes are gradually accumulated in specific chromatin environments as cell differentiation progresses and reflect different features and functional states of B cells. Promoters and enhancers of B-cell transcription factors acquire activation-related epigenetic marks and are sequentially expressed in particular maturation windows. These transcription factors further reconfigure the epigenetic marks and activity state of their target sites to regulate the expression of genes related to B-cell functions. Together with this observation, extensive DNA methylation changes in areas outside gene regulatory elements such as hypomethylation of heterochromatic regions and hypermethylation of CpG-rich regions, also take place in mature B cells, which intriguingly have been described as hallmarks of cancer. This process starts in germinal center B cells, a highly proliferative cell type, and becomes particularly apparent in long-lived cells such as memory and plasma cells. Overall, the characterization of the DNA methylome during B-cell differentiation not only provides insights into the complex epigenetic network of regulatory elements that mediate the maturation process but also suggests that late B cells also passively accumulate epigenetic changes related to cell proliferation and longevity.

Introduction to epigenetics The term epigenetics was coined back in 1942 by Conrad Hal Waddington to designate all those changes in the phenotype that are not caused by changes in the genotype [1]. In modern times, epigenetics is reformulated as the heritable changes in the chromatin structure that modify gene expression without changes in the DNA sequence itself, and it permeates basically every branch of life sciences, from evolution to development, cell differentiation and pathophysiology of diseases. During development and cell differentiation, epigenetics endows the ability to firstly establish a novel cellular state and then maintain that state to stabilize cell identity that is necessary for multicellular organisms. At the molecular level, epigenetics deals with the dynamic modulation of the chromatin structure, which modifies access to the information contained in the DNA, and it is made of multiple molecular layers of functional information such as DNA methylation, histone modifications, chromatin accessibility and nuclear dynamics [2-4]. DNA methylation is the most widely studied epigenetic layer. At the biochemical level, it consists on the covalent addition of a methyl group (CH3) at the 5 position of cytosines, mostly in the context of CpG dinucleotides, but some cell types such as stem cells and neurons also show cytosine methylation in non-CpG contexts [5-6]. Typically, these dinucleotides are 2

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concentrated in large clusters, called CpG islands (CGIs) that are enriched in promoter and/or first exon regions of approximately 60% of all human promoters [7]. Overall the genome of stem cells is highly methylated in regions with low-CpG content and unmethylated CGIs,and this global pattern can change in the context of cell differentiation and diseases such as cancer [2, 8-10]. DNA methylation is mediated by a class of enzymes called DNA methyltransferases (DNMTs) being DNMT1, DNMT3A and DNMT3B the most active members. DNA methylation can be either passively lost through high proliferation [11] or actively. Active demethylation is mediated by enzymes of the TET family, which are able to deaminate methylated cytosines, which are then replaced by unmethylated cytosines by DNA repair mechanisms [12]. There is also evidence that AID, an enzyme essential in the somatic hypermutation machinery in germinal center B cells, can also induce active demethylation [13-14]. Based on a large body of publications, it is widely accepted that main role of DNA methylation is gene repression. However, recent studies of the DNA methylome and transcriptome are revealing that this association is more nuanced than previously appreciated, and that DNA methylation can have multiple context-dependent roles [15]. In particular, it is essential to realize that a particular functional state of the genome is achieved by the interplay among various epigenetic layers, and in this context specific histone modifications play a key role [16]. The aminoacids of N-terminal tails of histones can be modified in distinct ways such as methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, among others [1719]. These chemical modifications are introduced or removed from particular aminoacid residues of histone tails by a long list of specific enzymes, including histone methyltranferases and demethylases, histone acetyltransferases and deacetylases, etc [20]. Combination of different histone modifications form a specific “histone code” that determines the structure and function of a particular chromatin region [21]. Current efforts from international consortia are generating genome-wide profiles of multiple histone modifications with non-overlapping functions (e.g. H3K4me3 for promoters, H3K4me1 for enhancers, H3K27ac for active regulatory elements, H3K36me3 for transcribed regions, H3K27me3 for polycomb-mediated repression and H3K9me3 for heterochromatin) [16, 22]. Integration of overlapping and mutually-exclusive patterns of these key histone marks allow for segmentation of the genome into functional elements called chromatin states, including e.g. poised, weak or active promoters, weak or strong enhancers, transcriptional elongation or different types of repressed/heterochromatic regions [23]. In addition to histone modifications, chromatin accessibility determines nucleosome-free regions in which transcription factors are binding and represents an important epigenetic layer as well [24]. Finally, the recent years are witnessing the emergence of the 3D structure of the genome and DNA looping between distant regions as a new key element in the regulation of gene expression [25]. In particular, 3D interactions among elements such as promoters and enhancers are part of the epigenetic network of gene regulation [26].

A summary of B-cell development Before focusing on the epigenetic landscape of B cells, we will first succinctly review the B-cell differentiation process, which has been thoroughly reviewed elsewhere [27-30]. B-cell 3

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maturation is a multi-step process governed by the interplay between expression of maturation stage-specific transcription factors and proteins, and external influences from the different microenvironments in which B cells develop [31-33]. The tissue where the process starts is the bone marrow, where hematopoietic precursor cells differentiate into common lymphoid progenitors. Then, such progenitor cells commit to the B-cell lineage due to the expression of lineage-specific transcription factors such as PAX5, EBF1 and TCF3 (also known as E2A) [31] to give rise to precursor B cells, which gradually rearrange their immunoglobulin genes. In this differentiation window, three stages of early B cells can be distinguished in the bone marrow: 1) pro-B cells, which undergo a VDJ rearrangement of the IGH locus, 2) pre-B cells, that express membrane  chains with surrogate light chains in the pre-B receptor, and 3) immature B cells in which heavy and light chains are combined to form an immunoglobulin (i.e. IgM) that is expressed on the cell surface. Immature cells further mature into transitional B cells, which leave the bone marrow and fully mature into naive B cells in the peripheral blood. These naive B cells circulate through secondary lymphoid organs until, eventually, they are activated in the lymph node follicle by exogenous antigens and interactions with antigenspecific T cells. Then, they become germinal center founder B cells, which enter the dark zone of the germinal center to become highly-proliferating cells (called centroblasts). In this context, upregulation of the BCL6 transcription factor is essential for the germinal center formation. A key step at this stage is the induction of somatic hypermutation of the immunoglobulin genes through the action of activation-induced cytidine deaminase (AICDA locus encoding for the AID enzyme). Centroblasts further differentiate into centrocytes (i.e. light zone B cells) that undergo positive and negative selection depending on their increased or decreased affinity of their B-cell receptors, respectively. Upon positive selection, a fraction of the centrocytes is instructed to recirculate to the dark zone to generate antibody variants with even higher affinity. Centrocytes undergo immunoglobulin class-switch recombination and eventually, they differentiate into high-affinity memory and plasma cells. Plasma cells producing large amounts of high-affinity antibodies are generated thanks to the upregulation of transcription factors such as IRF4 and BLIMP1, and downregulation of PAX5. Plasma cells migrate to the bone marrow, where they can turn into long-lived plasma cells. In contrast, the mechanisms that differentiate centrocytes into memory B cells are less clear, as key transcription factors for memory cell fate have not been identified. The prevailing model is that memory B cells stochastically originate from germinal centre B cells and that a survival advantage is sufficient for their differentiation [34]. Memory B cells can also live for extended periods of time, and ensure a rapid antibody-mediated immune response upon a secondary exposure to the same antigen. This summary represents the canonical pathway for B-cell differentiation. However, other modes of maturation outside the germinal center are also possible and able to generate memory B cells both in a T cell-independent and T cell-dependent fashion, which have been reviewed elsewhere [35-36]. B-cell lymphopoiesis represents an excellent model to investigate how the epigenome is physiologically modulated during normal cell differentiation. On the one hand, different B-cell maturation stages are characterized by distinct phenotypic and transcriptional features, and each of these subpopulations can be sorted for molecular studies using specific surface markers from bone marrow, peripheral blood or lymphoid tissues of healthy donors. On the other hand, different B-cell maturation stages have multiple cellular behaviors (e.g. 4

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proliferative vs. quiescent), life spans (short vs. long-lived) and are influenced by external cues from distinct microenvironments. Various previous review articles have focused on the epigenetic regulation of specific genes and processes important for B-cell development [3742]. Here, these aspects will only be briefly reviewed and we will focus mostly on the lessons learnt from genome-wide analysis of the epigenome during B-cell differentiation.

Epigenetic regulation of specific B cell-related genes and processes Over the last two decades, extensive mechanistic studies have revealed that epigenetic modifiers affect various aspects of B-cell development, and that key B-cell differentiation genes and processes are epigenetically regulated. Unless otherwise specified, the functional studies summarized below are based on mouse models. Loss of function studies in transgenic mice have revealed several epigenetic modifiers to be essential for the regulation of cell differentiation in general [43] and particular windows of B-cell maturation [44-45]. Specific members of the polycomb complexes 1 and 2, related to gene repression through methylation of H3K27, affect B-cell maturation. Deficiencies in e.g. BMI1 or MEL18 lead to blocks in B-cell development [46-47], CBX2 alters splenic B-cell responses to lipopolysaccharide [48], and EZH2 affects both early B-cell development [49] and the formation of the germinal center [50]. Histone deubiquitinases are also involved in B-cell differentiation, e.g. MYSM1, a H2A deubiquitinase, orchestrates a variety of histone modifications and recruitment to TFs to the EBF1 locus, controlling thus early B-cell differentiation [51]. Histone acetyltransferanses (HATs) and histone deacetylases (HDACs) have also been involved in B-cell differentiation. For instance, deficiency of the acetyltransferase MOZ impairs the generation of centroblasts in the germinal center [52]. In the case of HDACs, proper B-cell development requires either HDAC1 or HDAC2, as deletion of both enzymes blocks early B-cell differentiation [53], HDAC7 deficiency impairs early B-cell development [54] and treatment with HDAC inhibitors affects Bcell proliferation, survival and differentiation, and reduces primary antibody responses [55]. Finally, DNA methyltransferases are also essential for hematopoiesis and B cells. Mice with hypomorphic DNMT1 are skewed towards myeloid lineages in early hematopoiesis [56] and in mature B cells, they show deficient germinal center formation [57]. Collectively, the studies mentioned above highlight that specific chromatin modifiers are essential at key steps of B-cell differentiation through controlling the expression of specific genes required for particular maturation stage transitions. The epigenetic mechanisms regulating the expression of several genes essential for B-cell development have also been studied. A large body of evidence indicates that the epigenetic landscape of the immunoglobulin loci, including histone modifications, DNA methylation, DNA looping and non-coding RNAs, plays a crucial role in V(D)J, class-switch recombination and somatic hypermutation (reviewed in [42, 58-61]). Furthermore, the epigenetic regulation of B cell TFs and proteins associated with particular maturation transitions, such as EBF1 and PAX5 (uncommitted progenitors to early B cells), AICDA/AID and BCL6 (naive B cells to germinal center B cells) and PRDM1 (germinal center B cells to plasma cells), among others, has been investigated. EBF1 has been shown to be regulated by the concerted action of TFs such as ETS1, PAX5, PU.1, and the RUNX1/CBF-complex, and epigenetic changes mediated by 5

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nucleosome remodeling and histone modifications [51, 62-64]. Interestingly, the Ebf1 locus is located at the heterochromatic nuclear lamina in progenitor cells whereas in pro-B cells, this locus and other B-cell specific loci become active through switching compartments and establishing new 3-dimensional interactions [65].In the case of the Pax5 gene, its stepwise epigenetic regulation in progenitor cells and early B cells has been studied in detail. The Pax5 enhancer becomes demethylated in hematopoietic progenitors whereas the Pax5 promoter is demethylated and activated only at the onset of pro-B cell development through binding of EBF1 [66]. Aicda activation is mediated by binding to several transcription factors [67-68] and is associated with demethylation of promoter and intronic regions of the locus [69]. The epigenetic configuration of Bcl6 changes in germinal center B cells through the action IRF8 and histone modifiers such as PRMT7 and MOZ [52, 70-72]. In the case of Prdm1/Blimp1, E2A and E2-2 transcription factors have been recently been described to be involved in its activation through binding and activation of distant enhancers and modifying the 3D architecture of the locus [73], and the BACH2 TF together with HDAC3 seem to also be involved in Prdm1 activation [74]. Additionally, in vitro differentiation models of human naive B cells into plasma cells reveal that regulatory elements of PRDM1 gain hydroxymethylation and overall lose methylation in early plasma cells [75]. In addition to being themselves epigenetically regulated, once expressed, these proteins in turn can induce a widespread reconfiguration of the epigenome and transcriptional program of B cells through binding to their target sites, and in the case of AID by active demethylation (it will be discussed later within this review).

An overview of genome-wide DNA methylation studies in B cells The differentiation of hematopoietic cells into general cell lineages has been related to DNA methylation changes, and several studies have provided general guidelines on the importance of epigenetic processes in lineage priming and commitment [76-80]. In B cells, several largescale studies over the last few years using next generation sequencing or microarrays have analyzed the DNA methylome of particular windows of differentiation, i.e. early B cells [81-82], mature B cells [57, 83-84] and plasma cells [75, 85], and a study in 2015 described the B-cell epigenome from uncommitted progenitors to long-lived bone marrow plasma cells [86]. In contrast to the mechanistic studies, which have been performed in mouse models, most of the studies profiling the DNA methylome have been carried out using sorted human B cells. Although these reports partially used different surface markers to sort B-cell subpopulations and the DNA methylome was measured by different techniques, they allow to draw a general overview on how the DNA methylation landscape is modulated in B cells. Whole-genome bisulfite sequencing studies in B cells have revealed that approximately 30% of the entire DNA methylome is modified during B-cell differentiation, affecting several million CpG sites [84, 86]. In this context, a previous whole-genome study of multiple healthy human cell lineages and tissues revealed 22% of the DNA methylome to be susceptible to changes [87]. In spite of the different analytical approaches of these studies, they suggest that B cells may be the human cell lineage with the most extensive changes in the DNA methylome. The detailed analysis of 6

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hematopoietic cell lineages also indicates that this vast DNA methylation reconfiguration seems to be specific for B cells and, to a lesser extent also for T cells, but not in the differentiation program of neutrophils or monocytes, as recently demonstrated by a recent report including 112 samples spanning multiple hematopoietic cell lineages and maturation stages [88]. This massive reconfiguration of the DNA methylome in B cells mostly affects heterochromatin/nuclear lamina associated domains, DNA repeats and polycomb repressed regions. Embedded into these global changes, the DNA methylation status of maturation-stage specific enhancers and promoters appears to be a key element in the differentiation program, which widely confirms and extends previous analyses of specific genes. These aspects will be revised in further detail in the following sections.

Transcriptions factors shape the enhancer DNA methylation landscape of B cells DNA methylation studies on the differentiation of hematopoietic stem cells into general cell lineages have provided general guidelines on the importance of epigenetic processes in lineage priming and commitment [4]. Transcriptional programs activated upon cell lineage commitment and during differentiation are induced by binding of key TF binding to their target regulatory elements. This is tightly related to chromatin changes in promoters, and a widespread modification of the epigenetic status of enhancer elements [89-91], including modulation of the 3D structure of the genome [92]. DNA methylation profiling during B-cell differentiation has revealed a major, step-wise epigenetic configuration of regulatory elements, in part of promoters, but most prominently of enhancers [81-82, 86]. In early B-cell differentiation, the sharp increase in the expression of B cell TFs upon B-cell commitment shows a highly significant inverse correlation with the average DNA methylation level of their binding sites [86]. As enhancer elements that had not yet been activated during differentiation are generally methylated, a given TF should be in the first instance able to bind to methylated DNA regions. Although it is generally accepted that TFs cannot bind methylated DNA, a recent high-throughput study on the binding abilities of 542 TFs to methylated or unmethylated target sites indicates that some TFs actually prefer methylated DNA [93]. Once a TF has bound its target site, it can become demethylated either passively through abolishing the action of DNMTs in active regions upon DNA replication, or actively through the action of the TET family of demethylases [94]. This latter process of active demethylation has been the subject of several studies demonstrating TET enzymes are associated with a cell division-independent demethylation and activation of enhancers [95-98]. In mice, loss of Tet2 and Tet3 impairs early B-cell commitment by blocking demethylation of IgK enhancers and globally decreasing chromatin accessibility to enhancers of B cell TFs [99-100]. Furthermore, conditional knockout of Tet2 and Tet3 prevents lineage-specific demethylation in regulatory elements of genes related to the B-cell lineage, causing alterations in B-cell differentiation [101]. Out of the entire B-cell differentiation process, enhancer demethylation seems to be of particular importance in the transition from uncommitted progenitors to early B cells. In this transition, half of all the observed DNA methylation changes can be mapped to enhancers of genes involved in leukocyte activation and B-cell signaling, which in turn are binding sites of B cell-specific TFs such as EBF1, PAX5, E2F and BATF, among others [81-82, 86]. In the case of EBF1, it has been shown to be involved in DNA demethylation and nucleosome remodeling at the promoter of 7

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Cd79a, which is part of the B-cell receptor complex, and thus enhances the subsequent Cd79a activation by PAX5 [102]. Similarly, an upstream enhancer of the Cd19 gene, encoding for a Bcell receptor adaptor protein, is first remodeled by E2A and EBF1, and then by PAX5, but the Cd19 promoter is only activated after PAX5 binding [103]. A recent large-scale report has deepened into the EBF1 pioneering activity and identified that it is dependent on its C-terminal domain, which promotes chromatin accessibility and DNA demethylation in previously inactive chromatin [104]. Although not yet demonstrated in B cells, the ability of EBF1 to demethylate the DNA of regulatory elements is most likely associated with recruitment of TET2, as their interaction has been demonstrated in the context of tumor cells [105]. In later stages of B-cell maturation, DNA methylation changes in class-switched memory B cells as compared to naive B cells are also enriched in promoters and enhancers [84, 106], although overall a large fraction of the changes in these cell types are observed in non-functional DNA such as heterochromatin and polycomb-repressed regions [106]. Hypomethylated regulatory regions in class-switched memory B cells were enriched for binding sites of NF-κB, OCT2, IRF4, AP-1, EBF1 and RUNX3 [84]. As DNA methylation mostly shows an accumulative pattern during B-cell maturation [86], part of the changes observed between naive and class-switched memory B cells represent an imprint of changes taking place in the naive B cell to germinal center B cell transition. In that transition, loss of methylation in germinal center B cells overlapped with regulatory elements marked by DNAseI hypersensitive sites and was enriched in binding sites of MYC and BCL11A [83]. In plasma cells, an epigenomic study in mice has revealed that, as compared to naive B cells, demethylation in both plasmablasts and plama cells was enriched for IRF, POU homeobox domain and bZIPTF families whereas those regions hypomethylated only in plasma cells were associated with binding motifs for transcription factors of the Rel-homology domain, NFATc1 and E2A TF families [85]. However, this study did not include the analysis of germinal center B cells and in part, the changes observed in plasmablasts and plasma cells may be an epigenetic imprint of the extensive wave of demethylation in germinal center B cells.

Extensive epigenetic remodeling of B cells at the germinal center Several publications over the last few years have reported that the transition from naive to germinal-center B cells involves a massive reconfiguration of the DNA methylome, mostly in the form of hypomethylation, although hypermethylation is also present [13, 57, 83-84, 86]. A detailed epigenetic mapping of pre-germinal center and germinal center cell subpopulations from two whole DNA methylome studies (i.e. naive B cells in blood and tonsils, germinal center founder B cells, centroblasts and centrocytes) indicates that the centroblast stage in the darkzone of the germinal center is the target of extensive epigenetic remodeling and diversification [84, 86]. This statement is supported by the fact that the DNA methylome of naive B cells and germinal center founder cells is similar, but extensively differs from that of centroblasts, which in turn are epigenetically similar to more differentiated centrocytes [84]. As part of the process of maturation of antigen affinity in adaptive immune responses, centroblasts undergo clonal expansion and somatic hypermutation through AID [107]. These two physiological processes together with the downregulation of DNMT3A [83] may underlie the epigenetic lability of 8

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germinal center B cells. On the one hand proliferation may be in charge of a large fraction of the demethylation events observed in germinal center B cells, which take place in CpG-poor late-replicating regions such as heterochromatin, lamina-associated domains and DNA repeats such as Alu elements [83, 86]. It has been described that DNA methylation in late-replicating regions passively decreases in relationship with cell replication cycles [11], and therefore it may contain information on the proliferative history of the cell. In fact, this hypomethylation of heterochromatic regions is one of the hallmarks of highly proliferative cells such as cancer cells [108-109]. At the same time, germinal center B cells gain methylation in CGIs that are silenced by the H3K27me3 mark deposited by the polycomb repressive complex 2 [86]. This hypermethylation of regions already repressed by histone modifications may represent a means to reduce epigenetic plasticity, as suggested for cancer cells [110]. In normal B cells, this hypermethylation of polycomb regions may also be related to reduced plasticity, as more mature B cells are more difficult to reprogram into induced pluripotent stem cells than precursor B cells [111]. Furthermore, changes in heterochromatin and polycomb-repressed regions in germinal center B cells seem to be acquired following a stochastic process leading to epigenetic diversification. Therefore, we speculate that another function of random DNA methylation changes introduced in individual cells within the pool of germinal center B cells may lead to increased phenotypic diversity of memory and plasma cells. In addition to passive changes in heterochromatic and repressed regions, DNA demethylation in germinal center B cells can also be induced by the very same protein that induces somatic hypermutation [14]. Hypomethylation in germinal center B cells as compared to naive B cells is enriched in AID binding sites [57]. A more recent article using AID-deficient mice revealed that germinal center B cells lack most of the DNA methylation changes observed in wild-type animals, and that AID-dependent hypomethylated sites are enriched at somatic hypermutation hotspots [13]. The germinal center B cells from AID-negative mice are also highly proliferative, which argues against a role of proliferation in inducing passive demethylation in this cell type. This lack of association between proliferation and epigenetic changes in germinal center B cells of AID-negative mice may in part be related to differences between mice and humans, the different methods used to measure methylation and bioinformatic approaches to detect methylation changes, as well as potential indirect effects of AID silencing. In this regard, AIDnegative mice unexpectedly also show a decrease in hypermethylated sites in germinal center B cells, suggesting that AID may also affect global DNA methylation landscape through indirect mechanisms. Furthermore, the off-target effects outside immunoglobulin genes of AID have been described to target within super-enhancers and regulatory clusters [112], which do not overlap with heterochromatic regions. Although the precise mechanisms leading to extensive modulation of the DNA methylome in the germinal center are still a matter of controversy, high proliferation and AID activity are likely to cooperate in this process. Interestingly, as it will be discussed in the next section, long-lived memory B cells and plasma cells, which lack AID expression and are not proliferative, further alter their DNA methylome in (partially) the same sites that start acquiring changes in germinal center B cells, suggesting additional causes for DNA methylation alterations.

Accumulation of epigenetic changes in memory B cells and plasma cells 9

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The final output of the germinal center reaction is the creation of plasma cells, which produce vast amounts of high-affinity antibodies and memory B cells, which can rapidly differentiate into antibody secreting plasma cells upon a second antigen exposure. From the DNA methylation and transcriptional perspective, the transition from germinal center B cells to memory and plasma cells is an intriguing phenomenon. Germinal center, memory and plasma cells from tonsils have a similar DNA methylome despite having vastly differing transcriptional programs [83, 86], which highlights that the widely reported correlation between DNA methylation, although valid for some genes, cannot be considered a general rule in the gene regulation field. A deeper understanding of gene regulation requires the analysis of chromatin marks, as reported by studies integrating the analysis multiple epigenetic marks and gene expression in the same cell types [16]. Early plasma cell commitment has been epigenetically studied in further detail and identified that it is related to cell-cycle dependent demethylation of specific regulatory sites. This phenomenon was initially described using an in vitro differentiation of human naive B cells into plasma cells [75] and then also observed sorted B cells and plasma cells from mice [85]. An important aspect in the life of memory and plasma cells is that, although they are born in secondary lymphoid organs, they circulate through peripheral blood (memory B cells) or migrate to the bone marrow (plasma cells), where they can live for extended periods of time. The DNA methylation analysis of class-switched memory B cells from peripheral blood and bone marrow plasma cells has revealed that these two cell types show extensive epigenetic changes in genomic elements apparently not related to gene regulation [84, 86], which is more pronounced in bone marrow plasma cells [86]. This difference is very clear when comparing tonsillar and bone marrow plasma cells, which are transcriptionally similar but show widely different DNA methylomes [86]. The epigenetic changes in long-lived memory and plasma cells are mostly found in late-replicating heterochromatic regions (hypomethylation) and polycombrepressed regions (hypermethylation). Although the cause of this epigenetic profile is not yet well understood, we would like to discuss them from different perspectives that may be useful to design novel experiments. First, it has been described that the pool of germinal center B cells is epigenetically heterogeneous [13] and similar to newly generated memory and plasma cells [83]. Therefore, we may speculate that only a fraction of cells from the initial pool of heterogeneous memory and plasma cells are selected (e.g. those with more epigenetic changes) to give rise to long-lived cells. Second, the epigenetic changes observed in memory and plasma cells may be related to a reduced plasticity. These cell types are physiologically programmed to live for extended periods of time without changing and therefore, their epigenetic signature, which is diametrically different from that of stem cells (i.e. highly methylated heterochromatin and unmethylated polycomb-repressed regions), may be related with this lack of plasticity. In this context, recent studies in T-cell differentiation have reported the presence of a similar effect. Long-lived memory T cells show extensive DNA methylation changes as compared to naive T cells [88, 113-114], in part overlapping with those observed in B cells (own unpublished studies from the authors, data not shown). In contrast, the differentiation program of myeloid cells, which are short-lived, does not show this massive epigenetic modulation [88]. Finally, there is a large body of published evidence indicating that the epigenetic landscape of long-lived memory and plasma cells has also been observed in cancer cells [108-109, 115-117], chronological age [118-122] and senescence [123], although 10

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the later has been recently described to be related to cellular age rather than senescence itself [124]. Thus, DNA methylation can be seen as an epigenetic mark related to both cellular and organismal age [124-125]. The observation of this signature of long-lived memory and plasma cells may reflect their cellular longevity, as highlighted by the fact that young cells exiting the germinal center reaction show clearly less epigenetic changes than those that have lived longer in blood and bone marrow [83, 86]. The association among the acquisition of epigenetic changes in normal cells, epigenetic clocks for both organismal and cellular aging, and the epigenetic changes observed in cancer is becoming clear [126], and there is need to clarify the mechanisms and implications of these DNA methylation changes.

Linking B-cell epigenetics with the biological features and clinical behavior of lymphoid neoplasms The field of cancer epigenetics has traditionally focused on the comparison between tumor and lineage-matched normal cells. In the case of B-cell neoplams, most published studies used the easily accessible B cells from peripheral blood as negative control. However, considering the broad reconfiguration of the DNA methylome in normal B cells and the fact that different B-cell neoplasms are originated from particular maturation stages, it is essential to use maturation-stage specific B cells as negative controls for specific B-cell neoplams entities. In some instances, such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), inter-patient epigenetic heterogeneity has been linked to similarities to specific B-cell maturation states [84, 106, 127]. CLLs can be categorized into three different entities with different clinical behavior based on epigenetic imprints of mature B cells at different developmental states; those CLLs associated with a signature of less mature B cells have a worse prognosis than those with an intermediate signature, which in turn are clinically worse than those related to a pattern of more mature B cells [84, 106, 128-129]. These three groups can be detected using high-throughput approaches of few epigenetic biomarkers [84, 106, 128-129], paving the way to clinical applications using various technologies [130]. Furthermore, although overall patients can be generally categorized into three groups, a recent report has observed that leukemic cells from different patients seem to be derived from a continuum of B-cell maturation states [84]. In the case of MCL, two subtypes of MCLs can be distinguished based on whether their cell of origin has experienced or not the germinal center reaction [127]. These two subtypes have a distinct epigenetic signature of their cellular origin and also show a different clinical behavior, being the germinal center-independent MCLs of worse prognosis than those originated from germinal center-experienced B cells. As shown above using CLL and MCL as examples, B-cell tumors seem maintain an imprint of the cell from which they originate, and this knowledge is essential to use the appropriate normal counterpart in the detection of tumor-specific DNA methylation changes occurring during development of the neoplastic B-cell clone. However, this comparison may not be as simple as it may seem. If a particular B-cell tumor subtype is compared versus its matched maturation stage, a large proportion of these changes are actually shared with the normal B-cell maturation process. For instance, if acute lymphoblastic leukemias (ALLs) are compared with their cellular origin (i.e precursor B cells), two thirds of the CpG sites that change in the ALLs are shared with the process of normal B-cell differentiation, especially with changes 11

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introduced in late maturation stages such as long-lived memory and plasma cells[86]. This intriguing finding suggests that part of the DNA methylation changes observed in tumors may be the passive result of e.g. proliferation history and cellular longevity, which is shared both by tumors and long-lived memory/plasma cells. Thus, to identify tumor-specific changes with a functional impact on tumor transformation, it may be relevant to separate those changes shared with normal B-cell differentiation from those that occur only in the context of neoplastic transformation. This strategy was used in a recent article in MCL linking DNA methylation changes to histone modifications associated with active chromatin. The authors identified that, as compared with the fraction shared with normal B cells, the fraction of the changes independent from B-cell differentiation was enriched in simultaneous changes in the activity of promoters and enhancers [127]. This finding underlines the importance of studying the DNA methylome of tumors in the context of the complete maturation program. Furthermore, it suggests that epigenetic changes with a functional oncogenic impact in tumors are more likely to be found in the fraction of the DNA methylome that changes independently from the physiological process of B-cell differentiation.

Future directions The role of epigenetics during B-cell development has revealed new insights into the molecular determinants of specific B-cell maturation states as well as unexpected links to cancer and aging. However, the analysis of the epigenome of the entire B-cell development program is far from being complete. DNA methylation is just one layer of the epigenetic portfolio. In the context of the Blueprint Epigenome Project under the umbrella of the International Cancer Epigenome Consortium [22], a broad range of reference epigenomes of cells from the hematopoietic system have been generated, including various B-cell maturation stages. Such reference epigenomes comprise multiple layers such as DNA methylation, different histone modifications, chromatin accessibility and gene expression. This integrative epigenetic datasets of B-cell maturation represents a rich resource for the scientific community and shall lead to multiple novel discoveries in the near future. This approach, however, is limited by the fact that it is analyzing a limited set of differentiation states that most likely show intrasubpopulation heterogeneity, as they are defined only by the expression of a restricted set of surface markers. In the recent years, we have witnessed the development of epigenetic technologies allowing the analysis of small numbers of cells [89, 131] and even of genomewide single cell transcriptomes [132-133], DNA methylomes [134-136], histone modification maps [137], chromatin accessibility maps [138-139] and 3D genomic architecture [140]. Furthermore, technologies to study more than one information layer from the same single cells are being developed [141]. At the time of writing this review, we are not aware of any publication using these single cell technologies in the context of B-cell differentiation. However, these techniques are already helping to redefine the hematopoietic tree based on single cell transcriptomics [142]. The application of these technologies to single B cells from different compartments such as bone marrow, peripheral blood and lymphoid organs shall reveal an unprecedented view of B-cell differentiation.

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Acknowledgements The author’s own studies on epigenomics are funded by the Worldwide Cancer Research (grant agreement 16-1285, to J.I.M.-S.), European Hematology Association (Non-Clinical Advanced Research Grant to J.I.M.-S.), Leukemia Research Foundation (Research Grant to C.C.O), and Gabrielle's Angel Foundation for Cancer Research (Research Grant to C.C.O.)

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Invited review Seminars in Cancer Biology Special issue on "Epigenetics on hematological malignancies"

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Invited review Seminars in Cancer Biology Special issue on "Epigenetics on hematological malignancies"

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Invited review Seminars in Cancer Biology Special issue on "Epigenetics on hematological malignancies"

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Invited review Seminars in Cancer Biology Special issue on "Epigenetics on hematological malignancies"

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Invited review Seminars in Cancer Biology Special issue on "Epigenetics on hematological malignancies"

A. Datta, P. Flicek, R. Royo, J. Martens, E. Gine, A. Lopez-Guillermo, H.G. Stunnenberg, W. Klapper, C. Pott, S. Heath, I.G. Gut, R. Siebert, E. Campo, J.I. Martin-Subero, Decoding the DNA Methylome of Mantle Cell Lymphoma in the Light of the Entire B Cell Lineage, Cancer Cell 30(5) (2016) 806-821. [128] S. Bhoi, V. Ljungstrom, P. Baliakas, M. Mattsson, K.E. Smedby, G. Juliusson, R. Rosenquist, L. Mansouri, Prognostic impact of epigenetic classification in chronic lymphocytic leukemia: The case of subset #2, Epigenetics 11(6) (2016) 449-55. [129] A.C. Queiros, N. Villamor, G. Clot, A. Martinez-Trillos, M. Kulis, A. Navarro, E.M. Penas, S. Jayne, A. Majid, J. Richter, A.K. Bergmann, J. Kolarova, C. Royo, N. Russinol, G. Castellano, M. Pinyol, S. Bea, I. Salaverria, M. Lopez-Guerra, D. Colomer, M. Aymerich, M. Rozman, J. Delgado, E. Gine, M. Gonzalez-Diaz, X.S. Puente, R. Siebert, M.J. Dyer, C. Lopez-Otin, C. Rozman, E. Campo, A. Lopez-Guillermo, J.I. Martin-Subero, A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact, Leukemia 29(3) (2015) 598-605. [130] Blueprint Consortium, Quantitative comparison of DNA methylation assays for biomarker development and clinical applications, Nat Biotechnol 34(7) (2016) 726-37. [131] C. Schmidl, A.F. Rendeiro, N.C. Sheffield, C. Bock, ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors, Nat Methods 12(10) (2015) 963-5. [132] D.A. Jaitin, E. Kenigsberg, H. Keren-Shaul, N. Elefant, F. Paul, I. Zaretsky, A. Mildner, N. Cohen, S. Jung, A. Tanay, I. Amit, Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types, Science 343(6172) (2014) 776-9. [133] D. Ramskold, S. Luo, Y.C. Wang, R. Li, Q. Deng, O.R. Faridani, G.A. Daniels, I. Khrebtukova, J.F. Loring, L.C. Laurent, G.P. Schroth, R. Sandberg, Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells, Nat Biotechnol 30(8) (2012) 777-82. [134] M. Farlik, N.C. Sheffield, A. Nuzzo, P. Datlinger, A. Schonegger, J. Klughammer, C. Bock, Single-cell DNA methylome sequencing and bioinformatic inference of epigenomic cell-state dynamics, Cell Rep 10(8) (2015) 1386-97. [135] H. Guo, P. Zhu, X. Wu, X. Li, L. Wen, F. Tang, Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing, Genome Res 23(12) (2013) 2126-35. [136] S.A. Smallwood, H.J. Lee, C. Angermueller, F. Krueger, H. Saadeh, J. Peat, S.R. Andrews, O. Stegle, W. Reik, G. Kelsey, Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity, Nat Methods 11(8) (2014) 817-20. [137] A. Rotem, O. Ram, N. Shoresh, R.A. Sperling, A. Goren, D.A. Weitz, B.E. Bernstein, Singlecell ChIP-seq reveals cell subpopulations defined by chromatin state, Nat Biotechnol 33(11) (2015) 1165-72. [138] J.D. Buenrostro, B. Wu, U.M. Litzenburger, D. Ruff, M.L. Gonzales, M.P. Snyder, H.Y. Chang, W.J. Greenleaf, Single-cell chromatin accessibility reveals principles of regulatory variation, Nature 523(7561) (2015) 486-90. [139] D.A. Cusanovich, R. Daza, A. Adey, H.A. Pliner, L. Christiansen, K.L. Gunderson, F.J. Steemers, C. Trapnell, J. Shendure, Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing, Science 348(6237) (2015) 910-4. [140] T. Nagano, Y. Lubling, T.J. Stevens, S. Schoenfelder, E. Yaffe, W. Dean, E.D. Laue, A. Tanay, P. Fraser, Single-cell Hi-C reveals cell-to-cell variability in chromosome structure, Nature 502(7469) (2013) 59-64. [141] I.C. Macaulay, C.P. Ponting, T. Voet, Single-Cell Multiomics: Multiple Measurements from Single Cells, Trends Genet 33(2) (2017) 155-168. [142] F.E. Mercier, D.T. Scadden, Not All Created Equal: Lineage Hard-Wiring in the Production of Blood, Cell 163(7) (2015) 1568-70.

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Invited review Seminars in Cancer Biology Special issue on "Epigenetics on hematological malignancies"

Figure 1. Known components of epigenetic regulation related to B cell differentiation. These include epigenetic modifiers whose disruption alters B cell differentiation, key B cell transcription factors or proteins modulating the B cell epigenome, and regulatory/non-regulatory chromatin states undergoing differential DNA methylation in B cell differentiation.

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Invited review Seminars in Cancer Biology Special issue on "Epigenetics on hematological malignancies"

Figure 2. Survey of human B cell subpopulations whose DNA methylation profile has been studied, including the source of the cells and the technique used to analyze them. GC: Germinal center, MIRA: methylated-CpG island recovery assay, HELP: HpaII tiny fragment enrichment by ligation-mediated PCR, WGBS: Whole-genome bisulfite sequencing, TWGBS: Tagmentation-based WGBS.

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Invited review Seminars in Cancer Biology Special issue on "Epigenetics on hematological malignancies"

Figure 3. Integrative scheme of B cell differentiation including key B cell transcription factors, microenvironmental shifts and global DNA methylation dynamics in different chromatin states. * Expression of these transcription factors is plasma cell specific (not related to long-lived memory B cells).

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