Cancer progression involves genetic and epigenetic changes that disrupt chromatin 3D organization, affecting enhancer-promoter interactions and promoting growth. Here, we provide an integrative approach, combining chromatin conformation, accessibility, and transcription analysis, validated by in silico and CRISPR-interference screens, to identify relevant 3D topologies in pediatric T cell leukemia (T-ALL and ETP-ALL). We characterize 3D hubs as regulatory centers for oncogenes and disease markers, linking them to biological processes like cell division, inflammation, and stress response. Single-cell mapping reveals heterogeneous gene activation in discrete epigenetic clones, aiding in patient stratification for relapse risk after chemotherapy. Finally, we identify MYB as a 3D hub regulator in leukemia cells and show that the targeting of key regulators leads to hub dissolution, thereby providing a novel and effective anti-leukemic strategy. Overall, our work demonstrates the relevance of studying oncogenic 3D hubs to better understand cancer biology and tumor heterogeneity and to propose novel therapeutic strategies.

Two distinct lineages, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common inner cell mass (ICM) progenitors in mammalian embryos. To study how these sister identities are forged, we leveraged mouse embryonic stem (ES) cells and extra-embryonic endoderm (XEN) stem cells—in vitro counterparts of the EPI and PrE. Bidirectional reprogramming between ES and XEN coupled with single-cell RNA and ATAC-seq analyses showed distinct rates, efficiencies, and trajectories of state conversions, identifying drivers and roadblocks of reciprocal conversions. While GATA4-mediated ES-to-iXEN conversion was rapid and nearly deterministic, OCT4-, KLF4-, and SOX2-induced XEN-to-induced pluripotent stem (iPS) reprogramming progressed with diminished efficiency and kinetics. A dominant PrE transcriptional program, safeguarded by GATA4, alongside elevated chromatin accessibility and reduced DNA methylation of the EPI underscored the differential plasticities of the two states. Mapping in vitro to embryo trajectories tracked reprogramming cells in either direction along EPI and PrE in vivo states, without transitioning through the ICM.

Transposable elements (TEs) are abundant in the human genome, and they provide the source for genetic and functional diversity. Previous studies have suggested that TEs are repressed by DNA methylation and chromatin modifications. Here through integrating transcriptome and 3D genome architecture studies, we showed that haploinsufficient loss of NIPBL selectively activates alternative promoters (altPs) at the long terminal repeats (LTRs) of the TE subclasses. This activation occurs through the reorganization of topologically associating domain (TAD) hierarchical structures and the recruitment of proximal enhancers. These observations indicate that TAD hierarchy restricts transcriptional activation of LTRs that already possess open chromatin features. Perturbation of hierarchical chromatin topology can lead to co-option of LTRs as functional altPs, driving aberrant transcriptional activation of oncogenes. These data uncovered a new layer of regulatory mechanisms of TE expression and posit TAD hierarchy dysregulation as a new mechanism for altP-mediated oncogene activation and transcriptional diversity in cancer.

Mouse embryonic stem cells (mESCs), in addition to differentiating into the three germ layers, can reverse typical developmental trajectories, as exemplified by their ability to de-differentiate into 2-cell-like cells (2CLCs) that resemble the mammalian embryo during zygotic genome activation (ZGA). This unique property offers the opportunity to elucidate the molecular principles that govern the pre-implantation stages of mammalian development. Here, we dissect the functions of the chromatin repressor EHMT2, a candidate antagonist of the mESC-to-2CLC transition, by leveraging a multipurpose allele for acute protein depletion and efficient immunoprecipitation. Our experiments revealed distinct principles of EHMT2-mediated gene repression in mESCs based on specific chromatin binding patterns and protein co-factors. Most notably, EHMT2 directly represses large clusters of co-regulated gene loci that comprise a significant fraction of the 2CLC-specific transcriptome by initiating H3K9me2 spreading from distal LINE-1 elements. EHMT2 counteracts the recruitment of the activator DPPA2/4 to promoter-proximal endogenous retroviral elements (ERVs) at 2CLC genes. EHMT2 depletion enhances the expression of ZGA-associated transcripts in 2CLCs and synergizes with spliceosome inhibition and retinoic acid signaling to facilitate the mESC-to-2CLC transition. In contrast to ZGA-associated genes, the repression of germ layer-associated transcripts by EHMT2 occurs outside of gene clusters, in collaboration with ZFP462, and involves binding to non-repetitive candidate enhancers. Our observations provide novel mechanistic insight into how pluripotent cells achieve attenuation of their bidirectional differentiation potential and reveal unique transcriptional features of murine totipotent cells.

The molecular mechanisms that drive essential patterning events in the mammalian embryo remain poorly understood. Analysis of transcription factor expression kinetics at peri-gastrulation stages of development suggest Otx2 as a candidate regulator of the definitive endoderm, the precursor of all gut-derived organs. Accordingly, timed OTX2 depletion in gastruloids or during directed differentiation results in abnormal definitive endoderm specification in mouse and human, characterized by altered expression of components and transcriptional targets of the canonical WNT signaling pathway, perturbed adhesion and migration programs, and de-repression of regulators of other lineages. These defects cumulate in impaired foregut formation. Mechanistically, OTX2 is required to activate a subset of endoderm-specific enhancers and to suppress select enhancers of other lineages, allowing timely exit from the primitive streak and correct specification of anterior endoderm. Our results establish OTX2 as an early gut regulator and suggest molecular principles underlying spatiotemporal cell identity conserved across germ layers and species.

Dysregulation of enhancer-promoter communication in the three-dimensional (3D) nucleus is increasingly recognized as a potential driver of oncogenic programs. Here, we profiled the 3D enhancer-promoter networks of patient-derived glioblastoma stem cells to identify central regulatory nodes. We focused on hyperconnected 3D hubs and demonstrated that hub-interacting genes exhibit high and coordinated expression at the single-cell level and are associated with oncogenic programs that distinguish glioblastoma from low-grade glioma. Epigenetic silencing of a recurrent hub-with an uncharacterized role in glioblastoma-was sufficient to cause downregulation of hub-connected genes, shifts in transcriptional states, and reduced clonogenicity. Integration of datasets across 16 cancers identified "universal" and cancer-type-specific 3D hubs that enrich for oncogenic programs and factors associated with worse prognosis. Genetic alterations could explain only a small fraction of hub hyperconnectivity and increased activity. Overall, our study provides strong support for the potential central role of 3D regulatory hubs in controlling oncogenic programs and properties.

Naive pluripotent stem cells (nPSCs) frequently undergo pathological loss of DNA methylation at imprinted gene loci, posing a hurdle for biomedical applications and underscoring the need to identify underlying causes. We show that nPSCs from inbred mouse strains exhibit strain-specific susceptibility to locus-specific deregulation of imprinting marks during reprogramming and upon exposure to a mitogen-activated protein kinase (MAPK) inhibitor, a common approach to maintain naive pluripotency. Analysis of genetically diverse nPSCs from the Diversity Outbred (DO) stock confirms the impact of genetic variation on epigenome stability, which we leverage to identify trans-acting quantitative trait loci (QTLs) that modulate DNA methylation levels at specific targets or genome-wide. Analysis of multi-target QTLs on chromosomes 4 and 17 suggests candidate transcriptional regulators contributing to DNA methylation maintenance in nPSCs. We propose that genetic variants represent biomarkers to identify pluripotent cell lines with desirable properties and may allow the targeted engineering of nPSCs with stable epigenomes.

Cellular reprogramming converts differentiated cells into a pluripotent state through extensive chromatin remodeling. The histone variant macroH2A has classically viewed as an epigenetic barrier stabilizing somatic identity and restricting pluripotency gene activation. Here, we show that during reprogramming, macroH2A1 nucleosomes undergo functional repurposing. Early in the process, mH2A1 nucleosomes act as a barrier to cellular plasticity, but later facilitate the establishment and maintenance of pluripotency by reshaping the epigenetic landscape. High-resolution chromatin profiling reveals that mH2A1.2 nucleosomes undergo rapid, large-scale repositioning, dissociating from promoters and reassembling ~30 bp away, frequently near NRF-1 binding sites. This repositioning occludes E2F4 binding, relieving cell-cycle arrest thereby enabling reprogramming. Likewise, mH2A1.1 nucleosomes display extensive mobility that culminates in deposition at pluripotency genes in ESCs, where they sustain their expression by assembling promoter transcriptional hubs. In this later role, mH2A1.1 functions as a chromatin bookmark stabilizing the Nanog, Sox2, and Oct4 network. These findings suggest that mH2A1 nucleosomal mobility underlines its context-dependent functional repurposing from reprogramming inhibitor to facilitator, illustrating how chromatin components evolve dynamic roles to coordinate cell-state transitions.

The molecular mechanisms that drive essential developmental patterning events in the mammalian embryo remain poorly understood. To generate a conceptual framework for gene regulatory processes during germ layer specification, we analyzed transcription factor (TF) expression kinetics around gastrulation and during in vitro differentiation. This approach identified Otx2 as a candidate regulator of definitive endoderm (DE), the precursor of all gut-derived tissues. Analysis of multipurpose degron alleles in gastruloid and directed differentiation models revealed that loss of OTX2 before or after DE specification alters the expression of core components and targets of specific cellular signaling pathways, perturbs adhesion and migration programs as well as de-represses regulators of other lineages, resulting in impaired foregut specification. Key targets of OTX2 are conserved in human DE. Mechanistically, OTX2 is required to establish chromatin accessibility at candidate enhancers, which regulate genes critical to establishing an anterior cell identity in the developing gut. Our results provide a working model for the progressive establishment of spatiotemporal cell identity by developmental TFs across germ layers and species, which may facilitate the generation of gut cell types for regenerative medicine applications.

Transposable elements (TEs) are abundant in the human genome, and they provide the sources for genetic and functional diversity. The regulation of TEs expression and their functional consequences in physiological conditions and cancer development remain to be fully elucidated. Previous studies suggested TEs are repressed by DNA methylation and chromatin modifications. The effect of 3D chromatin topology on TE regulation remains elusive. Here, by integrating transcriptome and 3D genome architecture studies, we showed that haploinsufficient loss of NIPBL selectively activates alternative promoters at the long terminal repeats (LTRs) of the TE subclasses. This activation occurs through the reorganization of topologically associating domain (TAD) hierarchical structures and recruitment of proximal enhancers. These observations indicate that TAD hierarchy restricts transcriptional activation of LTRs that already possess open chromatin features. In cancer, perturbation of the hierarchical chromatin topology can lead to co-option of LTRs as functional alternative promoters in a context-dependent manner and drive aberrant transcriptional activation of novel oncogenes and other divergent transcripts. These data uncovered a new layer of regulatory mechanism of TE expression beyond DNA and chromatin modification in human genome. They also posit the TAD hierarchy dysregulation as a novel mechanism for alternative promoter-mediated oncogene activation and transcriptional diversity in cancer, which may be exploited therapeutically.

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages: the trophectoderm, the epiblast and the primitive endoderm. Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements through which transcriptional regulators enact these fates remain understudied. Here, we characterize, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observe extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although distinct groups of genes are irresponsive to topological changes. In each lineage, a high degree of connectivity, or ‘hubness’, positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a predictive model for transcriptional regulation (3D-HiChAT) that outperforms models using only 1D promoter or proximal variables to predict levels and cell-type specificity of gene expression. Using 3D-HiChAT, we identify, in silico, candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments, we validate several enhancers that control gene expression in their respective lineages. Our study identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to comprehensively understand lineage-specific transcriptional behaviors.

Phenotypic plasticity diminishes as epigenetic barriers steer cellular differentiation, yet Germinal Center (GC) B cells exhibit transient properties highly reminiscent of stem cells and highly plastic populations. While most B lymphoid malignancies arise from mature GC B cells, it's remains unclear whether this GC plasticity could be hijacked and influence lymphoma progression upon specific mutations.To understand how differentiated GC B cells regain stem-like plasticity features, we isolated and reprogrammed murine GC B cells to measure their ability to acquire an induced pluripotent stem cell (iPSC) state and used single-cell multiomic analyses and small molecule inhibitors to probe underlying mechanisms.Contrary to other mature B cells, GC B cells showed an exceptionally high reprogramming efficiency to iPSCs, strictly dependent on T Follicular Helper (TFH) cells. TFH-activated GC B cells displayed upregulated pluripotent signatures along with increased chromatin accessibility on stem cell genes/super-enhancers and dampening of B-cell programs. Disrupting TFH help reception in GC B cells using the Ezh2Y641F lymphoma-associated mutation, decreased iPSC formation on the contrary to the Btg1Q36H mutation which increases TFH help, hence pluripotency acquisition. Moreover, lymphoma mutations in Histone 1 subunits, increased reprogramming efficiency for all GC B cell populations, bypassing the need for TFH help. Finally, high expression of GC stemness signatures in B-cell lymphoma patients correlated with poorer outcomes, suggesting an association with lymphoma fitness.In summary, mature GC B cells physiologically reacquire a non cell-autonomous stem-like plasticity. Confining plasticity of TFH-selected B cells could potentially restrain lymphoma development and relapse in specific contexts.

Cellular reprogramming of somatic cells towards induced pluripotency is a multistep stochastic process mediated by the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), which orchestrate global epigenetic and transcriptional changes. We performed a large-scale analysis of integrated ChIP-seq, ATAC-seq and RNA-seq data and revealed the spatiotemporal highly dynamic pattern of OSKM DNA binding during reprogramming. We found that OSKM show distinct temporal patterns of binding to different classes of pluripotency-related enhancers. Genes involved in reprogramming are regulated by the coordinated activity of multiple enhancers, which are sequentially bound by OSKM for strict transcriptional control. Based on these findings, we developed an unbiased approach to identify Reprogramming-Inducible Enhancers (RIEs), constructed enhancer-traps and isolated cells undergoing reprogramming in real time. We used a representative RIE taken from the Upp1 gene fused to Gfp and isolated cells at different time-points during reprogramming and found that they have unique developmental capacities as they are reprogrammed with high efficiency due to their distinct molecular signatures. In conclusion, our experiments have led to the development of an unbiased method to identify and isolate reprogrammable cells in real time by exploiting the functional dynamics of OSKM, which can be used as efficient reprogramming biomarkers.

ABSTRACT Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages, the trophectoderm (TE), the epiblast (EPI) and the primitive endoderm (PrE). Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements via which transcriptional regulators enact these fates remain understudied. To address this gap, we have characterized, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observed extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although there are distinct groups of genes that are irresponsive to topological changes. In each lineage, a high degree of connectivity or “hubness” positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages, compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a novel predictive model for transcriptional regulation (3D-HiChAT), which outperformed models that use only 1D promoter or proximal variables in predicting levels and cell-type specificity of gene expression. Using 3D-HiChAT, we performed genome-wide in silico perturbations to nominate candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments we validated several novel enhancers that control expression of one or more genes in their respective lineages. Our study comprehensively identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to understand lineage-specific transcriptional behaviors. HIGHLIGHTS - Cell lines representing early embryonic lineages undergo drastic enhancer remodeling and fine-scale 3D chromatin reorganization - Highly interacting 3D hubs strongly enrich for highly expressed, cell-type specific and essential genes - 3D chromatin features greatly improve prediction of cell-type specific gene expression compared to 1D promoter features - In silico and experimental perturbations identify novel enhancers regulating the expression of two or more genes in early embryonic lineages

Chemo-immunotherapy can prolong life expectancy of patients with Diffuse Large B-cell Lymphomas (DLBCL). However, the emergence of donor-derived lymphoma cases after stem cell transplantation and the relapse of a third of DLBCL patients, strongly suggests the existence of persisting lymphoma repopulating cells (LRC) with stem-like functionality. This apparently controversial concept arises from the fact that DLBCLs originate from mature rather than progenitor cells. Identifying the biological and epigenetic features of these elusive LRC, and how they arise from normal germinal center (GC) B cells, is therefore critical and remains underexplored. Although fully mature, GC B cells naturally manifest remarkable phenotypic plasticity as compared to other cell types. We hypothesized that this plasticity reflects stem-like programming and functionality. Along these lines, we found that GC B cells are uniquely enriched for pluripotent stem cell transcriptional signatures, and increased chromatin accessibility at stem cell enhancers. We reasoned that the capacity to undergo induced Pluripotent Stem Cells (iPSC) reprogramming could serve as a surrogate assay for stem-like plasticity. Therefore, we investigated the potential of GC B cells to form iPSCs using a doxycycline-dependent mouse strain that enables the inducible expression of Yamanaka transcription factors (TF) in any given cell. Strikingly, GC B cells isolated from our mice manifested a 10-fold (p<0.0001) increase in iPSC reprogramming as compared to other mature B cells. GC B cells are a heterogenous mix of functionally distinct subpopulations. Using single cell RNA-seq and iPSC assays in sorted cells, we noted that the rare population (~3%) of GC B cells selected by T-cell help manifested the stem-like transcriptional profile and iPSC reprogramming phenotype. Moreover, blocking T-cell help during the GC reaction in vivo using CD40 blockade abrogated iPSC forming capacity. Lymphoma-associated EZH2 Y641F mutations impair GC B-cell interactions with T FH cells due to aberrant repression of immune synapse genes. Accordingly, EZH2 Y641F GC B cells manifested loss of the iPSC phenotype. T-cell help induces MYC in GC B cells. However, induction of MYC in mature naïve B cells did not confer them with iPSC potential indicating that other mechanisms must be at play. Lymphoma-associated Btg1 Q36Hmutations confer fitness in GC B cells by enhancing their response to T-cell help. Reciprocal to EZH2 Y641F, Btg1 Q36Hmutant GC B cells manifested further significant increase in iPSC formation. Performing multi-ome (simultaneous single-cell RNAseq and ATACseq) studies in GC B cells revealed massive gain in chromatin accessibility of bona fide stem cell super-enhancers in GC B cells that received T-cell help. There was also a reduced accessibility at genes that maintain B-cell lineage (e.g. Pax5) and GC phenotypes (e.g. Foxo1). Finally, lymphoma-associated Histone 1 mutations induce upregulation of stem cell programs in GCs and could enhance iPSC reprogramming in fibroblasts. We therefore hypothesized that H1 deficiency would overcome the GC dependency on T-cells for their plasticity phenotype. Indeed, increased iPSC formation occurred across all GC subpopulations in H1 C/E-/- (HIST1C/HIST1E knockout) mice. We speculate that restricting plasticity to B cells under selection by T cells, limits the potential for GC B cells to acquire high levels of plasticity, hence reducing the potential for these cells to initiate lymphomas. Consistent with this notion, we found that DLBCL patients enriched for these stem cell signatures manifested inferior clinical outcomes.

The introduction of chromosome conformation capture (3C)-based technologies coupled with next-generation sequencing have significantly advanced our understanding of how the genetic material is organized within the eukaryotic nucleus. Three-dimensional (3D) genomic organization occurs at hierarchical levels, ranging from chromosome territories and subnuclear compartments to smaller self-associated domains and fine-scale chromatin interactions. The latter can be further categorized into different subtypes, such as structural or regulatory, based either on their presumed functionality and/or the factors that mediate their formation. Various enrichment strategies coupled with 3C-based technologies have been developed to prospectively isolate and quantify chromatin interactions around regions occupied by specific proteins or marks of interest. These approaches not only enable high-resolution characterization of the selected chromatin contacts at a cost-effective manner, but also offer important biological insights into their organizational principles and regulatory function. In this chapter, we will focus on the recently developed HiChIP technology with an emphasis on the discovery of putative active enhancers and promoter interactions in cell types of interest. We will describe the specific steps for designing, performing and analyzing successful HiChIP experiments as well as important limitations and considerations.

Introduction: The pathogenesis of thymic epithelial tumors remains largely unknown. We previously identified GTF2I L424H as the most frequently recurrent mutation in thymic epithelial tumors. Nevertheless, the precise role of this mutation in tumorigenesis of thymic epithelial cells is unclear.Methods: To investigate the role of GTF2I L424H mutation in thymic epithelial cells in vivo, we generated and charac-terized a mouse model in which the Gtf2i L424H mutation was conditionally knocked-in in the Foxn1 thorn thymic epithelial cells. Digital spatial profiling was performed on thymomas and normal thymic tissues with GeoMx-mouse whole transcriptome atlas. Immunohistochemistry staining was performed using both mouse tissues and human thymic epithelial tumors.Results: We observed that the Gtf2i mutation impairs development of the thymic medulla and maturation of medullary thymic epithelial cells in young mice and causes tumor formation in the thymus of aged mice. Cell cycle -related pathways, such as E2F targets and MYC targets, are enriched in the tumor epithelial cells. Results of gene set variation assay analysis revealed that gene signatures of cortical thymic epithelial cells and thymic epithelial pro-genitor cells are also enriched in the thymomas of the knock-in mice, which mirrors the human counterparts in The Cancer Genome Atlas database. Immunohistochemistry results revealed similar expression pattern of epithelial cell markers between mouse and human thymomas.Conclusions: We have developed and characterized a novel thymoma mouse model. This study improves knowledge of the molecular drivers in thymic epithelial cells and provides a tool for further study of the biology of thymic epithelial tumors and for development of novel therapies.(c) 2022 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

An amendment to this paper has been published and can be accessed via the original article.

During self-renewal, cell-type-defining features are drastically perturbed in mitosis and must be faithfully re-established upon G1 entry, a process that remains largely elusive. Here, we characterized at a genome-wide scale the dynamic transcriptional and architectural resetting of mouse pluripotent stem cells (PSCs) upon mitotic exit. We captured distinct waves of transcriptional reactivation with rapid induction of stem cell genes and transient activation of lineage-specific genes. Topological reorganization at different hierarchical levels also occurred in an asynchronous manner and showed partial coordination with transcriptional resetting. Globally, rapid transcriptional and architectural resetting associated with mitotic retention of H3K27 acetylation, supporting a bookmarking function. Indeed, mitotic depletion of H3K27ac impaired the early reactivation of bookmarked, stem-cell-associated genes. However, 3D chromatin reorganization remained largely unaffected, suggesting that these processes are driven by distinct forces upon mitotic exit. This study uncovers principles and mediators of PSC molecular resetting during self-renewal.

Loss of imprinting (LOI) results in severe developmental defects, but the mechanisms preventing LOI remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) in pluripotent stem cells. We demonstrate that the IG-DMR consists of two antagonistic elements: a paternally methylated CpG island that prevents recruitment of TET dioxygenases and a maternally unmethylated non-canonical enhancer that ensures expression of the Gtl2 lncRNA by counteracting de novo DNA methyltransferases. Genetic or epigenetic editing of these elements leads to distinct LOI phenotypes with characteristic alternations of allele-specific gene expression, DNA methylation, and 3D chromatin topology. Although repression of the Gtl2 promoter results in dysregulated imprinting, the stability of LOI phenotypes depends on the IG-DMR, suggesting a functional hierarchy. These findings establish the IG-DMR as a bipartite control element that maintains imprinting by allele-specific restriction of the DNA (de)methylation machinery.