Author Correction: Regulation of transposable elements by DNA modifications.

The originally published article contained an error in Figure 2a: for the left side of the figure part (showing piRNA-directed DNA methylation of mouse transposable elements), DNMT3A/B should have been DNMT3C. The article has now been corrected online.

Regulation of transposable elements by DNA modifications.

Maintenance of genome stability requires control over the expression of transposable elements (TEs), whose activity can have substantial deleterious effects on the host. Chemical modification of DNA is a commonly used strategy to achieve this, and it has long been argued that the emergence of 5-methylcytosine (5mC) in many species was driven by the requirement to silence TEs. Potential roles in TE regulation have also been suggested for other DNA modifications, such as N6-methyladenine and oxidation derivatives of 5mC, although the underlying mechanistic relationships are poorly understood. Here, we discuss current evidence implicating DNA modifications and DNA-modifying enzymes in TE regulation across different species.

Locus-specific chromatin profiling of evolutionarily young transposable elements.

Despite a vast expansion in the availability of epigenomic data, our knowledge of the chromatin landscape at interspersed repeats remains highly limited by difficulties in mapping short-read sequencing data to these regions. In particular, little is known about the locus-specific regulation of evolutionarily young transposable elements (TEs), which have been implicated in genome stability, gene regulation and innate immunity in a variety of developmental and disease contexts. Here we propose an approach for generating locus-specific protein-DNA binding profiles at interspersed repeats, which leverages information on the spatial proximity between repetitive and non-repetitive genomic regions. We demonstrate that the combination of HiChIP and a newly developed mapping tool (PAtChER) yields accurate protein enrichment profiles at individual repetitive loci. Using this approach, we reveal previously unappreciated variation in the epigenetic profiles of young TE loci in mouse and human cells. Insights gained using our method will be invaluable for dissecting the molecular determinants of TE regulation and their impact on the genome.

Syncytiotrophoblast Markers Are Downregulated in Placentas from Idiopathic Stillbirths.

The trophoblast cells are responsible for the transfer of nutrients between the mother and the foetus and play a major role in placental endocrine function by producing and releasing large amounts of hormones and growth factors. Syncytiotrophoblast cells (STB), formed by the fusion of mononuclear cytotrophoblasts (CTB), constitute the interface between the foetus and the mother and are essential for all of these functions. We performed transcriptome analysis of human placental samples from two control groups-live births (LB), and stillbirths (SB) with a clinically recognised cause-and from our study group, idiopathic stillbirths (iSB). We identified 1172 DEGs in iSB, when comparing with the LB group; however, when we compared iSB with the SB group, only 15 and 12 genes were down- and upregulated in iSB, respectively. An assessment of these DEGs identified 15 commonly downregulated genes in iSB. Among these, several syncytiotrophoblast markers, like genes from the PSG and CSH families, as well as ALPP, KISS1, and CRH, were significantly downregulated in placental samples from iSB. The transcriptome analysis revealed underlying differences at a molecular level involving the syncytiotrophoblast. This suggests that defects in the syncytial layer may underlie unexplained stillbirths, therefore offering insights to improve clinical obstetrics practice.

Complex multi-enhancer contacts captured by genome architecture mapping.

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. Here we report a genome-wide method, genome architecture mapping (GAM), for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify enrichment for specific interactions between active genes and enhancers across very large genomic distances using a mathematical model termed SLICE (statistical inference of co-segregation). GAM also reveals an abundance of three-way contacts across the genome, especially between regions that are highly transcribed or contain super-enhancers, providing a level of insight into genome architecture that, owing to the technical limitations of current technologies, has previously remained unattainable. Furthermore, GAM highlights a role for gene-expression-specific contacts in organizing the genome in mammalian nuclei.

Tet3 ablation in adult brain neurons increases anxiety-like behavior and regulates cognitive function in mice.

TET3 is a member of the ten-eleven translocation (TET) family of enzymes which oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Tet3 is highly expressed in the brain, where 5hmC levels are most abundant. In adult mice, we observed that TET3 is present in mature neurons and oligodendrocytes but is absent in astrocytes. To investigate the function of TET3 in adult postmitotic neurons, we crossed Tet3 floxed mice with a neuronal Cre-expressing mouse line, Camk2a-CreERT2, obtaining a Tet3 conditional KO (cKO) mouse line. Ablation of Tet3 in adult mature neurons resulted in increased anxiety-like behavior with concomitant hypercorticalism, and impaired hippocampal-dependent spatial orientation. Transcriptome and gene-specific expression analysis of the hippocampus showed dysregulation of genes involved in glucocorticoid signaling pathway (HPA axis) in the ventral hippocampus, whereas upregulation of immediate early genes was observed in both dorsal and ventral hippocampal areas. In addition, Tet3 cKO mice exhibit increased dendritic spine maturation in the ventral CA1 hippocampal subregion. Based on these observations, we suggest that TET3 is involved in molecular alterations that govern hippocampal-dependent functions. These results reveal a critical role for epigenetic modifications in modulating brain functions, opening new insights into the molecular basis of neurological disorders.

Correction to: Tet3 regulates cellular identity and DNA methylation in neural progenitor cells.

The article Tet3 regulates cellular identity and DNA methylation in neural progenitor cells, written by Miguel R. Branco and C. Joana Marques, was originally published electronically on the publisher's internet portal.

Tet3 regulates cellular identity and DNA methylation in neural progenitor cells.

TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here, we interrogated the function of Tet3 in neural precursor cells (NPCs), using a stable and inducible knockdown system and an in vitro neural differentiation protocol. We show that Tet3 is upregulated during neural differentiation, whereas Tet1 is downregulated. Surprisingly, Tet3 knockdown led to a de-repression of pluripotency-associated genes such as Oct4, Nanog or Tcl1, with concomitant hypomethylation. Moreover, in Tet3 knockdown NPCs, we observed the appearance of OCT4-positive cells forming cellular aggregates, suggesting de-differentiation of the cells. Notably, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a smaller number of CpGs that are located at neurogenesis-related genes and at imprinting control regions (ICRs) of Peg10, Zrsr1 and Mcts2 imprinted genes. Overall, our results suggest that TET3 is necessary to maintain silencing of pluripotency genes and consequently neural stem cell identity, possibly through regulation of DNA methylation levels in neural precursor cells.

TET2- and TDG-mediated changes are required for the acquisition of distinct histone modifications in divergent terminal differentiation of myeloid cells.

The plasticity of myeloid cells is illustrated by a diversity of functions including their role as effectors of innate immunity as macrophages (MACs) and bone remodelling as osteoclasts (OCs). TET2, a methylcytosine dioxygenase highly expressed in these cells and frequently mutated in myeloid leukemias, may be a key contributor to this plasticity. Through transcriptomic and epigenomic analyses, we investigated 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and gene expression changes in two divergent terminal myeloid differentiation processes, namely MAC and OC differentiation. MACs and OCs undergo highly similar 5hmC and 5mC changes, despite their wide differences in gene expression. Many TET2- and thymine-DNA glycosylase (TDG)-dependent 5mC and 5hmC changes directly activate the common terminal myeloid differentiation programme. However, the acquisition of differential features between MACs and OCs also depends on TET2/TDG. In fact, 5mC oxidation precedes differential histone modification changes between MACs and OCs. TET2 and TDG downregulation impairs the acquisition of such differential histone modification and expression patterns at MAC-/OC-specific genes. We prove that the histone H3K4 methyltransferase SETD1A is differentially recruited between MACs and OCs in a TET2-dependent manner. We demonstrate a novel role of these enzymes in the establishment of specific elements of identity and function in terminal myeloid differentiation.

Uncovering the role of 5-hydroxymethylcytosine in the epigenome.

Just over 2 years ago, TET1 was found to catalyse the oxidation of 5-methylcytosine, a well-known epigenetic mark, into 5-hydroxymethylcytosine in mammalian DNA. The exciting prospect of a novel epigenetic modification that may dynamically regulate DNA methylation has led to the rapid accumulation of publications from a wide array of fields, from biochemistry to stem cell biology. Although we have only started to scratch the surface, interesting clues on the role of 5-hydroxymethylcytosine are quickly emerging.

Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation.

Methylation at the 5' position of cytosine in DNA has important roles in genome function and is dynamically reprogrammed during early embryonic and germ cell development. The mammalian genome also contains 5-hydroxymethylcytosine (5hmC), which seems to be generated by oxidation of 5-methylcytosine (5mC) by the TET family of enzymes that are highly expressed in embryonic stem (ES) cells. Here we use antibodies against 5hmC and 5mC together with high throughput sequencing to determine genome-wide patterns of methylation and hydroxymethylation in mouse wild-type and mutant ES cells and differentiating embryoid bodies. We find that 5hmC is mostly associated with euchromatin and that whereas 5mC is under-represented at gene promoters and CpG islands, 5hmC is enriched and is associated with increased transcriptional levels. Most, if not all, 5hmC in the genome depends on pre-existing 5mC and the balance between these two modifications is different between genomic regions. Knockdown of Tet1 and Tet2 causes downregulation of a group of genes that includes pluripotency-related genes (including Esrrb, Prdm14, Dppa3, Klf2, Tcl1 and Zfp42) and a concomitant increase in methylation of their promoters, together with an increased propensity of ES cells for extraembryonic lineage differentiation. Declining levels of TETs during differentiation are associated with decreased hydroxymethylation levels at the promoters of ES cell-specific genes together with increased methylation and gene silencing. We propose that the balance between hydroxymethylation and methylation in the genome is inextricably linked with the balance between pluripotency and lineage commitment.

Promoter DNA methylation couples genome-defence mechanisms to epigenetic reprogramming in the mouse germline.

Mouse primordial germ cells (PGCs) erase global DNA methylation (5mC) as part of the comprehensive epigenetic reprogramming that occurs during PGC development. 5mC plays an important role in maintaining stable gene silencing and repression of transposable elements (TE) but it is not clear how the extensive loss of DNA methylation impacts on gene expression and TE repression in developing PGCs. Using a novel epigenetic disruption and recovery screen and genetic analyses, we identified a core set of germline-specific genes that are dependent exclusively on promoter DNA methylation for initiation and maintenance of developmental silencing. These gene promoters appear to possess a specialised chromatin environment that does not acquire any of the repressive H3K27me3, H3K9me2, H3K9me3 or H4K20me3 histone modifications when silenced by DNA methylation. Intriguingly, this methylation-dependent subset is highly enriched in genes with roles in suppressing TE activity in germ cells. We show that the mechanism for developmental regulation of the germline genome-defence genes involves DNMT3B-dependent de novo DNA methylation. These genes are then activated by lineage-specific promoter demethylation during distinct global epigenetic reprogramming events in migratory (~E8.5) and post-migratory (E10.5-11.5) PGCs. We propose that genes involved in genome defence are developmentally regulated primarily by promoter DNA methylation as a sensory mechanism that is coupled to the potential for TE activation during global 5mC erasure, thereby acting as a failsafe to ensure TE suppression and maintain genomic integrity in the germline.

In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases.

The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs.

Poised transcription factories prime silent uPA gene prior to activation.

The position of genes in the interphase nucleus and their association with functional landmarks correlate with active and/or silent states of expression. Gene activation can induce chromatin looping from chromosome territories (CTs) and is thought to require de novo association with transcription factories. We identify two types of factory: "poised transcription factories," containing RNA polymerase II phosphorylated on Ser5, but not Ser2, residues, which differ from "active factories" associated with phosphorylation on both residues. Using the urokinase-type plasminogen activator (uPA) gene as a model system, we find that this inducible gene is predominantly associated with poised (S5p(+)S2p(-)) factories prior to activation and localized at the CT interior. Shortly after induction, the uPA locus is found associated with active (S5p(+)S2p(+)) factories and loops out from its CT. However, the levels of gene association with poised or active transcription factories, before and after activation, are independent of locus positioning relative to its CT. RNA-FISH analyses show that, after activation, the uPA gene is transcribed with the same frequency at each CT position. Unexpectedly, prior to activation, the uPA loci internal to the CT are seldom transcriptionally active, while the smaller number of uPA loci found outside their CT are transcribed as frequently as after induction. The association of inducible genes with poised transcription factories prior to activation is likely to contribute to the rapid and robust induction of gene expression in response to external stimuli, whereas gene positioning at the CT interior may be important to reinforce silencing mechanisms prior to induction.

Inferring Protein-DNA Binding Profiles at Interspersed Repeats Using HiChIP and PAtChER.

Alignment of short-read sequencing data to interspersed genomic repeats, such as transposable elements, can be problematic. This is especially true for evolutionarily young elements, which have not sufficiently diverged from each other to produce distinct and uniquely mappable reads. Mapping difficulties pose a challenge for studying the portfolio of epigenetic modifications and other chromatin regulators that bind to transposons and dictate their activity, which are typically studied using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Since ChIP-seq requires chromatin fragmentation to achieve appropriate resolution, longer reads do not appreciably improve mappability. Here, we present an experimental and computational protocol that couples ChIP-seq with 3D genome folding information to produce protein binding profiles with dramatically increased coverage at interspersed repeats.

One-carbon metabolism is required for epigenetic stability in the mouse placenta.

One-carbon metabolism, including the folate cycle, has a crucial role in fetal development though its molecular function is complex and unclear. The hypomorphic Mtrr gt allele is known to disrupt one-carbon metabolism, and thus methyl group availability, leading to several developmental phenotypes (e.g., neural tube closure defects, fetal growth anomalies). Remarkably, previous studies showed that some of the phenotypes were transgenerationally inherited. Here, we explored the genome-wide epigenetic impact of one-carbon metabolism in placentas associated with fetal growth phenotypes and determined whether specific DNA methylation changes were inherited. Firstly, methylome analysis of Mtrr gt/gt homozygous placentas revealed genome-wide epigenetic instability. Several differentially methylated regions (DMRs) were identified including at the Cxcl1 gene promoter and at the En2 gene locus, which may have phenotypic implications. Importantly, we discovered hypomethylation and ectopic expression of a subset of ERV elements throughout the genome of Mtrr gt/gt placentas with broad implications for genomic stability. Next, we determined that known spermatozoan DMRs in Mtrr gt/gt males were reprogrammed in the placenta with little evidence of direct or transgenerational germline DMR inheritance. However, some spermatozoan DMRs were associated with placental gene misexpression despite normalisation of DNA methylation, suggesting the inheritance of an alternative epigenetic mechanism. Integration of published wildtype histone ChIP-seq datasets with Mtrr gt/gt spermatozoan methylome and placental transcriptome datasets point towards H3K4me3 deposition at key loci. These data suggest that histone modifications might play a role in epigenetic inheritance in this context. Overall, this study sheds light on the mechanistic complexities of one-carbon metabolism in development and epigenetic inheritance.

The N-terminus of Stag1 is required to repress the 2C program by maintaining rRNA expression and nucleolar integrity.

Our understanding of how STAG proteins contribute to cell identity and disease have largely been studied from the perspective of chromosome topology and protein-coding gene expression. Here, we show that STAG1 is the dominant paralog in mouse embryonic stem cells (mESCs) and is required for pluripotency. mESCs express a wide diversity of naturally occurring Stag1 isoforms, resulting in complex regulation of both the levels of STAG paralogs and the proportion of their unique terminal ends. Skewing the balance of these isoforms impacts cell identity. We define a novel role for STAG1, in particular its N-terminus, in regulating repeat expression, nucleolar integrity, and repression of the two-cell (2C) state to maintain mESC identity. Our results move beyond protein-coding gene regulation via chromatin loops to new roles for STAG1 in nucleolar structure and function, and offer fresh perspectives on how STAG proteins, known to be cancer targets, contribute to cell identity and disease.

H4K16ac activates the transcription of transposable elements and contributes to their cis-regulatory function.

Mammalian genomes harbor abundant transposable elements (TEs) and their remnants, with numerous epigenetic repression mechanisms enacted to silence TE transcription. However, TEs are upregulated during early development, neuronal lineage, and cancers, although the epigenetic factors contributing to the transcription of TEs have yet to be fully elucidated. Here, we demonstrate that the male-specific lethal (MSL)-complex-mediated histone H4 acetylation at lysine 16 (H4K16ac) is enriched at TEs in human embryonic stem cells (hESCs) and cancer cells. This in turn activates transcription of subsets of full-length long interspersed nuclear elements (LINE1s, L1s) and endogenous retrovirus (ERV) long terminal repeats (LTRs). Furthermore, we show that the H4K16ac-marked L1 and LTR subfamilies display enhancer-like functions and are enriched in genomic locations with chromatin features associated with active enhancers. Importantly, such regions often reside at boundaries of topologically associated domains and loop with genes. CRISPR-based epigenetic perturbation and genetic deletion of L1s reveal that H4K16ac-marked L1s and LTRs regulate the expression of genes in cis. Overall, TEs enriched with H4K16ac contribute to the cis-regulatory landscape at specific genomic locations by maintaining an active chromatin landscape at TEs.

Regulation of human trophoblast gene expression by endogenous retroviruses.

The placenta is a fast-evolving organ with large morphological and histological differences across eutherians, but the genetic changes driving placental evolution have not been fully elucidated. Transposable elements, through their capacity to quickly generate genetic variation and affect host gene regulation, may have helped to define species-specific trophoblast gene expression programs. Here we assess the contribution of transposable elements to human trophoblast gene expression as enhancers or promoters. Using epigenomic data from primary human trophoblast and trophoblast stem-cell lines, we identified multiple endogenous retrovirus families with regulatory potential that lie close to genes with preferential expression in trophoblast. These largely primate-specific elements are associated with inter-species gene expression differences and are bound by transcription factors with key roles in placental development. Using genetic editing, we demonstrate that several elements act as transcriptional enhancers of important placental genes, such as CSF1R and PSG5. We also identify an LTR10A element that regulates ENG expression, affecting secretion of soluble endoglin, with potential implications for preeclampsia. Our data show that transposons have made important contributions to human trophoblast gene regulation, and suggest that their activity may affect pregnancy outcomes.

Vitamin C activates young LINE-1 elements in mouse embryonic stem cells via H3K9me3 demethylation.

BACKGROUND: Vitamin C (vitC) enhances the activity of 2-oxoglutarate-dependent dioxygenases, including TET enzymes, which catalyse DNA demethylation, and Jumonji-domain histone demethylases. The epigenetic remodelling promoted by vitC improves the efficiency of induced pluripotent stem cell derivation, and is required to attain a ground-state of pluripotency in embryonic stem cells (ESCs) that closely mimics the inner cell mass of the early blastocyst. However, genome-wide DNA and histone demethylation can lead to upregulation of transposable elements (TEs), and it is not known how vitC addition in culture media affects TE expression in pluripotent stem cells. RESULTS: Here we show that vitC increases the expression of several TE families, including evolutionarily young LINE-1 (L1) elements, in mouse ESCs. We find that TET activity is dispensable for L1 upregulation, and that instead it occurs largely as a result of H3K9me3 loss mediated by KDM4A/C histone demethylases. Despite increased L1 levels, we did not detect increased somatic insertion rates in vitC-treated cells. Notably, treatment of human ESCs with vitC also increases L1 protein levels, albeit through a distinct, post-transcriptional mechanism. CONCLUSION: VitC directly modulates the expression of mouse L1s and other TEs through epigenetic mechanisms, with potential for downstream effects related to the multiple emerging roles of L1s in cellular function.