TERT activation targets DNA methylation and multiple aging hallmarks.

Insufficient telomerase activity, stemming from low telomerase reverse transcriptase (TERT) gene transcription, contributes to telomere dysfunction and aging pathologies. Besides its traditional function in telomere synthesis, TERT acts as a transcriptional co-regulator of genes pivotal in aging and age-associated diseases. Here, we report the identification of a TERT activator compound (TAC) that upregulates TERT transcription via the MEK/ERK/AP-1 cascade. In primary human cells and naturally aged mice, TAC-induced elevation of TERT levels promotes telomere synthesis, blunts tissue aging hallmarks with reduced cellular senescence and inflammatory cytokines, and silences p16INK4a expression via upregulation of DNMT3B-mediated promoter hypermethylation. In the brain, TAC alleviates neuroinflammation, increases neurotrophic factors, stimulates adult neurogenesis, and preserves cognitive function without evident toxicity, including cancer risk. Together, these findings underscore TERT's critical role in aging processes and provide preclinical proof of concept for physiological TERT activation as a strategy to mitigate multiple aging hallmarks and associated pathologies.

Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss.

Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.

Single substitution in H3.3G34 alters DNMT3A recruitment to cause progressive neurodegeneration.

Germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated H3.3G34R/V/W knock-in mice and identified strikingly distinct developmental defects for each mutation. H3.3G34R-mutants exhibited progressive microcephaly and neurodegeneration, with abnormal accumulation of disease-associated microglia and concurrent neuronal depletion. G34R severely decreased H3K36me2 on the mutant H3.3 tail, impairing recruitment of DNA methyltransferase DNMT3A and its redistribution on chromatin. These changes were concurrent with sustained expression of complement and other innate immune genes possibly through loss of non-CG (CH) methylation and silencing of neuronal gene promoters through aberrant CG methylation. Complement expression in G34R brains may lead to neuroinflammation possibly accounting for progressive neurodegeneration. Our study reveals that H3.3G34-substitutions have differential impact on the epigenome, which underlie the diverse phenotypes observed, and uncovers potential roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating synaptic pruning and neuroinflammation in post-natal brains.

Role of Mammalian DNA Methyltransferases in Development.

DNA methylation at the 5-position of cytosine (5mC) plays vital roles in mammalian development. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), and the two DNMT families, DNMT3 and DNMT1, are responsible for methylation establishment and maintenance, respectively. Since their discovery, biochemical and structural studies have revealed the key mechanisms underlying how DNMTs catalyze de novo and maintenance DNA methylation. In particular, recent development of low-input genomic and epigenomic technologies has deepened our understanding of DNA methylation regulation in germ lines and early stage embryos. In this review, we first describe the methylation machinery including the DNMTs and their essential cofactors. We then discuss how DNMTs are recruited to or excluded from certain genomic elements. Lastly, we summarize recent understanding of the regulation of DNA methylation dynamics in mammalian germ lines and early embryos with a focus on both mice and humans.

Evolutionary Persistence of DNA Methylation for Millions of Years after Ancient Loss of a De Novo Methyltransferase.

Cytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles. In the yeast Cryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 mya. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 million years through a process analogous to Darwinian evolution of the genome.

CpG Island Hypermethylation Mediated by DNMT3A Is a Consequence of AML Progression.

DNMT3A mutations occur in ∼25% of acute myeloid leukemia (AML) patients. The most common mutation, DNMT3AR882H, has dominant negative activity that reduces DNA methylation activity by ∼80% in vitro. To understand the contribution of DNMT3A-dependent methylation to leukemogenesis, we performed whole-genome bisulfite sequencing of primary leukemic and non-leukemic cells in patients with or without DNMT3AR882 mutations. Non-leukemic hematopoietic cells with DNMT3AR882H displayed focal methylation loss, suggesting that hypomethylation antedates AML. Although virtually all AMLs with wild-type DNMT3A displayed CpG island hypermethylation, this change was not associated with gene silencing and was essentially absent in AMLs with DNMT3AR882 mutations. Primary hematopoietic stem cells expanded with cytokines were hypermethylated in a DNMT3A-dependent manner, suggesting that hypermethylation may be a response to, rather than a cause of, cellular proliferation. Our findings suggest that hypomethylation is an initiating phenotype in AMLs with DNMT3AR882, while DNMT3A-dependent CpG island hypermethylation is a consequence of AML progression.

Early-Life Gene Expression in Neurons Modulates Lasting Epigenetic States.

In mammals, the environment plays a critical role in promoting the final steps in neuronal development during the early postnatal period. While epigenetic factors are thought to contribute to this process, the underlying molecular mechanisms remain poorly understood. Here, we show that in the brain during early life, the DNA methyltransferase DNMT3A transiently binds across transcribed regions of lowly expressed genes, and its binding specifies the pattern of DNA methylation at CA sequences (mCA) within these genes. We find that DNMT3A occupancy and mCA deposition within the transcribed regions of genes is negatively regulated by gene transcription and may be modified by early-life experience. Once deposited, mCA is bound by the methyl-DNA-binding protein MECP2 and functions in a rheostat-like manner to fine-tune the cell-type-specific transcription of genes that are critical for brain function.

Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.

Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.

Editing DNA Methylation in the Mammalian Genome.

Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.

DNA methylation controls stemness of astrocytes in health and ischaemia.

Astrocytes are the most abundant cell type in the mammalian brain and provide structural and metabolic support to neurons, regulate synapses and become reactive after injury and disease. However, a small subset of astrocytes settles in specialized areas of the adult brain where these astrocytes instead actively generate differentiated neuronal and glial progeny and are therefore referred to as neural stem cells1-3. Common parenchymal astrocytes and quiescent neural stem cells share similar transcriptomes despite their very distinct functions4-6. Thus, how stem cell activity is molecularly encoded remains unknown. Here we examine the transcriptome, chromatin accessibility and methylome of neural stem cells and their progeny, and of astrocytes from the striatum and cortex in the healthy and ischaemic adult mouse brain. We identify distinct methylation profiles associated with either astrocyte or stem cell function. Stem cell function is mediated by methylation of astrocyte genes and demethylation of stem cell genes that are expressed later. Ischaemic injury to the brain induces gain of stemness in striatal astrocytes7. We show that this response involves reprogramming the astrocyte methylome to a stem cell methylome and is absent if the de novo methyltransferase DNMT3A is missing. Overall, we unveil DNA methylation as a promising target for regenerative medicine.

Selective chemical tracking of Dnmt1 catalytic activity in live cells.

Enzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. DNA methylation is brought about by collective action of three AdoMet-dependent DNA methyltransferases, whose catalytic interactions and temporal interplay are poorly understood. We used structure-guided engineering of the Dnmt1 methyltransferase to enable catalytic transfer of azide tags onto DNA from a synthetic cofactor analog, Ado-6-azide, in vitro. We then CRISPR-edited the Dnmt1 locus in mouse embryonic stem cells to install the engineered codon, which, following pulse internalization of the Ado-6-azide cofactor by electroporation, permitted selective azide tagging of Dnmt1-specific genomic targets in cellulo. The deposited covalent tags were exploited as "click" handles for reading adjoining sequences and precise genomic mapping of the methylation sites. The proposed approach, Dnmt-TOP-seq, enables high-resolution temporal tracking of the Dnmt1 catalysis in mammalian cells, paving the way to selective studies of other methylation pathways in eukaryotic systems.

DNA Methylation and Histone H1 Jointly Repress Transposable Elements and Aberrant Intragenic Transcripts.

DNA methylation and histone H1 mediate transcriptional silencing of genes and transposable elements, but how they interact is unclear. In plants and animals with mosaic genomic methylation, functionally mysterious methylation is also common within constitutively active housekeeping genes. Here, we show that H1 is enriched in methylated sequences, including genes, of Arabidopsis thaliana, yet this enrichment is independent of DNA methylation. Loss of H1 disperses heterochromatin, globally alters nucleosome organization, and activates H1-bound genes, but only weakly de-represses transposable elements. However, H1 loss strongly activates transposable elements hypomethylated through mutation of DNA methyltransferase MET1. Hypomethylation of genes also activates antisense transcription, which is modestly enhanced by H1 loss. Our results demonstrate that H1 and DNA methylation jointly maintain transcriptional homeostasis by silencing transposable elements and aberrant intragenic transcripts. Such functionality plausibly explains why DNA methylation, a well-known mutagen, has been maintained within coding sequences of crucial plant and animal genes.

Genomic Profiling by ALaP-Seq Reveals Transcriptional Regulation by PML Bodies through DNMT3A Exclusion.

The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with ∼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.

ATP Hydrolysis by the SNF2 Domain of Dnmt5 Is Coupled to Both Specific Recognition and Modification of Hemimethylated DNA.

C.neoformans Dnmt5 is an unusually specific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain. We find that the SNF2 domain couples substrate specificity to an ATPase step essential for DNA methylation. Coupling occurs independent of nucleosomes. Hemimethylated DNA preferentially stimulates ATPase activity, and mutating Dnmt5's ATP-binding pocket disproportionately reduces ATPase stimulation by hemimethylated versus unmethylated substrates. Engineered DNA substrates that stabilize a reaction intermediate by mimicking a "flipped-out" conformation of the target cytosine bypass the SNF2 domain's requirement for hemimethylation. This result implies that ATP hydrolysis by the SNF2 domain is coupled to the DNMT domain conformational changes induced by preferred substrates. These findings establish a new role for a SNF2 ATPase: controlling an adjoined enzymatic domain's substrate recognition and catalysis. We speculate that this coupling contributes to the exquisite specificity of Dnmt5 via mechanisms related to kinetic proofreading.

The inactive Dnmt3b3 isoform preferentially enhances Dnmt3b-mediated DNA methylation.

The de novo DNA methyltransferases Dnmt3a and Dnmt3b play crucial roles in developmental and cellular processes. Their enzymatic activities are stimulated by a regulatory protein Dnmt3L (Dnmt3-like) in vitro. However, genetic evidence indicates that Dnmt3L functions predominantly as a regulator of Dnmt3a in germ cells. How Dnmt3a and Dnmt3b activities are regulated during embryonic development and in somatic cells remains largely unknown. Here we show that Dnmt3b3, a catalytically inactive Dnmt3b isoform expressed in differentiated cells, positively regulates de novo methylation by Dnmt3a and Dnmt3b with a preference for Dnmt3b. Dnmt3b3 is equally potent as Dnmt3L in stimulating the activities of Dnmt3a2 and Dnmt3b2 in vitro. Like Dnmt3L, Dnmt3b3 forms a complex with Dnmt3a2 with a stoichiometry of 2:2. However, rescue experiments in Dnmt3a/3b/3l triple-knockout (TKO) mouse embryonic stem cells (mESCs) reveal that Dnmt3b3 prefers Dnmt3b2 over Dnmt3a2 in remethylating genomic sequences. Dnmt3a2, an active isoform that lacks the N-terminal uncharacterized region of Dnmt3a1 including a nuclear localization signal, has very low activity in TKO mESCs, indicating that an accessory protein is absolutely required for its function. Our results suggest that Dnmt3b3 and perhaps similar Dnmt3b isoforms facilitate de novo DNA methylation during embryonic development and in somatic cells.

miR-137 confers robustness to the territorial restriction of the neural plate border.

The neural plate border (NPB) of vertebrate embryos is segregated from the neural plate (NP) and epidermal regions, and comprises an intermingled group of progenitors with multiple fate potential. Recent studies have shown that, during the gastrula stage, TFAP2A acts as a pioneer factor in remodeling the epigenetic landscape required to activate components of the NPB induction program. Here, we show that chick Tfap2a has two highly conserved binding sites for miR-137, and both display a reciprocal expression pattern at the NPB and NP, respectively. In addition, ectopic miR-137 expression reduced TFAP2A, whereas its functional inhibition expanded their territorial distribution overlapping with PAX7. Furthermore, we demonstrate that loss of the de novo DNA methyltransferase DNMT3A expanded miR-137 expression to the NPB. Bisulfite sequencing revealed a markedly elevated presence of non-canonical CpH methylation within the miR-137 promoter region when comparing NPB and NP samples. Our findings show that miR-137 contributes to the robustness of NPB territorial restriction in vertebrate development.

Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages.

During development, progenitor cells with binary potential give rise to daughter cells that have distinct functions. Heritable epigenetic mechanisms then lock in gene-expression programs that define lineage identity. Regulation of the gene encoding the T cell-specific coreceptor CD4 in helper and cytotoxic T cells exemplifies this process, with enhancer- and silencer-regulated establishment of epigenetic memory for stable gene expression and repression, respectively. Using a genetic screen, we identified the DNA-methylation machinery as essential for maintaining silencing of Cd4 in the cytotoxic lineage. Furthermore, we found a requirement for the proximal enhancer in mediating the removal of DNA-methylation marks from Cd4, which allowed stable expression of Cd4 in helper T cells. Our findings suggest that stage-specific methylation and demethylation events in Cd4 regulate its heritable expression in response to the distinct signals that dictate lineage 'choice' during T cell development.

Active turnover of DNA methylation during cell fate decisions.

DNA methylation is a key layer of epigenetic regulation. The deposition of methylation marks relies on the catalytic activity of DNA methyltransferases (DNMTs), and their active removal relies on the activity of ten-eleven translocation (TET) enzymes. Paradoxically, in important biological contexts these antagonistic factors are co-expressed and target overlapping genomic regions. The ensuing cyclic biochemistry of cytosine modifications gives rise to a continuous, out-of-thermal equilibrium transition through different methylation states. But what is the purpose of this intriguing turnover of DNA methylation? Recent evidence demonstrates that methylation turnover is enriched at gene distal regulatory elements, including enhancers, and can give rise to large-scale oscillatory dynamics. We discuss this phenomenon and propose that DNA methylation turnover might facilitate key lineage decisions.

Dynamically adjusted cell fate decisions and resilience to mutant invasion during steady-state hematopoiesis revealed by an experimentally parameterized mathematical model.

A major next step in hematopoietic stem cell (HSC) biology is to enhance our quantitative understanding of cellular and evolutionary dynamics involved in undisturbed hematopoiesis. Mathematical models have been and continue to be key in this respect, and are most powerful when parameterized experimentally and containing sufficient biological complexity. In this paper, we use data from label propagation experiments in mice to parameterize a mathematical model of hematopoiesis that includes homeostatic control mechanisms as well as clonal evolution. We find that nonlinear feedback control can drastically change the interpretation of kinetic estimates at homeostasis. This suggests that short-term HSC and multipotent progenitors can dynamically adjust to sustain themselves temporarily in the absence of long-term HSCs, even if they differentiate more often than they self-renew in undisturbed homeostasis. Additionally, the presence of feedback control in the model renders the system resilient against mutant invasion. Invasion barriers, however, can be overcome by a combination of age-related changes in stem cell differentiation and evolutionary niche construction dynamics based on a mutant-associated inflammatory environment. This helps us understand the evolution of e.g., TET2 or DNMT3A mutants, and how to potentially reduce mutant burden.

The histone chaperone CAF-1 cooperates with the DNA methyltransferases to maintain Cd4 silencing in cytotoxic T cells.

The transcriptional repression of alternative lineage genes is critical for cell fate commitment. Mechanisms by which locus-specific gene silencing is initiated and heritably maintained during cell division are not clearly understood. To study the maintenance of silent gene states, we investigated how the Cd4 gene is stably repressed in CD8+ T cells. Through CRISPR and shRNA screening, we identified the histone chaperone CAF-1 as a critical component for Cd4 repression. We found that the large subunit of CAF-1, Chaf1a, requires the N-terminal KER domain to associate with the histone deacetylases HDAC1/2 and the histone demethylase LSD1, enzymes that also participate in Cd4 silencing. When CAF-1 was lacking, Cd4 derepression was markedly enhanced in the absence of the de novo DNA methyltransferase Dnmt3a but not the maintenance DNA methyltransferase Dnmt1. In contrast to Dnmt1, Dnmt3a deficiency did not significantly alter levels of DNA methylation at the Cd4 locus. Instead, Dnmt3a deficiency sensitized CD8+ T cells to Cd4 derepression mediated by compromised functions of histone-modifying factors, including the enzymes associated with CAF-1. Thus, we propose that the heritable silencing of the Cd4 gene in CD8+ T cells exploits cooperative functions among the DNA methyltransferases, CAF-1, and histone-modifying enzymes.