Maternal obesity associates with an increased risk of offspring neurodevelopmental disorders. Although the underlying mechanism(s) remain unclear, evidence suggests a role for altered DNA methylation. We utilized a murine model of diet-induced obesity to investigate the impact of maternal obesity on the offspring brain transcriptome and DNA methylation. C57Bl/6 dams were fed high-fat high-sugar (HFD, n = 7) or control (CON, n = 7) diets. Maternal obesity/hyperglycemia associated with offspring growth restriction, with brain-sparing specifically in females. Postnatal hypoglycemia was seen in HFD males, but not females. The 3' RNA-sequencing revealed perturbations in metabolic and cell differentiation pathways in neonatal male and female offspring frontal cortex and cerebellum. Compared with controls, HFD males, but not females, had lower cortical and cerebellar DNMT gene and protein expression, and reduced cerebellar TET enzyme mRNA. Whilst female offspring had lower cerebellar 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) than males, there were no effects of HFD on 5mC/5hmC in cortex or cerebellum in either sex. Our data suggest that maternal obesity has sex-specific effects on fetal neurodevelopment, including enzymes involved in DNA methylation/demethylation. These mechanisms may play a role in the increased risk of neurodevelopmental disorders following obese/diabetic pregnancies, including increased male susceptibility to these disorders.

Rett syndrome (RTT) is a neurodevelopmental disorder that is mainly caused by mutations in the methyl-DNA-binding protein MECP2. MECP2 is an important epigenetic regulator that plays a pivotal role in neuronal gene regulation, where it has been reported to function as both a repressor and an activator. Despite extensive efforts in mechanistic studies over the past two decades, a clear consensus on how MECP2 dysfunction impacts molecular mechanisms and contributes to disease progression has not been reached. Here, we review recent insights from epigenomic, transcriptomic and proteomic studies that advance our understanding of MECP2 as an interacting hub for DNA, RNA and transcription factors, orchestrating diverse processes that are crucial for neuronal function. By discussing findings from different model systems, we identify crucial epigenetic details and cofactor interactions, enriching our understanding of the multifaceted roles of MECP2 in transcriptional regulation and chromatin structure. These mechanistic insights offer potential avenues for rational therapeutic design for RTT.

The diversity of mammalian neurons is delineated by subtle gene expression differences that may require specialized mechanisms to be maintained. Neurons uniquely express the longest genes in the genome and use non-CG DNA methylation (mCA), together with the Rett syndrome protein methyl-CpG-binding protein 2 (MeCP2), to control gene expression. However, whether these distinctive gene structures and molecular machinery regulate neuronal diversity remains unexplored. Here, we use genomic and spatial transcriptomic analyses to show that MeCP2 maintains transcriptomic diversity across closely related neuron types. We uncover differential susceptibility of neuronal populations to MeCP2 loss according to global mCA levels and dissect methylation patterns driving shared and distinct MeCP2 gene regulation. We show that MeCP2 regulates long, mCA-enriched, 'repeatedly tuned' genes, that is, genes differentially expressed between many closely related neuron types, including across spatially distinct, vision-dependent gene programs in the visual cortex. Thus, MeCP2 maintains neuron type-specific gene programs to facilitate cellular diversity in the brain.

Aging is accompanied by widespread DNA methylation changes across the genome. While age-related methylation studies typically focus on individual CpGs, cluster analysis provides more robust data and improved interpretation. We characterized age-associated CpG-cluster methylation changes in mouse spleens, peripheral blood mononuclear cells, and livers. We identified a novel signature termed blanket methylations (BMs), fully methylated CpG clusters absent in young tissues but appearing in aged tissues. BM formation was locus- and cell-dependent, with minimal overlap among tissues. Statistical analysis, heterogeneity assessment, and random modeling demonstrated that BMs arise through nonrandom mechanisms and correlate with accelerated aging. Notably, BMs appeared in chronologically young mice with progeroid or disease-driven aging, including in 4-month-old Zmpste24-/- (lifespan ∼5 months) and 3-month-old Huntington's disease model mice (lifespan ∼4 months). The detection of BMs in purified CD4+ T cells demonstrated that their occurrence is intrinsic to aging cells rather than a result of infiltration from other tissues. Further investigation revealed age-related downregulation of zinc-finger-CxxC-domain genes, including Tet1 and Tet3, which protect CpG islands from methylation. Importantly, TET1 or TET3 depletion induced BM formation, linking their loss to age-associated methylation drift. These findings establish BMs as a robust marker of epigenomic aging, providing insight into age-related methylation changes.

Neuronal activity, including sensory-evoked and spontaneous firing, regulates the expression of a subset of genes known as activity-dependent genes. A key issue in this process is the activation and accumulation of transcription factors (TFs), which bind to cis-elements at specific enhancers and promoters, ultimately driving RNA synthesis through transcription machinery. Epigenetic factors such as histone modifiers also play a crucial role in facilitating the specific binding of TFs. Recent evidence from epigenome analyses and imaging studies have revealed intriguing mechanisms: the default chromatin structure at activity-dependent genes is formed independently of neuronal activity, while neuronal activity modulates spatiotemporal dynamics of TFs and their interactions with epigenetic factors (EFs). In this article we review new insights into activity-dependent gene regulation that affects brain development and plasticity.

DNA methylation is a critical epigenetic mechanism involved in numerous physiological processes. Alterations in DNA methylation patterns are associated with various brain disorders, including dementias such as Alzheimer's disease (AD) and frontotemporal dementia (FTD). Investigating these alterations is essential for understanding the pathogenesis and progression of these disorders. Among the various methods for detecting DNA methylation, DNA sequencing is one of the most widely employed. Specifically, two main sequencing approaches are commonly used for DNA methylation analysis: bisulfite sequencing and single-molecule long-read sequencing. In this review, we compared the performances of CpG methylation detection obtained using two popular sequencing platforms, Illumina for bisulfite sequencing and Oxford Nanopore (ON) for long-read sequencing. Our comparison considers several factors, including accuracy, efficiency, genomic regions, costs, wet-lab protocols, and bioinformatics pipelines. We provide insights into the strengths and limitations of both methods with a particular focus on their application in research on AD and FTD.

The mammalian nucleus is compartmentalized by diverse subnuclear structures. These subnuclear structures, marked by nuclear bodies and histone modifications, are often cell-type specific and affect gene regulation and 3D genome organization1-3. Understanding their relationships rests on identifying the molecular constituents of subnuclear structures and mapping their associations with specific genomic loci and transcriptional levels in individual cells, all in complex tissues. Here, we introduce two-layer DNA seqFISH+, which enables simultaneous mapping of 100,049 genomic loci, together with the nascent transcriptome for 17,856 genes and subnuclear structures in single cells. These data enable imaging-based chromatin profiling of diverse subnuclear markers and can capture their changes at genomic scales ranging from 100-200 kilobases to approximately 1 megabase, depending on the marker and DNA locus. By using multi-omics datasets in the adult mouse cerebellum, we showed that repressive chromatin regions are more variable by cell type than are active regions across the genome. We also discovered that RNA polymerase II-enriched foci were locally associated with long, cell-type-specific genes (bigger than 200 kilobases) in a manner distinct from that of nuclear speckles. Furthermore, our analysis revealed that cell-type-specific regions of heterochromatin marked by histone H3 trimethylated at lysine 27 (H3K27me3) and histone H4 trimethylated at lysine 20 (H4K20me3) are enriched at specific genes and gene clusters, respectively, and shape radial chromosomal positioning and inter-chromosomal interactions in neurons and glial cells. Together, our results provide a single-cell high-resolution multi-omics view of subnuclear structures, associated genomic loci and their effects on gene regulation, directly within complex tissues.

Higher-order chromatin structure and DNA methylation are critical for gene regulation, but how these vary across the human body remains unclear. We performed multi-omic profiling of 3D genome structure and DNA methylation for 86,689 single nuclei across 16 human tissues, identifying 35 major and 206 cell subtypes. We revealed extensive changes in CG and non-CG methylation across almost all cell types and characterized 3D chromatin structure at an unprecedented cellular resolution. Intriguingly, extensive discrepancies exist between cell types delineated by DNA methylation and genome structure, indicating that the role of distinct epigenomic features in maintaining cell identity may vary by lineage. This study expands our understanding of the diversity of DNA methylation and chromatin structure and offers an extensive reference for exploring gene regulation in human health and disease.

Animal models are commonly used to investigate developmental processes and disease risk, but humans and model systems (e.g., mice) differ substantially in the pace of development and aging. The timeline of human developmental circuits is well known, but it is unclear how such timelines compare with those in mice. We lack age alignments across the lifespan of mice and humans. Here, we build upon our Translating Time resource, which is a tool that equates corresponding ages during development. We collected 1,125 observations from age-related changes in body, bone, dental, and brain processes to equate corresponding ages across humans, mice, and rats to boost power for comparison across humans and mice. We acquired high-resolution diffusion MR scans of mouse brains (n = 16) of either sex at sequential stages of postnatal development [postnatal day (P)3, 4, 12, 21, 60] to track brain circuit maturation (e.g., olfactory association, transcallosal pathways). We found heterogeneity in white matter pathway growth. Corpus callosum growth largely ceases days after birth, while the olfactory association pathway grows through P60. We found that a P3-4, mouse equates to a human at roughly GW24 and a P60 mouse equates to a human in teenage years. Therefore, white matter pathway maturation is extended in mice as it is in humans, but there are species-specific adaptations. For example, olfactory-related wiring is protracted in mice, which is linked to their reliance on olfaction. Our findings underscore the importance of translational tools to map common and species-specific biological processes from model systems to humans.

Retinoblastoma, the most common pediatric intraocular malignancy, arises from RB1 inactivation, leading to uncontrolled proliferation of retinal progenitor cells. Epigenetic dysregulation a key driver of retinoblastoma progression, yet the underlying mechanisms are poorly understood. UHRF1, a regulator of DNA methylation and chromatin remodeling, has been implicated in oncogenesis but its role in retinoblastoma has not been fully characterized. To investigate Uhrf1's role in tumor initiation and progression, we generated a genetically engineered mouse model of retinoblastoma with conditional Uhrf1 knockout. Remarkably, Uhrf1 loss completely blocked tumor formation, despite persistent early oncogenic events. Transcriptomic and epigenomic profiling revealed that Uhrf1 is essential for tumor progression, facilitating oncogenic transcription, chromatin accessibility, and aberrant DNA methylation. Beyond its epigenetic role, we found Uhrf1 modulates the tumor immune microenvironment, promoting chemokine secretion and microglial infiltration, suggesting that Uhrf1 promotes immune cell recruitment. Mechanistically, Uhrf1 enhances chemokine expression through NF-κB signaling, establishing a novel connection between epigenetic regulation and tumor-associated immune responses. These findings establish Uhrf1 as a critical driver of retinoblastoma progression, essential for tumor maintenance but not initiation. By sustaining oncogenic transcriptional programs and shaping a pro-tumor immune microenvironment, Uhrf1 emerges as a promising therapeutic target for inhibiting tumor growth and immune evasion.

Sorghum is an increasingly popular topic of research in elucidating survival and adaptation approaches to augmented salinity. Nonetheless, little is known about the outcome and modulatory networks involved in the gibberellic acid (GA3)-induced salt stress alleviation in sorghum. Here, we identified 50 mg/L GA3 as the optimal concentration for sorghum ('Jitian 3') development under salt stress. Based on genome-wide DNA methylation analysis, CpG sites displayed the most abundant methylation statuses among all stages, with a mean value of 68.2 %, then CHG (57.9 %), and CHH (21.2 %). We identified 18,032 differentially methylated regions (DMRs) in the GA3-exposed groups. In particular, we recognized 5943 DMR genes and 269 DMR-promoter genes. Using conjoint transcriptome and DNA methylation analyses, we identified 337 important methylated-genes, which were involved in "phenylpropanoid biosynthesis", "arginine and proline metabolism" and "tyrosine metabolism". Together, the aforementioned data provides an in-depth understanding of the epigenetic modulation of gene expression during GA3 treatment.

DNA methylation, a dynamic epigenetic mark influencing gene expression, is regulated by DNA demethylases that remove methylated cytosines at genomic regions marked by the INCREASED DNA METHYLATION (IDM) complex. In Arabidopsis, IDM3, a small α-crystalline domain-containing protein, stabilises the IDM complex. To investigate its role in tomato, we generated slidm3 mutants using genome editing. These mutants displayed a 'hairy' phenotype with increased glandular trichomes, resembling the hairplus (hap) mutant. Affinity purification of SlIDM3-GFP associated proteins identified several chromatin remodelling factors, including HAP. Genome-wide DNA methylation analysis revealed sequence context dependent alterations in the slidm3-1 plants, similar to the hap mutant. CHH methylation was predominantly increased, while CG methylation, particularly in intergenic regions, was decreased in both mutants. This imbalanced methylation suggests the presence of a 'methylstat' mechanism attempting to restore methylation levels at abnormally demethylated sites in the mutants. Comparative functional analysis of differentially methylated regions in the slidm3-1 and hap mutants identified potential methylation-regulated genes that could be linked to the hairy phenotype. Our findings indicate that SlIDM3 may form a chromatin remodelling complex with HAP, epigenetically regulating trichome development.

Members of the DNA methyltransferase (DNMT) family have been recognized as major epigenetic regulators of altered gene expression during tumor development. They establish and maintain DNA methylation of the CpG island of promoter and non-CpG region of the genome. The abnormal methylation status of tumor suppressor genes (TSGs) has been associated with tumorigenesis, leading to genomic instability, improper gene silence, and immune evasion. DNMT1 helps preserve methylation patterns during DNA replication, whereas the DNMT3 family is responsible for de novo methylation, creating new methylation patterns. Altered DNA methylation significantly supports tumor growth by changing gene expression patterns. FDA-approved DNMT inhibitors reverse hypermethylation-induced gene repression and improve therapeutic outcomes for cancer. Recent studies indicate that combining DNMT inhibitors with chemotherapies and immunotherapies can have synergistic effects, especially in aggressive metastatic tumors. Improving the treatment schedules, increasing isoform specificity, reducing toxicity, and utilizing genome-wide analyses of CRISPR-based editing to create personalized epigenetic therapies tailored to individual patient needs are promising strategies for enhancing therapeutic outcomes. This review discusses the interaction between DNMT regulators and DNMT1, its binding partners, the connection between DNA methylation and tumors, how these processes contribute to tumor development, and DNMT inhibitors' advancements and pharmacological properties.

Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene cause Rett syndrome, a severe childhood neurological disorder. MeCP2 is a well-established transcriptional repressor, yet upon its loss, hundreds of genes are dysregulated in both directions. To understand what drives such dysregulation, we deleted Mecp2 in adult mice, circumventing developmental contributions and secondary pathogenesis. We performed time series transcriptional, chromatin, and phenotypic analyses of the hippocampus to determine the immediate consequences of MeCP2 loss and the cascade of pathogenesis. We find that loss of MeCP2 causes immediate and bidirectional progressive dysregulation of the transcriptome. To understand what drives gene downregulation, we profiled genome-wide histone modifications and found that a decrease in histone H3 acetylation (ac) at downregulated genes is among the earliest molecular changes occurring well before any measurable deficiencies in electrophysiology and neurological function. These data reveal a molecular cascade that drives disease independent of any developmental contributions or secondary pathogenesis.

Age-related changes in DNA methylation (DNAm) play a critical role in regulating gene expression. However, most epigenome-wide association studies have predominantly focused on individuals of European descent. This study aims to characterize longitudinal changes in DNAm patterns in a cohort of elderly Brazilian participants.

DNAm profiles were collected approximately nine years apart from 23 elderly Brazilian individuals using the Illumina Infinium MethyationEPIC BeadChip. Using mixed-effects models, we examined changes in DNAm patterns using both quantitative age and binary timepoint (e.g., baseline vs. follow-up) as predictors of interest to identify differentially methylated positions (DMPs). Significant DMPs were compared with a list of previously identified age-related DMPs. Differentially methylated regions (DMRs) were also identified using DMRcate. Gene ontology (GO) pathway enrichment analyses were performed to explore the functional significance of identified DMPs and DMRs.

Of the 586,229 autosomal probes included in the differential methylation analyses, 2768 significant (FDR < 0.05) age-associated DMPs (aDMPs) and 2757 significant (FDR < 0.05) timepoint-associated DMPs (tpDMPs) were identified. Of the 2768 aDMPs, 1471 were replicated from previous studies. Of the 1297 non-replicated CpGs, 77.4% were exclusive to the EPIC array. The DMR analyses identified 305 age-associated DMRs (aDMRs) and 372 timepoint-associated DMRs (tpDMRs). Both aDMPs and aDMRs exhibited age-related hypermethylation within CpG islands and promoter regions of the genome, whereas hypomethylation predominantly occurred in interCGI and intergenic regions and introns. The GO enrichment analyses identified several neurological and cognition-related pathways enriched for hypermethylated CpG islands, many of which were mapped near transcription start sites and first exon regions.

This longitudinal study identified age-associated and timepoint-associated DMPs and DMRs in a sample of elderly Brazilians. Most of the non-replicated CpGs were found to be on the new EPIC array, suggesting that more age-related studies using the EPIC array are required to validate these CpGs. The GO pathway enrichment analyses identified age-related enrichment of several gene sets related to cognitive and physical decline in elderly populations. The enrichment of these sites could provide evidence for age-related neurodegeneration and cognitive decline in elderly populations.

Cancer stem cells (CSCs) have a crucial function in the initiation, advancement, and resistance to therapy of tumors. Recent findings indicate that non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play a complex role in controlling the features of cancer stem cells (CSCs). Non-coding RNAs (ncRNAs) play a crucial role in controlling important characteristics of stem cells, such as their ability to renew themselves, differentiate into distinct cell types, and resist therapy. This article provides an overview of the current understanding of the complex relationship between non-coding RNAs (ncRNAs), namely microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), and cancer stem cells (CSCs). Particular microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are involved in regulating important signaling pathways like as Wnt, Notch, and Hedgehog, which control stem cell-like characteristics. The miR-34, miR-200, and let-7 families specifically aim at inhibiting the process of self-renewal and epithelial-to-mesenchymal transition. On the other hand, long non-coding RNAs (lncRNAs) such as H19, HOTAIR, and MALAT1 play a role in modifying the epigenetic landscape, hence enhancing the characteristics of stemness. This article also offers a thorough examination of the role of non-coding RNAs (ncRNAs) in regulating cancer stemness, emphasizing their impact on crucial biochemical pathways, epigenetic changes, and therapeutic implications. Comprehending the interaction between non-coding RNAs (ncRNAs) and cancer stem cells (CSCs) provides fresh perspectives on possible focused treatments for fighting aggressive and resistant malignancies. Gaining a comprehensive understanding of the connection between non-coding RNA (ncRNA) and cancer stem cells (CSC) offers valuable insights for the development of novel and precise treatments to combat aggressive cancers that are resistant to conventional therapies. In addition, the combination of ncRNA therapies with conventional methods like as chemotherapy or epigenetic medicines could result in synergistic effects. Nevertheless, there are still obstacles to overcome in terms of delivery, effectiveness, and safety. In summary, the interaction between non-coding RNA and cancer stemness shows potential as a targeted treatment approach in the field of precision oncology. This calls for additional investigation and use in clinical settings.

Single-cell transcriptomic studies have identified distinct microglial subpopulations with shared and divergent gene signatures across development, aging and disease. Whether these microglial subsets represent ontogenically separate lineages of cells, or they are manifestations of plastic changes of microglial states downstream of some converging signals is unknown. Furthermore, despite the well-established role of enhancer landscapes underlying the identity of microglia, to what extent histone modifications and DNA methylation regulate microglial state switches at enhancers have not been defined. Here, using genetic fate mapping, we demonstrate the common embryonic origin of proliferative-region-associated microglia (PAM) enriched in developing white matter, and track their dynamic transitions into disease-associated microglia (DAM) and white matter-associated microglia (WAM) states in disease and aging contexts, respectively. This study links spatiotemporally discrete microglial states through their transcriptomic and epigenomic plasticity, while revealing state-specific histone modification profiles that govern state switches in health and disease.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare inherited condition described worldwide and characterized by a wide spectrum of heterogeneity in terms of genotype and phenotype. How sacsin loss leads to neurodegeneration is still unclear, and current knowledge indicates that sacsin is involved in multiple functional mechanisms. We hence hypothesized the existence of epigenetic factors, in particular alterations in methylation patterns, that could contribute to ARSACS pathogenesis and explain the pleiotropic effects of SACS further than pathogenic mutations. To investigate this issue, we recruited eight patients affected by ARSACS, four characterized by early onset of the disease and four with late onset. We performed Whole Genome Bisulfite Sequencing using DNA from peripheral blood to define the methylome of patients and compared them with a control group. Our analysis showed that patients with ARSACS exhibit an altered methylation pattern and that the observed differences exist also among affected individuals with different age of onset. Our study provides valuable insights for employing epigenetic biomarkers to assess the severity and progression of this disorder and propels further investigations into the role of epigenetic processes in ARSACS pathogenesis.

DNA methylation, catalyzed by DNA methyltransferases (DNMT), plays pivotal role in regulating embryonic development, gene expression, adaption to environmental stress, and maintaining genome integrity. DNMT family consists of DNMT1, DNMT3A, DNMT3B, and the enzymatically inactive DNMT3L. DNMT3A and DNMT3B establish novel methylation patterns maintained by DNMT1 during replication. Genetic variants of DNMT3A and DNMT3B cause rare diseases such as Tatton-Brown-Rahman and ICF syndromes. Additionally, somatic mutations cause common conditions such as osteoarthritis, osteoporosis, clonal hematopoiesis of indeterminate potential (CHIP), hematologic malignancies, and cancer. While DNMTs have been extensively studied in vitro, in early development and in disease, their detailed physiologic roles remain less understood as in vivo investigations are hindered by the embryonic or perinatal lethality of the knockout mice. To circumvent this problem, tissue-specific Dnmt3a and Dnmt3b knockouts were engineered. This review explores their diverse molecular roles across various organs and cell types and characterizes the phenotype of the knockout mice. We provide a comprehensive collection of over forty tissue-specific knockout models generated by cre recombinase. We highlight the distinct functions of DNMT3A and DNMT3B in germ cells, early development, uterus, hematopoietic differentiation, musculoskeletal development, visceral organs, and nervous system. Our findings indicate that DNMT3A primarily regulates hematopoietic differentiation, while DNMT3B is crucial for cartilage homeostasis and ossification. We emphasize the context-dependent roles of DNMT3A and DNMT3B and demonstrate that they also complement DNMT1 maintenance methyltransferase activity. Overall, the expression patterns of DNMTs across tissues provide insights into potential therapeutic applications for treating neurologic diseases, cancer, and osteoporosis.

Single-cell methods to assess DNA methylation have not achieved the same level of cell throughput per experiment compared to other modalities, with large-scale datasets requiring extensive automation, time, and other resources. Here, we describe sciMETv3, a combinatorial indexing-based technique that enables atlas-scale libraries to be produced in a single experiment. To reduce the sequencing burden, we demonstrate the compatibility of sciMETv3 with capture techniques to enrich regulatory regions, as well as the ability to leverage enzymatic conversion, which can yield higher library diversity. We showcase the throughput of sciMETv3 by producing a >140,000 cell library from human middle frontal gyrus split across four multiplexed individuals using both Illumina and Ultima sequencing instrumentation. Finally, we introduce sciMET+ATAC to enable high-throughput exploration of the interplay between chromatin accessibility and DNA methylation within the same cell.

Selective C-H activation on complex biological macromolecules is a key goal in the field of organic chemistry. It requires thermodynamically challenging chemical transformations to be delivered under mild, aqueous conditions. 5-Methylcytosine (5mC) is a fundamentally important epigenetic modification in DNA that has major implications for biology and has emerged as a vital biomarker. Selective functionalisation of 5mC would enable new chemical approaches to tag, detect and map DNA methylation to enhance the study and exploitation of this epigenetic feature. We demonstrate the first example of direct and selective chemical oxidation of 5mC to 5-formylcytosine (5fC) in DNA, employing a photocatalytic system. This transformation was used to selectively tag 5mC. We also provide proof-of-concept for deploying this chemistry for single-base resolution sequencing of 5mC and genetic bases adenine (A), cytosine (C), guanine (G), thymine (T) in DNA on a next-generation sequencing system. This work exemplifies how photocatalysis has the potential to transform the analysis of DNA.

Brain disorders, especially neurodegenerative diseases, affect millions of people worldwide. There is no causal treatment available; therefore, there is an unmet clinical need for finding therapeutic options for these diseases. Epigenetic research has resulted in identification of various genomic loci with differential disease-specific epigenetic modifications, mainly DNA methylation. These biomarkers, although not yet translated into clinically approved options, offer therapeutic targets as epigenetic modifications are reversible. Indeed, clinical trials are designed to inhibit epigenetic writers, erasers, or readers using epigenetic drugs to interfere with epigenetic dysregulation in brain disorders. However, since such drugs elicit genome-wide effects and potentially cause toxicity, the recent developments in the field of epigenetic editing are gaining widespread attention. In this review, we provide examples of epigenetic biomarkers and epi-drugs, while describing efforts in the field of epigenetic editing, to eventually make a difference for the currently incurable brain disorders.

Mutations in methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome. MeCP2 is thought to regulate gene transcription by binding to methylated DNA broadly across the genome. Here, using cleavage under target and release under nuclease (CUT&RUN) assays in the adult mouse cortex, we show that MeCP2 strongly binds to specific gene enhancers that we call MeCP2-binding hotspots (MBHs). Unexpectedly, we find that MeCP2 binding to MBHs occurs in a DNA methylation-independent manner at MBHs. Multiple MBH sites surrounding genes mediate the transcriptional repression of genes enriched for neuronal functions. We show that MBHs regulate genes irrespective of genic methylation levels, suggesting that MeCP2 controls transcription via an intragenic methylation-independent mechanism. Hence, disruption of intragenic methylation-independent gene regulation by MeCP2 may in part underlie Rett syndrome.

Methyl CpG binding protein 2 (MeCP2) is a chromatin-associated protein that remains enigmatic despite more than 30 years of research, primarily due to the ever-growing list of its molecular functions, and, consequently, its related pathologies. Loss of function MECP2 mutations cause the neurodevelopmental disorder Rett syndrome (RTT); in addition, dysregulation of MeCP2 expression and/ or function are involved in numerous other pathologies, but the mechanisms of MeCP2 regulation are unclear. Advancing technologies and burgeoning mechanistic theories assist our understanding of the complexity of MeCP2 but may inadvertently cloud it if not rigorously tested. Here, rather than focus on RTT, we examine relatively underexplored aspects of MeCP2, such as its dosage homeostasis at the gene and protein levels, its controversial participation in phase separation, and its overlooked role in depression and oxidative stress. All these factors may be essential to understanding the full scope of MeCP2 function in healthy and diseased states, but are relatively infrequently studied and require further criticism. The aim of this review is to discuss the esoteric facets of MeCP2 at the molecular and pathological levels and to consider to what extent they may be necessary for general MeCP2 function.

The DNA methylation field has matured from a phase of discovery and genomic characterization to one seeking deeper functional understanding of how this modification contributes to development, ageing and disease. In particular, the past decade has seen many exciting mechanistic discoveries that have substantially expanded our appreciation for how this generic, evolutionarily ancient modification can be incorporated into robust epigenetic codes. Here, we summarize the current understanding of the distinct DNA methylation landscapes that emerge over the mammalian lifespan and discuss how they interact with other regulatory layers to support diverse genomic functions. We then review the rising interest in alternative patterns found during senescence and the somatic transition to cancer. Alongside advancements in single-cell and long-read sequencing technologies, the collective insights made across these fields offer new opportunities to connect the biochemical and genetic features of DNA methylation to cell physiology, developmental potential and phenotype.

Significant links between aging and DNA methylation are emerging from recent studies. On the one hand, DNA methylation undergoes changes with age, a process termed as epigenetic drift. On the other hand, DNA methylation serves as a readily accessible and accurate biomarker for aging. A key missing piece of information, however, is the molecular mechanisms underlying these processes and how they are related, if any. Addressing the limitations of previous research due to the limited number of investigated CpGs and the heterogeneous nature of tissue samples, here, we have examined DNA methylation of over 20 million CpGs across a broad age span in neurons and non-neuronal cells, primarily oligodendrocytes. We show that aging is a primary predictor of DNA methylation variation, surpassing the influence of factors such as sex and schizophrenia diagnosis, among others. On the genome-wide scale, epigenetic drift manifests as significant yet subtle trends that are influenced by the methylation level of individual CpGs. We reveal that CpGs that are highly differentiated between cell types are especially prone to age-associated DNA methylation alterations, leading to the divergence of epigenetic cell type identities as individuals age. On the other hand, CpGs that are included in commonly used epigenetic clocks tend to be those sites that are not highly cell type differentiated. Therefore, dysregulation of epigenetic cell type identities and current DNA epigenetic clocks represent distinct features of age-associated DNA methylation alterations.

Cardiomyocyte (CM) nuclei are constantly exposed to mechanical stress, but how they maintain their nuclear shape remains unknown. In this study, we found that ventricular CM nuclei acquire characteristic prominent spatial organizations of heterochromatin (SOH), which are disrupted by high-level expression of H2B-mCherry in mice. SOH disruption was associated with nuclear softening, leading to extreme elongation and rupture under unidirectional mechanical stress. Loosened chromatin then leaks into the cytosol, causing severe inflammation and cardiac dysfunction. Although SOH disruption was accompanied by loosened higher-order genomic structures, the change in gene expression before nuclear deformation was mild, suggesting that SOH play major roles in nuclear structural integrity. Aged CM nuclei consistently exhibited scattered SOH and marked elongation. Furthermore, we provide mechanistic insight into the development and maintenance of SOH driven by chromatin compaction and condensate formation. These results highlight SOH as a safeguard of nuclear shape and genomic integrity against mechanical stress.

Over the past decade, single-cell genomics technologies have allowed scalable profiling of cell-type-specific features, which has substantially increased our ability to study cellular diversity and transcriptional programs in heterogeneous tissues. Yet our understanding of mechanisms of gene regulation or the rules that govern interactions between cell types is still limited. The advent of new computational pipelines and technologies, such as single-cell epigenomics and spatially resolved transcriptomics, has created opportunities to explore two new axes of biological variation: cell-intrinsic regulation of cell states and expression programs and interactions between cells. Here, we summarize the most promising and robust technologies in these areas, discuss their strengths and limitations and discuss key computational approaches for analysis of these complex datasets. We highlight how data sharing and integration, documentation, visualization and benchmarking of results contribute to transparency, reproducibility, collaboration and democratization in neuroscience, and discuss needs and opportunities for future technology development and analysis.

Epigenetic regulation of the genome is required for cell-type differentiation during organismal development and is especially important to generate the panoply of specialized cell types that comprise the brain. Here, we review how progressive changes in the chromatin landscape, both in neural progenitors and in postmitotic neurons, orchestrate the timing of gene expression programs that underlie first neurogenesis and then functional neuronal maturation. We discuss how disease-associated mutations in chromatin regulators can change brain composition by impairing the timing of neurogenesis. Further, we highlight studies that are beginning to show how chromatin modifications are integrated at the level of chromatin architecture to coordinate changing transcriptional programs across developmental including in postmitotic neurons.

DNA methyltransferase 3A (DNMT3A) plays a critical role in establishing and maintaining DNA methylation patterns in vertebrates. Here we structurally and biochemically explore the interaction of DNMT3A1 with diverse modified nucleosomes indicative of different chromatin environments. A cryo-EM structure of the full-length DNMT3A1-DNMT3L complex with a H2AK119ub nucleosome reveals that the DNMT3A1 ubiquitin-dependent recruitment (UDR) motif interacts specifically with H2AK119ub and makes extensive contacts with the core nucleosome histone surface. This interaction facilitates robust DNMT3A1 binding to nucleosomes, and previously unexplained DNMT3A disease-associated mutations disrupt this interface. Furthermore, the UDR-nucleosome interaction synergises with other DNMT3A chromatin reading elements in the absence of histone ubiquitylation. H2AK119ub does not stimulate DNMT3A DNA methylation activity, as observed for the previously described H3K36me2 mark, which may explain low levels of DNA methylation on H2AK119ub marked facultative heterochromatin. This study highlights the importance of multivalent binding of DNMT3A to histone modifications and the nucleosome surface and increases our understanding of how DNMT3A1 chromatin recruitment occurs.

Brain development and neuronal cell specification are accompanied with epigenetic changes to achieve diverse gene expression regulation. Interacting with cell-type specific epigenetic marks, transcription factors bind to different sets of cis-regulatory elements in different types of cells. Currently, it remains largely unclear how cell-type specific gene regulation is achieved for neurons. In this study, we generated epigenetic maps to perform comparative histone modification analysis between excitatory and inhibitory neurons. We found that neuronal cell-type specific histone modifications are enriched in super enhancer regions containing abundant EGR1 motifs. Further CUT&RUN data validated that more EGR1 binding sites can be detected in excitatory neurons and primarily located in enhancers. Integrative analysis revealed that EGR1 binding is strongly correlated with various epigenetic markers for open chromatin regions and associated with distinct gene pathways with neuronal subtype-specific functions. In inhibitory neurons, the majority of genomic regions hosting EGR1 binding sites become accessible at early embryonic stages. In contrast, the super enhancers in excitatory neurons hosting EGR1 binding sites gained their accessibility during postnatal stages. This study highlights the significance of transcription factor binding to enhancer regions, which may play a crucial role in establishing cell-type specific gene regulation in neurons.

Background: Neuronal loss is a major pathological feature of neurodegenerative diseases. The analysis of plasma cell-free DNA (cfDNA) is an emerging approach to track cell death events in a minimally invasive way and from inaccessible areas of the body, such as the brain. Previous studies showed that DNA methylation (DNAm) profiles can be used to map the tissue of origin of cfDNA and to identify molecules released from the brain upon cell death. The aim of the present study is to contribute to this research field, presenting the development and validation of an assay for the detection of brain-derived cfDNA (bcfDNA). Methods: To identify CpG sites with brain-specific DNAm, we compared brain and non-brain tissues for their chromatin state profiles and genome-wide DNAm data, available in public datasets. The selected target genomic regions were experimentally validated by bisulfite sequencing on DNA extracted from 44 different autoptic tissues, including multiple brain regions. Sequencing data were analysed to identify brain-specific epihaplotypes. The developed assay was tested in plasma cfDNA from patients with immune effector cell-associated neurotoxicity syndrome (ICANS) following chimeric antigen receptor T (CAR-T) therapy. Results: We validated five genomic regions with brain-specific DNAm (four hypomethylated and one hypermethylated in the brain). DNAm analysis of the selected genomic regions in plasma samples from CAR-T patients revealed higher levels of bcfDNA in participants with ongoing neurotoxicity syndrome. Conclusions: We developed an assay for the analysis of bcfDNA in plasma. The assay is a promising tool for the early detection of neuronal loss in neurodegenerative diseases.

Multiple studies have demonstrated the significant effects of physical exercise on brain plasticity, the enhancement of memory and cognition, and mood improvement. Although the beneficial impact of exercise on brain functions and mental health is well established, the exact mechanisms underlying this phenomenon are currently under thorough investigation. Several hypotheses have emerged suggesting various possible mechanisms, including the effects of hormones, neurotrophins, neurotransmitters, and more recently also other compounds such as lactate or irisin, which are released under the exercise circumstances and act both locally or/and on distant tissues, triggering systemic body reactions. Nevertheless, none of these actually explain the long-lasting effect of exercise, which can persist for years or even be passed on to subsequent generations. It is believed that these long-lasting effects are mediated through epigenetic modifications, influencing the expression of particular genes and the translation and modification of specific proteins. This review explores the impact of regular physical exercise on brain function and brain plasticity and the associated occurrence of epigenetic modifications. It examines how these changes contribute to the prevention and treatment of neuropsychiatric and neurological disorders, as well as their influence on the natural aging process and mental health.

PHF21A is a histone-binding protein that recognizes unmethylated histone H3K4, the reaction product of LSD1 histone demethylase. PHF21A and LSD1 form a complex, and both undergo neuron-specific microexon splicing. The PHF21A neuronal microexon interferes with nucleosome binding, whereas the LSD1 neuronal microexon weakens H3K4 demethylation activity and can alter the substrate specificity to H3K9 or H4K20. However, the temporal expression patterns of PHF21A and LSD1 splicing isoforms during brain development and their biological roles remain unknown. In this work, we report that neuronal PHF21A isoform expression precedes neuronal LSD1 expression during human neuron differentiation and mouse brain development. The asynchronous splicing events resulted in stepwise deactivation of the LSD1-PHF21A complex in reversing H3K4 methylation. An unbiased proteomics survey revealed that the enzymatically inactive LSD1-PHF21A complex interacts with neuron-specific binding partners, including MYT1-family transcription factors and post-transcriptional mRNA processing proteins such as VIRMA. The interaction with the neuron-specific components, however, did not require the PHF21A microexon, indicating that the neuronal proteomic milieu, rather than the microexon-encoded PHF21A segment, is responsible for neuron-specific complex formation. Finally, by using two Phf21a mutant mouse models, we found that Phf21a neuronal splicing prevents excess synapse formation that otherwise would occur when canonical PHF21A is expressed in neurons. These results suggest that the role of the PHF21A microexon is to dampen LSD1-mediated H3K4 demethylation, thereby containing aberrant synaptogenesis.

Comprehensive maps of neuronal connectivity provide a foundation for understanding the structure of neural circuits. In a circuit, neurons are diverse in morphology, electrophysiology, gene expression, activity, and other neuronal properties. Thus, constructing a comprehensive connectivity map requires associating various properties of neurons, including their connectivity, at cellular resolution. A commonly used approach is to use the gene expression profiles as an anchor to which all other neuronal properties are associated. Recent advances in genomics and anatomical techniques dramatically improved the ability to determine and associate the long-range projections of neurons with their gene expression profiles. These studies revealed unprecedented details of the gene-projection relationship, but also highlighted conceptual challenges in understanding this relationship. In this article, I delve into the findings and the challenges revealed by recent studies using state-of-the-art neuroanatomical and transcriptomic techniques. Building upon these insights, I propose an approach that focuses on understanding the gene-projection relationship through basic features in gene expression profiles and projections, respectively, that associate with underlying cellular processes. I then discuss how the developmental trajectories of projections and gene expression profiles create additional challenges and necessitate interrogating the gene-projection relationship across time. Finally, I explore complementary strategies that, together, can provide a comprehensive view of the gene-projection relationship.

The human hippocampus and prefrontal cortex play critical roles in learning and cognition1,2, yet the dynamic molecular characteristics of their development remain enigmatic. Here we investigated the epigenomic and three-dimensional chromatin conformational reorganization during the development of the hippocampus and prefrontal cortex, using more than 53,000 joint single-nucleus profiles of chromatin conformation and DNA methylation generated by single-nucleus methyl-3C sequencing (snm3C-seq3)3. The remodelling of DNA methylation is temporally separated from chromatin conformation dynamics. Using single-cell profiling and multimodal single-molecule imaging approaches, we have found that short-range chromatin interactions are enriched in neurons, whereas long-range interactions are enriched in glial cells and non-brain tissues. We reconstructed the regulatory programs of cell-type development and differentiation, finding putatively causal common variants for schizophrenia strongly overlapping with chromatin loop-connected, cell-type-specific regulatory regions. Our data provide multimodal resources for studying gene regulatory dynamics in brain development and demonstrate that single-cell three-dimensional multi-omics is a powerful approach for dissecting neuropsychiatric risk loci.