Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome.

Short tandem repeat (STR) instability causes transcriptional silencing in several repeat expansion disorders. In fragile X syndrome (FXS), mutation-length expansion of a CGG STR represses FMR1 via local DNA methylation. Here, we find megabase-scale H3K9me3 domains on autosomes and encompassing FMR1 on the X chromosome in FXS patient-derived iPSCs, iPSC-derived neural progenitors, EBV-transformed lymphoblasts, and brain tissue with mutation-length CGG expansion. H3K9me3 domains connect via inter-chromosomal interactions and demarcate severe misfolding of TADs and loops. They harbor long synaptic genes replicating at the end of S phase, replication-stress-induced double-strand breaks, and STRs prone to stepwise somatic instability. CRISPR engineering of the mutation-length CGG to premutation length reverses H3K9me3 on the X chromosome and multiple autosomes, refolds TADs, and restores gene expression. H3K9me3 domains can also arise in normal-length iPSCs created with perturbations linked to genome instability, suggesting their relevance beyond FXS. Our results reveal Mb-scale heterochromatinization and trans interactions among loci susceptible to instability.

3D genome, on repeat: Higher-order folding principles of the heterochromatinized repetitive genome.

Nearly half of the human genome is comprised of diverse repetitive sequences ranging from satellite repeats to retrotransposable elements. Such sequences are susceptible to stepwise expansions, duplications, inversions, and recombination events which can compromise genome function. In this review, we discuss the higher-order folding mechanisms of compartmentalization and loop extrusion and how they shape, and are shaped by, heterochromatin. Using primarily mammalian model systems, we contrast mechanisms governing H3K9me3-mediated heterochromatinization of the repetitive genome and highlight emerging links between repetitive elements and chromatin folding.

Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence.

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate epigenetic states and subsequent gene expression. This metabolism-epigenome link sensitizes chromatin methylation to altered SAM abundance, yet the mechanisms that allow organisms to adapt and protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear mono-methylation of H3 Lys 9 (H3K9) at the expense of broad losses in histone di- and tri-methylation. Under SAM-depleted conditions, H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies provide evidence for an adaptive response that enables epigenetic persistence to metabolic stress.

Metabolism and the Epigenome: A Dynamic Relationship.

Many chromatin-modifying enzymes require metabolic cofactors to support their catalytic activities, providing a direct path for fluctuations in metabolite availability to regulate the epigenome. Over the past decade, our knowledge of this link has grown significantly. What began with studies showing that cofactor availability drives global abundances of chromatin modifications has transitioned to discoveries highlighting metabolic enzymes as loci-specific regulators of gene expression. Here, we cover our current understanding of mechanisms that facilitate the dynamic and complex relationship between metabolism and the epigenome, focusing on the roles of essential metabolic and chromatin associated enzymes. We discuss physiological conditions where availability of these epimetabolites is dynamically altered, highlighting known links to the epigenome and proposing other plausible connections.

Intrinsic catalytic properties of histone H3 lysine-9 methyltransferases preserve monomethylation levels under low S-adenosylmethionine.

S-adenosylmethionine (SAM) is the methyl donor for site-specific methylation reactions on histone proteins, imparting key epigenetic information. During SAM-depleted conditions that can arise from dietary methionine restriction, lysine di- and tri-methylation are reduced while sites such as Histone-3 lysine-9 (H3K9) are actively maintained, allowing cells to restore higher-state methylation upon metabolic recovery. Here, we investigated if the intrinsic catalytic properties of H3K9 histone methyltransferases (HMTs) contribute to this epigenetic persistence. We employed systematic kinetic analyses and substrate binding assays using four recombinant H3K9 HMTs (i.e., EHMT1, EHMT2, SUV39H1, and SUV39H2). At both high and low (i.e., sub-saturating) SAM, all HMTs displayed the highest catalytic efficiency (kcat/KM) for monomethylation compared to di- and trimethylation on H3 peptide substrates. The favored monomethylation reaction was also reflected in kcat values, apart from SUV39H2 which displayed a similar kcat regardless of substrate methylation state. Using differentially methylated nucleosomes as substrates, kinetic analyses of EHMT1 and EHMT2 revealed similar catalytic preferences. Orthogonal binding assays revealed only small differences in substrate affinity across methylation states, suggesting that catalytic steps dictate the monomethylation preferences of EHMT1, EHMT2, and SUV39H1. To link in vitro catalytic rates with nuclear methylation dynamics, we built a mathematical model incorporating measured kinetic parameters and a time course of mass spectrometry-based H3K9 methylation measurements following cellular SAM depletion. The model revealed that the intrinsic kinetic constants of the catalytic domains could recapitulate in vivo observations. Together, these results suggest catalytic discrimination by H3K9 HMTs maintains nuclear H3K9me1, ensuring epigenetic persistence after metabolic stress.

A practical guide for analysis of histone post-translational modifications by mass spectrometry: Best practices and pitfalls.

Advances in mass spectrometry (MS) have revolutionized the ability to measure global changes in histone post-translational modifications (PTMs). The method routinely quantifies over 60 modification states in a single sample, far exceeding the capabilities of traditional western blotting. Thus, MS-based histone analysis has become an increasingly popular tool for understanding how genetic and environmental factors influence epigenetic states. However, histone proteomics experiments exhibit unique challenges, such as batch-to-batch reproducibility, accurate peak integration, and noisy data. Here, we discuss the steps of histone PTM analysis, from sample preparation and peak integration to data analysis and validation. We outline a set of best practices for ensuring data quality, accurate normalization, and robust statistics. Using these practices, we quantify histone modifications in 5 human cell lines, revealing that each cell line exhibits a unique epigenetic signature. We also provide a reproducible workflow for histone PTM analysis in the form of an R script, which is freely available at https://github.com/DenuLab/HistoneAnalysisWorkflow.

General method for rapid purification of native chromatin fragments.

Biochemical, proteomic, and epigenetic studies of chromatin rely on the ability to efficiently isolate native nucleosomes in high yield and purity. However, isolation of native chromatin suitable for many downstream experiments remains a challenging task. This is especially true for the budding yeast Saccharomyces cerevisiae, which continues to serve as an important model organism for the study of chromatin structure and function. Here, we developed a time- and cost-efficient universal protocol for isolation of native chromatin fragments from yeast, insect, and mammalian cells. The resulting protocol preserves histone posttranslational modification in the native chromatin state and is applicable for both parallel multisample spin-column purification and large-scale isolation. This protocol is based on the efficient and stable purification of polynucleosomes and features a combination of optimized cell lysis and purification conditions, three options for chromatin fragmentation, and a novel ion-exchange chromatographic purification strategy. The procedure will aid chromatin researchers interested in isolating native chromatin material for biochemical studies and serve as a mild, acid- and detergent-free sample preparation method for MS analysis.

DOT1L is a barrier to histone acetylation during reprogramming to pluripotency.

Embryonic stem cells (ESCs) have transcriptionally permissive chromatin enriched for gene activation-associated histone modifications. A striking exception is DOT1L-mediated H3K79 dimethylation (H3K79me2) that is considered a positive regulator of transcription. We find that ESCs are depleted for H3K79me2 at shared locations of enrichment with somatic cells, which are highly and ubiquitously expressed housekeeping genes, and have lower RNA polymerase II (RNAPII) at the transcription start site (TSS) despite greater nascent transcription. Inhibiting DOT1L increases the efficiency of reprogramming of somatic to induced pluripotent stem cells, enables an ESC-like RNAPII pattern at the TSS, and functionally compensates for enforced RNAPII pausing. DOT1L inhibition increases H3K27 methylation and RNAPII elongation-enhancing histone acetylation without changing the expression of the causal histone-modifying enzymes. Only the maintenance of elevated histone acetylation is essential for enhanced reprogramming and occurs at loci that are depleted for H3K79me2. Thus, DOT1L inhibition promotes the hyperacetylation and hypertranscription pluripotent properties.

Dietary restriction of individual amino acids stimulates unique molecular responses in mouse liver.

Dietary protein and essential amino acid (EAA) restriction promotes favorable metabolic reprogramming, ultimately resulting in improvements to both health and lifespan. However, as individual EAAs have distinct catabolites and engage diverse downstream signaling pathways, it remains unclear to what extent shared or AA-specific molecular mechanisms promote diet-associated phenotypes. Here, we investigated the physiological and molecular effects of restricting either dietary methionine, leucine, or isoleucine (Met-R, Leu-R, and Ile-R) for 3 weeks in C57BL/6J male mice. While all 3 AA-depleted diets promoted fat and lean mass loss and slightly improved glucose tolerance, the molecular responses were more diverse; while hepatic metabolites altered by Met-R and Leu-R were highly similar, Ile-R led to dramatic changes in metabolites, including a 3-fold reduction in the oncometabolite 2-hydroxyglutarate. Pathways regulated in an EAA-specific manner included glycolysis, the pentose phosphate pathway (PPP), nucleotide metabolism, the TCA cycle and amino acid metabolism. Transcriptiome analysis and global profiling of histone post-translational modifications (PTMs) revealed different patterns of responses to each diet, although Met-R and Leu-R again shared similar transcriptional responses. While the pattern of global histone PTMs were largely unique for each dietary intervention, Met-R and Ile-R had similar changes in histone-3 methylation/acetylation PTMs at lysine-9. Few similarities were observed between the physiological or molecular responses to EAA restriction and treatment with rapamycin, an inhibitor of the mTORC1 AA-responsive protein kinase, indicating the response to EAA restriction may be largely independent of mTORC1. Together, these results demonstrate that dietary restriction of individual EAAs has unique, EAA-specific effects on the hepatic metabolome, epigenome, and transcriptome, and suggests that the specific EAAs present in dietary protein may play a key role at regulating health at the molecular level.

Fasting drives the metabolic, molecular and geroprotective effects of a calorie-restricted diet in mice.

Calorie restriction (CR) promotes healthy ageing in diverse species. Recently, it has been shown that fasting for a portion of each day has metabolic benefits and promotes lifespan. These findings complicate the interpretation of rodent CR studies, in which animals typically eat only once per day and rapidly consume their food, which collaterally imposes fasting. Here we show that a prolonged fast is necessary for key metabolic, molecular and geroprotective effects of a CR diet. Using a series of feeding regimens, we dissect the effects of calories and fasting, and proceed to demonstrate that fasting alone recapitulates many of the physiological and molecular effects of CR. Our results shed new light on how both when and how much we eat regulate metabolic health and longevity, and demonstrate that daily prolonged fasting, and not solely reduced caloric intake, is likely responsible for the metabolic and geroprotective benefits of a CR diet.

PGC-1a integrates a metabolism and growth network linked to caloric restriction.

Deleterious changes in energy metabolism have been linked to aging and disease vulnerability, while activation of mitochondrial pathways has been linked to delayed aging by caloric restriction (CR). The basis for these associations is poorly understood, and the scope of impact of mitochondrial activation on cellular function has yet to be defined. Here, we show that mitochondrial regulator PGC-1a is induced by CR in multiple tissues, and at the cellular level, CR-like activation of PGC-1a impacts a network that integrates mitochondrial status with metabolism and growth parameters. Transcriptional profiling reveals that diverse functions, including immune pathways, growth, structure, and macromolecule homeostasis, are responsive to PGC-1a. Mechanistically, these changes in gene expression were linked to chromatin remodeling and RNA processing. Metabolic changes implicated in the transcriptional data were confirmed functionally including shifts in NAD metabolism, lipid metabolism, and membrane lipid composition. Delayed cellular proliferation, altered cytoskeleton, and attenuated growth signaling through post-transcriptional and post-translational mechanisms were also identified as outcomes of PGC-1a-directed mitochondrial activation. Furthermore, in vivo in tissues from a genetically heterogeneous mouse population, endogenous PGC-1a expression was correlated with this same metabolism and growth network. These data show that small changes in metabolism have broad consequences that arguably would profoundly alter cell function. We suggest that this PGC-1a sensitive network may be the basis for the association between mitochondrial function and aging where small deficiencies precipitate loss of function across a spectrum of cellular activities.