Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism.

One-carbon metabolism is an essential branch of cellular metabolism that intersects with epigenetic regulation. In this work, we show how formaldehyde (FA), a one-carbon unit derived from both endogenous sources and environmental exposure, regulates one-carbon metabolism by inhibiting the biosynthesis of S-adenosylmethionine (SAM), the major methyl donor in cells. FA reacts with privileged, hyperreactive cysteine sites in the proteome, including Cys120 in S-adenosylmethionine synthase isoform type-1 (MAT1A). FA exposure inhibited MAT1A activity and decreased SAM production with MAT-isoform specificity. A genetic mouse model of chronic FA overload showed a decrease n SAM and in methylation on selected histones and genes. Epigenetic and transcriptional regulation of Mat1a and related genes function as compensatory mechanisms for FA-dependent SAM depletion, revealing a biochemical feedback cycle between FA and SAM one-carbon units.

Connecting copper and cancer: from transition metal signalling to metalloplasia.

Copper is an essential nutrient whose redox properties make it both beneficial and toxic to the cell. Recent progress in studying transition metal signalling has forged new links between researchers of different disciplines that can help translate basic research in the chemistry and biology of copper into clinical therapies and diagnostics to exploit copper-dependent disease vulnerabilities. This concept is particularly relevant in cancer, as tumour growth and metastasis have a heightened requirement for this metal nutrient. Indeed, the traditional view of copper as solely an active site metabolic cofactor has been challenged by emerging evidence that copper is also a dynamic signalling metal and metalloallosteric regulator, such as for copper-dependent phosphodiesterase 3B (PDE3B) in lipolysis, mitogen-activated protein kinase kinase 1 (MEK1) and MEK2 in cell growth and proliferation and the kinases ULK1 and ULK2 in autophagy. In this Perspective, we summarize our current understanding of the connection between copper and cancer and explore how challenges in the field could be addressed by using the framework of cuproplasia, which is defined as regulated copper-dependent cell proliferation and is a representative example of a broad range of metalloplasias. Cuproplasia is linked to a diverse array of cellular processes, including mitochondrial respiration, antioxidant defence, redox signalling, kinase signalling, autophagy and protein quality control. Identifying and characterizing new modes of copper-dependent signalling offers translational opportunities that leverage disease vulnerabilities to this metal nutrient.

Chromatin landscape signals differentially dictate the activities of mSWI/SNF family complexes.

Mammalian SWI/SNF (mSWI/SNF) adenosine triphosphate-dependent chromatin remodelers modulate genomic architecture and gene expression and are frequently mutated in disease. However, the specific chromatin features that govern their nucleosome binding and remodeling activities remain unknown. We subjected endogenously purified mSWI/SNF complexes and their constituent assembly modules to a diverse library of DNA-barcoded mononucleosomes, performing more than 25,000 binding and remodeling measurements. Here, we define histone modification-, variant-, and mutation-specific effects, alone and in combination, on mSWI/SNF activities and chromatin interactions. Further, we identify the combinatorial contributions of complex module components, reader domains, and nucleosome engagement properties to the localization of complexes to selectively permissive chromatin states. These findings uncover principles that shape the genomic binding and activity of a major chromatin remodeler complex family.

Single-molecule and in silico dissection of the interaction between Polycomb repressive complex 2 and chromatin.

Polycomb repressive complex 2 (PRC2) installs and spreads repressive histone methylation marks on eukaryotic chromosomes. Because of the key roles that PRC2 plays in development and disease, how this epigenetic machinery interacts with DNA and nucleosomes is of major interest. Nonetheless, the mechanism by which PRC2 engages with native-like chromatin remains incompletely understood. In this work, we employ single-molecule force spectroscopy and molecular dynamics simulations to dissect the behavior of PRC2 on polynucleosome arrays. Our results reveal an unexpectedly diverse repertoire of PRC2 binding configurations on chromatin. Besides reproducing known binding modes in which PRC2 interacts with bare DNA, mononucleosomes, and adjacent nucleosome pairs, our data also provide direct evidence that PRC2 can bridge pairs of distal nucleosomes. In particular, the "1-3" bridging mode, in which PRC2 engages two nucleosomes separated by one spacer nucleosome, is a preferred low-energy configuration. Moreover, we show that the distribution and stability of different PRC2-chromatin interaction modes are modulated by accessory subunits, oncogenic histone mutations, and the methylation state of chromatin. Overall, these findings have implications for the mechanism by which PRC2 spreads histone modifications and compacts chromatin. The experimental and computational platforms developed here provide a framework for understanding the molecular basis of epigenetic maintenance mediated by Polycomb-group proteins.

Distinct RNA N-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates.

The AlkB family of nonheme Fe(II)/2-oxoglutarate-dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases of N6-methyladenosine (m6A) but furnish different major products, N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a direct m6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for nonredundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems.

PRC2 engages a bivalent H3K27M-H3K27me3 dinucleosome inhibitor.

A lysine-to-methionine mutation at lysine 27 of histone 3 (H3K27M) has been shown to promote oncogenesis in a subset of pediatric gliomas. While there is evidence that this "oncohistone" mutation acts by inhibiting the histone methyltransferase PRC2, the details of this proposed mechanism nevertheless continue to be debated. Recent evidence suggests that PRC2 must simultaneously bind both H3K27M and H3K27me3 to experience competitive inhibition of its methyltransferase activity. In this work, we used PRC2 inhibitor treatments in a transgenic H3K27M cell line to validate this dependence in a cellular context. We further used designer chromatin inhibitors to probe the geometric constraints of PRC2 engagement of H3K27M and H3K27me3 in a biochemical setting. We found that PRC2 binds to a bivalent inhibitor unit consisting of an H3K27M and an H3K27me3 nucleosome and exhibits a distance dependence in its affinity for such an inhibitor, which favors closer proximity of the 2 nucleosomes within a chromatin array. Together, our data precisely delineate fundamental aspects of the H3K27M inhibitor and support a model wherein PRC2 becomes trapped at H3K27M-H3K27me3 boundaries.

Nucleation and Propagation of Heterochromatin by the Histone Methyltransferase PRC2: Geometric Constraints and Impact of the Regulatory Subunit JARID2.

Polycomb Repressive Complex 2 (PRC2) catalyzes mono-, di-, and trimethylation of lysine 27 on histone H3 (H3K27me1-3) to control expression of genes important for differentiation and maintenance of cell identity. PRC2 activity is regulated by a number of different inputs, including allosteric activation by its product, H3K27me3. This positive feedback loop is thought to be important for the establishment of large domains of condensed heterochromatin. In addition to other chromatin modifications, ancillary subunits of PRC2, foremost JARID2, affect the rate of H3K27 methylation. Many gaps remain in our understanding of how PRC2 integrates these various signals to determine where and when to deposit H3K27 methyl marks. In this study, we utilize designer chromatin substrates to demonstrate that propagation of H3K27 methylation by the PRC2 core complex has geometrically defined preferences that are overridden by the presence of JARID2. Our studies also show that phosphorylation of JARID2 can partially regulate its ability to stimulate PRC2 activity. Collectively, these biochemical insights further our understanding of the mechanisms that govern PRC2 activity, and highlight a role for JARID2 in de novo deposition of H3K27me3-containing repressive domains.

Histone H3 tail binds a unique sensing pocket in EZH2 to activate the PRC2 methyltransferase.

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressor Complex 2 (PRC2), the enzyme that catalyzes monomethylation, dimethylation, and trimethylation of lysine 27 on histone H3 (H3K27). Trimethylation at H3K27 (H3K27me3) is associated with transcriptional silencing of developmentally important genes. Intriguingly, H3K27me3 is mutually exclusive with H3K36 trimethylation on the same histone tail. Disruptions in this cross-talk result in aberrant H3K27/H3K36 methylation patterns and altered transcriptional profiles that have been implicated in tumorigenesis and other disease states. Despite their importance, the molecular details of how PRC2 "senses" H3K36 methylation are unclear. We demonstrate that PRC2 is activated in cis by the unmodified side chain of H3K36, and that this activation results in a fivefold increase in the kcat of its enzymatic activity catalyzing H3K27 methylation compared with activity on a substrate methylated at H3K36. Using a photo-cross-linking MS strategy and histone methyltransferase activity assays on PRC2 mutants, we find that EZH2 contains a specific sensing pocket for the H3K36 methylation state that allows the complex to distinguish between modified and unmodified H3K36 residues, altering enzymatic activity accordingly to preferentially methylate the unmodified nucleosome substrate. We also present evidence that this process may be disrupted in some cases of Weaver syndrome.

Molecular analysis of PRC2 recruitment to DNA in chromatin and its inhibition by RNA.

Many studies have revealed pathways of epigenetic gene silencing by Polycomb repressive complex 2 (PRC2) in vivo, but understanding the underlying molecular mechanisms requires biochemistry. Here we analyze interactions of reconstituted human PRC2 with nucleosome complexes. Histone modifications, the H3K27M cancer mutation, and inclusion of JARID2 or EZH1 in the PRC2 complex have unexpectedly minor effects on PRC2-nucleosome binding. Instead, protein-free linker DNA dominates the PRC2-nucleosome interaction. Specificity for CG-rich sequences is consistent with PRC2 occupying CG-rich DNA in vivo. PRC2 preferentially binds methylated DNA regulated by its AEBP2 subunit, suggesting how DNA and histone methylation collaborate to repress chromatin. We find that RNA, known to inhibit PRC2 activity, is not a methyltransferase inhibitor per se. Instead, RNA sequesters PRC2 from nucleosome substrates, because PRC2 binding requires linker DNA, and RNA and DNA binding are mutually exclusive. Together, we provide a model for PRC2 recruitment and an explanation for how actively transcribed genomic regions bind PRC2 but escape silencing.

Bisphosphoglycerate mutase controls serine pathway flux via 3-phosphoglycerate.

Lower glycolysis involves a series of reversible reactions, which interconvert intermediates that also feed anabolic pathways. 3-phosphoglycerate (3-PG) is an abundant lower glycolytic intermediate that feeds serine biosynthesis via the enzyme phosphoglycerate dehydrogenase, which is genomically amplified in several cancers. Phosphoglycerate mutase 1 (PGAM1) catalyzes the isomerization of 3-PG into the downstream glycolytic intermediate 2-phosphoglycerate (2-PG). PGAM1 needs to be histidine phosphorylated to become catalytically active. We show that the primary PGAM1 histidine phosphate donor is 2,3-bisphosphoglycerate (2,3-BPG), which is made from the glycolytic intermediate 1,3-bisphosphoglycerate (1,3-BPG) by bisphosphoglycerate mutase (BPGM). When BPGM is knocked out, 1,3-BPG can directly phosphorylate PGAM1. In this case, PGAM1 phosphorylation and activity are decreased, but nevertheless sufficient to maintain normal glycolytic flux and cellular growth rate. 3-PG, however, accumulates, leading to increased serine synthesis. Thus, one biological function of BPGM is controlling glycolytic intermediate levels and thereby serine biosynthetic flux.

Stimulation of ipt overexpression as a tool to elucidate the role of cytokinins in high temperature responses of Arabidopsis thaliana.

Cytokinins (CKs) are phytohormones regulating plant growth and development as well as response to the environment. In order to evaluate their function in heat stress (HS) responses, the effect of CK elevation was determined during three types of HS - targeted to shoots, targeted to roots and applied to the whole plant. The early (30min) and longer term (3h) responses were followed at the hormonal, transcriptomic and proteomic levels in Arabidopsis transformants with dexamethasone-inducible expression of the CK biosynthetic gene isopentenyltransferase (ipt) and the corresponding wild-type (Col-0). Combination of hormonal and phenotypic analyses showed transient up-regulation of the CK/abscisic acid ratio, which controls stomatal aperture, to be more pronounced in the transformant. HS responses of the root proteome and Rubisco-immunodepleted leaf proteome were followed using 2-D gel electrophoresis and MALDI-TOF/TOF. More than 100 HS-responsive proteins were detected, most of them being modulated by CK increase. Proteome and transcriptome analyses demonstrated that CKs have longer term positive effects on the stress-related proteins and transcripts, as well as on the photosynthesis-related ones. Transient accumulation of CKs and stimulation of their signal transduction in tissue(s) not exposed to HS indicate that they are involved in plant stress responses.

Temporally controlled targeting of 4-hydroxynonenal to specific proteins in living cells.

In-depth chemical understanding of complex biological processes hinges upon the ability to systematically perturb individual systems. However, current approaches to study impacts of biologically relevant reactive small molecules involve bathing of the entire cell or isolated organelle with excess amounts, leading to off-target effects. The resultant lack of biochemical specificity has plagued our understanding of how biological electrophiles mediate signal transduction or regulate responses that confer defense mechanisms to cellular electrophilic stress. Here we introduce a target-specific electrophile delivery platform that will ultimately pave the way to interrogate effects of reactive electrophiles on specific target proteins in cells. The new methodology is demonstrated by photoinducible targeted delivery of 4-hydroxynonenal (HNE) to the proteins Keap1 and PTEN. Covalent conjugation of the HNE-precursor to HaloTag fused to the target proteins enables directed HNE delivery upon photoactivation. The strategy provides proof of concept of selective delivery of reactive electrophiles to individual electrophile-responsive proteins in mammalian cells. It opens a new avenue enabling more precise determination of the pathophysiological consequences of HNE-induced chemical modifications on specific target proteins in cells.

Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression.

Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.

SIRT6 regulates TNF-α secretion through hydrolysis of long-chain fatty acyl lysine.

The Sir2 family of enzymes or sirtuins are known as nicotinamide adenine dinucleotide (NAD)-dependent deacetylases and have been implicated in the regulation of transcription, genome stability, metabolism and lifespan. However, four of the seven mammalian sirtuins have very weak deacetylase activity in vitro. Here we show that human SIRT6 efficiently removes long-chain fatty acyl groups, such as myristoyl, from lysine residues. The crystal structure of SIRT6 reveals a large hydrophobic pocket that can accommodate long-chain fatty acyl groups. We demonstrate further that SIRT6 promotes the secretion of tumour necrosis factor-α (TNF-α) by removing the fatty acyl modification on K19 and K20 of TNF-α. Protein lysine fatty acylation has been known to occur in mammalian cells, but the function and regulatory mechanisms of this modification were unknown. Our data indicate that protein lysine fatty acylation is a novel mechanism that regulates protein secretion. The discovery of SIRT6 as an enzyme that controls protein lysine fatty acylation provides new opportunities to investigate the physiological function of a protein post-translational modification that has been little studied until now.