Metabolic sinkholes: Histones as methyl repositories.

Perez and Sarkies uncover histones as methyl group repositories in normal and cancer human cells, shedding light on an intriguing function of histone methylation in optimizing the cellular methylation potential independently of gene regulation.

The role of histone H3 leucine 126 in fine-tuning the copper reductase activity of nucleosomes.

The copper reductase activity of histone H3 suggests undiscovered characteristics within the protein. Here, we investigated the function of leucine 126 (H3L126), which occupies an axial position relative to the copper binding. Typically found as methionine or leucine in copper-binding proteins, the axial ligand influences the reduction potential of the bound ion, modulating its tendency to accept or yield electrons. We found that mutation of H3L126 to methionine (H3L126M) enhanced the enzymatic activity of native yeast nucleosomes in vitro and increased intracellular levels of Cu1+, leading to improved copper-dependent activities including mitochondrial respiration and growth in oxidative media with low copper. Conversely, H3L126 to histidine (H3L126H) mutation decreased nucleosome's enzymatic activity and adversely affected copper-dependent activities in vivo. Our findings demonstrate that H3L126 fine-tunes the copper reductase activity of nucleosomes and highlights the utility of nucleosome enzymatic activity as a novel paradigm to uncover previously unnoticed features of histones.

Histone acetylation regulates intracellular pH.

Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pH(i)). As pH(i) decreases, histones are globally deacetylated by histone deacetylases (HDACs), and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pH(i). Conversely, global histone acetylation increases as pH(i) rises, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pH(i), particularly compromising pH(i) maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus, acetylation of chromatin functions as a rheostat to regulate pH(i) with important implications for mechanism of action and therapeutic use of HDAC inhibitors.

Endoplasmic reticulum-mitochondria junction is required for iron homeostasis.

The endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) is a protein complex that physically tethers the two organelles to each other and creates the physical basis for communication between them. ERMES functions in lipid exchange between the ER and mitochondria, protein import into mitochondria, and maintenance of mitochondrial morphology and genome. Here, we report that ERMES is also required for iron homeostasis. Loss of ERMES components activates an Aft1-dependent iron deficiency response even in iron-replete conditions, leading to accumulation of excess iron inside the cell. This function is independent of known ERMES roles in calcium regulation, phospholipid biosynthesis, or effects on mitochondrial morphology. A mutation in the vacuolar protein sorting 13 (VPS13) gene that rescues the glycolytic phenotype of ERMES mutants suppresses the iron deficiency response and iron accumulation. Our findings reveal that proper communication between the ER and mitochondria is required for appropriate maintenance of cellular iron levels.

A ncRNA modulates histone modification and mRNA induction in the yeast GAL gene cluster.

The extensively studied yeast GAL1-10 gene cluster is tightly regulated by environmental sugar availability. Unexpectedly, under repressive conditions the 3' region of the GAL10 coding sequence is trimethylated by Set1 on histone H3 K4, normally characteristic of 5' regions of actively transcribed genes. This reflects transcription of a long noncoding RNA (GAL10-ncRNA) that is reciprocal to GAL1 and GAL10 mRNAs and driven by the DNA-binding protein Reb1. Point mutations in predicted Reb1-binding sites abolished Reb1 binding and ncRNA synthesis. The GAL10-ncRNA is transcribed approximately once every 50 min and targeted for degradation by the TRAMP and exosome complexes, resulting in low steady-state levels (approximately one molecule per 14 cells). GAL10-ncRNA transcription recruits the methyltransferase Set2 and histone deacetylation activities in cis, leading to stable changes in chromatin structure. These chromatin modifications act principally through the Rpd3S complex to aid glucose repression of GAL1-10 at physiologically relevant sugar concentrations.

Stimulation of histone deacetylase activity by metabolites of intermediary metabolism.

Histone deacetylases (HDACs) function in a wide range of molecular processes, including gene expression, and are of significant interest as therapeutic targets. Although their native complexes, subcellular localization, and recruitment mechanisms to chromatin have been extensively studied, much less is known about whether the enzymatic activity of non-sirtuin HDACs can be regulated by natural metabolites. Here, we show that several coenzyme A (CoA) derivatives, such as acetyl-CoA, butyryl-CoA, HMG-CoA, and malonyl-CoA, as well as NADPH but not NADP(+), NADH, or NAD(+), act as allosteric activators of recombinant HDAC1 and HDAC2 in vitro following a mixed activation kinetic. In contrast, free CoA, like unconjugated butyrate, inhibits HDAC activity in vitro. Analysis of a large number of engineered HDAC1 mutants suggests that the HDAC activity can potentially be decoupled from "activatability" by the CoA derivatives. In vivo, pharmacological inhibition of glucose-6-phosphate dehydrogenase (G6PD) to decrease NADPH levels led to significant increases in global levels of histone H3 and H4 acetylation. The similarity in structures of the identified metabolites and the exquisite selectivity of NADPH over NADP(+), NADH, and NAD(+) as an HDAC activator reveal a previously unrecognized biochemical feature of the HDAC proteins with important consequences for regulation of histone acetylation as well as the development of more specific and potent HDAC inhibitors.

Zinc is Essential for the Copper Reductase Activity of Yeast Nucleosomes.

The histone H3-H4 tetramer is a copper reductase enzyme, facilitating the production of cuprous (Cu1+) ions for distribution to copper-dependent enzymes. It was, however, unknown if this enzymatic activity occurred within nucleosomes. To investigate this, we obtained native nucleosomes from Saccharomyces cerevisiae using micrococcal nuclease digestion of chromatin in isolated nuclei and ion-exchange chromatographic purification. The purified nucleosomal fragments robustly reduced Cu2+ to Cu1+ ions, with the optimal activity dependent on the presence of zinc ions. Mutation of the histone H3 histidine 113 (H3H113) residue at the active site substantially reduced the enzymatic activity of nucleosomes, underscoring the catalytic role of histone H3. Consistently, limiting zinc ions reduced intracellular Cu1+ levels and compromised growth, phenotypes that were mitigated by genetically enhancing the copper reductase activity of histone H3. These results indicate that yeast nucleosomes possess copper reductase activity, suggesting that the fundamental unit of eukaryotic chromatin is an enzyme complex.

A pathogenic role for histone H3 copper reductase activity in a yeast model of Friedreich's ataxia.

Disruptions to iron-sulfur (Fe-S) clusters, essential cofactors for a broad range of proteins, cause widespread cellular defects resulting in human disease. A source of damage to Fe-S clusters is cuprous (Cu1+) ions. Since histone H3 enzymatically produces Cu1+ for copper-dependent functions, we asked whether this activity could become detrimental to Fe-S clusters. Here, we report that histone H3­mediated Cu1+ toxicity is a major determinant of cellular functional pool of Fe-S clusters. Inadequate Fe-S cluster supply, due to diminished assembly as occurs in Friedreich's ataxia or defective distribution, causes severe metabolic and growth defects in Saccharomyces cerevisiae. Decreasing Cu1+ abundance, through attenuation of histone cupric reductase activity or depletion of total cellular copper, restored Fe-S cluster­dependent metabolism and growth. Our findings reveal an interplay between chromatin and mitochondria in Fe-S cluster homeostasis and a potential pathogenic role for histone enzyme activity and Cu1+ in diseases with Fe-S cluster dysfunction.

The histone H3-H4 tetramer is a copper reductase enzyme.

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3' dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.

Histone deacetylase inhibitors provoke a tumor supportive phenotype in pancreatic cancer associated fibroblasts.

Although histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs, thus far, they have been unsuccessful in early phase clinical trials for pancreatic ductal adenocarcinoma (PDAC). One potential reason for their poor efficacy is the tumor stroma, where cancer-associated fibroblasts (CAFs) are a prominent cell type and a source of resistance to cancer therapies. Here, we demonstrate that stromal fibroblasts contribute to the poor efficacy of HDACi's in PDAC. HDACi-treated fibroblasts show increased biological aggressiveness and are characterized by increased secretion of pro-inflammatory tumor-supportive cytokines and chemokines. We find that HDAC2 binds to the enhancer and promoter regions of pro-inflammatory genes specifically in CAFs and in silico analysis identified AP-1 to be the most frequently associated transcription factor bound in these regions. Pharmacologic inhibition of pathways upstream of AP-1 suppresses the HDACi-induced inflammatory gene expression and tumor-supportive responses in fibroblasts. Our findings demonstrate that the combination of HDACi's with chemical inhibitors of the AP-1 signaling pathway attenuate the inflammatory phenotype of fibroblasts and may improve the efficacy of HDACi in PDAC and, potentially, in other solid tumors rich in stroma.

Acetylation and accessibility of rDNA chromatin in Saccharomyces cerevisiae in (Delta)top1 and (Delta)sir2 mutants.

The insertion of reporter genes in the ribosomal DNA (rDNA) locus of Saccharomyces cerevisiae causes their transcriptional repression. This kind of transcriptional silencing depends on proteins such as Sir2p and Top1p, and has been shown to be mediated by chromatin. While Sir2p modifies nucleosomes directly through its histone deacetylase activity, little is known about changes in the chromatin structure that occur at the rDNA locus when TOP1 is deleted. Here, we show that the absence of Top1p causes increased histone acetylation at the rDNA locus. Moreover, rDNA chromatin becomes more accessible in a similar manner in both top1 and sir2 mutant strains.

Evolution of histone 2A for chromatin compaction in eukaryotes.

During eukaryotic evolution, genome size has increased disproportionately to nuclear volume, necessitating greater degrees of chromatin compaction in higher eukaryotes, which have evolved several mechanisms for genome compaction. However, it is unknown whether histones themselves have evolved to regulate chromatin compaction. Analysis of histone sequences from 160 eukaryotes revealed that the H2A N-terminus has systematically acquired arginines as genomes expanded. Insertion of arginines into their evolutionarily conserved position in H2A of a small-genome organism increased linear compaction by as much as 40%, while their absence markedly diminished compaction in cells with large genomes. This effect was recapitulated in vitro with nucleosomal arrays using unmodified histones, indicating that the H2A N-terminus directly modulates the chromatin fiber likely through intra- and inter-nucleosomal arginine-DNA contacts to enable tighter nucleosomal packing. Our findings reveal a novel evolutionary mechanism for regulation of chromatin compaction and may explain the frequent mutations of the H2A N-terminus in cancer.

H3 k36 methylation helps determine the timing of cdc45 association with replication origins.

BACKGROUND: Replication origins fire at different times during S-phase. Such timing is determined by the chromosomal context, which includes the activity of nearby genes, telomeric position effects and chromatin structure, such as the acetylation state of the surrounding chromatin. Activation of replication origins involves the conversion of a pre-replicative complex to a replicative complex. A pivotal step during this conversion is the binding of the replication factor Cdc45, which associates with replication origins at approximately their time of activation in a manner partially controlled by histone acetylation. METHODOLOGY/PRINCIPAL FINDINGS: Here we identify histone H3 K36 methylation (H3 K36me) by Set2 as a novel regulator of the time of Cdc45 association with replication origins. Deletion of SET2 abolishes all forms of H3 K36 methylation. This causes a delay in Cdc45 binding to origins and renders the dynamics of this interaction insensitive to the state of histone acetylation of the surrounding chromosomal region. Furthermore, a decrease in H3 K36me3 and a concomitant increase in H3 K36me1 around the time of Cdc45 binding to replication origins suggests opposing functions for these two methylation states. Indeed, we find K36me3 depleted from early firing origins when compared to late origins genomewide, supporting a delaying effect of this histone modification for the association of replication factors with origins. CONCLUSIONS/SIGNIFICANCE: We propose a model in which K36me1 together with histone acetylation advance, while K36me3 and histone deacetylation delay, the time of Cdc45 association with replication origins. The involvement of the transcriptionally induced H3 K36 methylation mark in regulating the timing of Cdc45 binding to replication origins provides a novel means of how gene expression may affect origin dynamics during S-phase.

Histone acetylation regulates the time of replication origin firing.

The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. We demonstrate here that the state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing.

Hyperacetylation of chromatin at the ADH2 promoter allows Adr1 to bind in repressed conditions.

We report that in vivo increased acetylation of the repressed Saccharomyces cerevisiae ADH2 promoter chromatin, as obtained by disrupting the genes for the two deacetylases HDA1 and RPD3, destabilizes the structure of the TATA box-containing nucleosome. This acetylation-dependent chromatin remodeling is not sufficient to allow the binding of the TATA box-binding protein, but facilitates the recruitment of the transcriptional activator Adr1 and induces faster kinetics of mRNA accumulation when the cells are shifted to derepressing conditions.