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.

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.

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.

Transient nuclear deformation primes epigenetic state and promotes cell reprogramming.

Cell reprogramming has wide applications in tissue regeneration, disease modelling and personalized medicine. In addition to biochemical cues, mechanical forces also contribute to the modulation of the epigenetic state and a variety of cell functions through distinct mechanisms that are not fully understood. Here we show that millisecond deformation of the cell nucleus caused by confinement into microfluidic channels results in wrinkling and transient disassembly of the nuclear lamina, local detachment of lamina-associated domains in chromatin and a decrease of histone methylation (histone H3 lysine 9 trimethylation) and DNA methylation. These global changes in chromatin at the early stage of cell reprogramming boost the conversion of fibroblasts into neurons and can be partially reproduced by inhibition of histone H3 lysine 9 and DNA methylation. This mechanopriming approach also triggers macrophage reprogramming into neurons and fibroblast conversion into induced pluripotent stem cells, being thus a promising mechanically based epigenetic state modulation method for cell engineering.

Chromatin as a metabolic organelle: Integrating the cellular flow of carbon with gene expression.

Hsieh et al. (2022) reveal that carbon starvation elicits an unexpected compensatory reallocation of histone acetylation to establish an adaptive gene expression program, demonstrating how chromatin may integrate cellular carbon flow via histone acetylation with gene regulation.

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.

MLLT3 governs human haematopoietic stem-cell self-renewal and engraftment.

Limited knowledge of the mechanisms that govern the self-renewal of human haematopoietic stem cells (HSCs), and why this fails in culture, have impeded the expansion of HSCs for transplantation1. Here we identify MLLT3 (also known as AF9) as a crucial regulator of HSCs that is highly enriched in human fetal, neonatal and adult HSCs, but downregulated in culture. Depletion of MLLT3 prevented the maintenance of transplantable human haematopoietic stem or progenitor cells (HSPCs) in culture, whereas stabilizing MLLT3 expression in culture enabled more than 12-fold expansion of transplantable HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Similar to endogenous MLLT3, overexpressed MLLT3 localized to active promoters in HSPCs, sustained levels of H3K79me2 and protected the HSC transcriptional program in culture. MLLT3 thus acts as HSC maintenance factor that links histone reader and modifying activities to modulate HSC gene expression, and may provide a promising approach to expand HSCs for transplantation.

EvoChromo: towards a synthesis of chromatin biology and evolution.

Over the past few years, interest in chromatin and its evolution has grown. To further advance these interests, we organized a workshop with the support of The Company of Biologists to debate the current state of knowledge regarding the origin and evolution of chromatin. This workshop led to prospective views on the development of a new field of research that we term 'EvoChromo'. In this short Spotlight article, we define the breadth and expected impact of this new area of scientific inquiry on our understanding of both chromatin and evolution.

Promoter-Enhancer Communication Occurs Primarily within Insulated Neighborhoods.

Metazoan chromosomes are sequentially partitioned into topologically associating domains (TADs) and then into smaller sub-domains. One class of sub-domains, insulated neighborhoods, are proposed to spatially sequester and insulate the enclosed genes through self-association and chromatin looping. However, it has not been determined functionally whether promoter-enhancer interactions and gene regulation are broadly restricted to within these loops. Here, we employed published datasets from murine embryonic stem cells (mESCs) to identify insulated neighborhoods that confine promoter-enhancer interactions and demarcate gene regulatory regions. To directly address the functionality of these regions, we depleted estrogen-related receptor ß (Esrrb), which binds the Mediator co-activator complex, to impair enhancers of genes within 222 insulated neighborhoods without causing mESC differentiation. Esrrb depletion reduces Mediator binding, promoter-enhancer looping, and expression of both nascent RNA and mRNA within the insulated neighborhoods without significantly affecting the flanking genes. Our data indicate that insulated neighborhoods represent functional regulons in mammalian genomes.

Reprogramming normal human epithelial tissues to a common, lethal neuroendocrine cancer lineage.

The use of potent therapies inhibiting critical oncogenic pathways active in epithelial cancers has led to multiple resistance mechanisms, including the development of highly aggressive, small cell neuroendocrine carcinoma (SCNC). SCNC patients have a dismal prognosis due in part to a limited understanding of the molecular mechanisms driving this malignancy and the lack of effective treatments. Here, we demonstrate that a common set of defined oncogenic drivers reproducibly reprograms normal human prostate and lung epithelial cells to small cell prostate cancer (SCPC) and small cell lung cancer (SCLC), respectively. We identify shared active transcription factor binding regions in the reprogrammed prostate and lung SCNCs by integrative analyses of epigenetic and transcriptional landscapes. These results suggest that neuroendocrine cancers arising from distinct epithelial tissues may share common vulnerabilities that could be exploited for the development of drugs targeting SCNCs.

Glucose inhibits cardiac muscle maturation through nucleotide biosynthesis.

The heart switches its energy substrate from glucose to fatty acids at birth, and maternal hyperglycemia is associated with congenital heart disease. However, little is known about how blood glucose impacts heart formation. Using a chemically defined human pluripotent stem-cell-derived cardiomyocyte differentiation system, we found that high glucose inhibits the maturation of cardiomyocytes at genetic, structural, metabolic, electrophysiological, and biomechanical levels by promoting nucleotide biosynthesis through the pentose phosphate pathway. Blood glucose level in embryos is stable in utero during normal pregnancy, but glucose uptake by fetal cardiac tissue is drastically reduced in late gestational stages. In a murine model of diabetic pregnancy, fetal hearts showed cardiomyopathy with increased mitotic activity and decreased maturity. These data suggest that high glucose suppresses cardiac maturation, providing a possible mechanistic basis for congenital heart disease in diabetic pregnancy.

Mot1, Ino80C, and NC2 Function Coordinately to Regulate Pervasive Transcription in Yeast and Mammals.

Pervasive transcription initiates from cryptic promoters and is observed in eukaryotes ranging from yeast to mammals. The Set2-Rpd3 regulatory system prevents cryptic promoter function within expressed genes. However, conserved systems that control pervasive transcription within intergenic regions have not been well established. Here we show that Mot1, Ino80 chromatin remodeling complex (Ino80C), and NC2 co-localize on chromatin and coordinately suppress pervasive transcription in S. cerevisiae and murine embryonic stem cells (mESCs). In yeast, all three proteins bind subtelomeric heterochromatin through a Sir3-stimulated mechanism and to euchromatin via a TBP-stimulated mechanism. In mESCs, the proteins bind to active and poised TBP-bound promoters along with promoters of polycomb-silenced genes apparently lacking TBP. Depletion of Mot1, Ino80C, or NC2 by anchor away in yeast or RNAi in mESCs leads to near-identical transcriptome phenotypes, with new subtelomeric transcription in yeast, and greatly increased pervasive transcription in both yeast and mESCs.

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.

Cbx3 maintains lineage specificity during neural differentiation.

Chromobox homolog 3 (Cbx3/heterochromatin protein 1γ [HP1γ]) stimulates cell differentiation, but its mechanism is unknown. We found that Cbx3 binds to gene promoters upon differentiation of murine embryonic stem cells (ESCs) to neural progenitor cells (NPCs) and recruits the Mediator subunit Med26. RNAi knockdown of either Cbx3 or Med26 inhibits neural differentiation while up-regulating genes involved in mesodermal lineage decisions. Thus, Cbx3 and Med26 together ensure the fidelity of lineage specification by enhancing the expression of neural genes and down-regulating genes specific to alternative fates.

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.

Exploitation of EP300 and CREBBP Lysine Acetyltransferases by Cancer.

p300 and CREB-binding protein (CBP), two homologous lysine acetyltransferases in metazoans, have a myriad of cellular functions. They exert their influence mainly through their roles as transcriptional regulators but also via nontranscriptional effects inside and outside of the nucleus on processes such as DNA replication and metabolism. The versatility of p300/CBP as molecular tools has led to their exploitation by viral oncogenes for cellular transformation and by cancer cells to achieve and maintain an oncogenic phenotype. How cancer cells use p300/CBP in their favor varies depending on the cellular context and is evident by the growing list of loss- and gain-of-function genetic alterations in p300 and CBP in solid tumors and hematological malignancies. Here, we discuss the biological functions of p300/CBP and how disruption of these functions by mutations and alterations in expression or subcellular localization contributes to the cancer phenotype.

MEF2C protects bone marrow B-lymphoid progenitors during stress haematopoiesis.

DNA double strand break (DSB) repair is critical for generation of B-cell receptors, which are pre-requisite for B-cell progenitor survival. However, the transcription factors that promote DSB repair in B cells are not known. Here we show that MEF2C enhances the expression of DNA repair and recombination factors in B-cell progenitors, promoting DSB repair, V(D)J recombination and cell survival. Although Mef2c-deficient mice maintain relatively intact peripheral B-lymphoid cellularity during homeostasis, they exhibit poor B-lymphoid recovery after sub-lethal irradiation and 5-fluorouracil injection. MEF2C binds active regulatory regions with high-chromatin accessibility in DNA repair and V(D)J genes in both mouse B-cell progenitors and human B lymphoblasts. Loss of Mef2c in pre-B cells reduces chromatin accessibility in multiple regulatory regions of the MEF2C-activated genes. MEF2C therefore protects B lymphopoiesis during stress by ensuring proper expression of genes that encode DNA repair and B-cell factors.

Reciprocal Regulation of the Cardiac Epigenome by Chromatin Structural Proteins Hmgb and Ctcf: IMPLICATIONS FOR TRANSCRIPTIONAL REGULATION.

Transcriptome remodeling in heart disease occurs through the coordinated actions of transcription factors, histone modifications, and other chromatin features at pathology-associated genes. The extent to which genome-wide chromatin reorganization also contributes to the resultant changes in gene expression remains unknown. We examined the roles of two chromatin structural proteins, Ctcf (CCCTC-binding factor) and Hmgb2 (high mobility group protein B2), in regulating pathologic transcription and chromatin remodeling. Our data demonstrate a reciprocal relationship between Hmgb2 and Ctcf in controlling aspects of chromatin structure and gene expression. Both proteins regulate each others' expression as well as transcription in cardiac myocytes; however, only Hmgb2 does so in a manner that involves global reprogramming of chromatin accessibility. We demonstrate that the actions of Hmgb2 on local chromatin accessibility are conserved across genomic loci, whereas the effects on transcription are loci-dependent and emerge in concert with histone modification and other chromatin features. Finally, although both proteins share gene targets, Hmgb2 and Ctcf, neither binds these genes simultaneously nor do they physically colocalize in myocyte nuclei. Our study uncovers a previously unknown relationship between these two ubiquitous chromatin proteins and provides a mechanistic explanation for how Hmgb2 regulates gene expression and cellular phenotype. Furthermore, we provide direct evidence for structural remodeling of chromatin on a genome-wide scale in the setting of cardiac disease.

MCT1 Modulates Cancer Cell Pyruvate Export and Growth of Tumors that Co-express MCT1 and MCT4.

Monocarboxylate transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here, we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that co-express MCT1 and MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest MCT1 expression is elevated in glycolytic cancers to promote pyruvate export that when inhibited, enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors, further supporting their use as anti-cancer therapeutics.