Lysine-36 of Drosophila histone H3.3 supports adult longevity.

Aging is a multifactorial process that disturbs homeostasis, increases disease susceptibility, and ultimately results in death. Although the definitive set of molecular mechanisms responsible for aging remain to be discovered, epigenetic change over time is proving to be a promising piece of the puzzle. Several post-translational histone modifications have been linked to the maintenance of longevity. Here, we focus on lysine-36 of the replication-independent histone protein, H3.3 (H3.3K36). To interrogate the role of this residue in Drosophila developmental gene regulation, we generated a lysine-to-arginine mutant that blocks the activity of its cognate-modifying enzymes. We found that an H3.3BK36R mutation causes a significant reduction in adult lifespan, accompanied by dysregulation of the genomic and transcriptomic architecture. Transgenic co-expression of wild-type H3.3B completely rescues the longevity defect. Because H3.3 is known to accumulate in nondividing tissues, we carried out transcriptome profiling of young vs aged adult fly heads. The data show that loss of H3.3K36 results in age-dependent misexpression of NF-κB and other innate immune target genes, as well as defects in silencing of heterochromatin. We propose H3.3K36 maintains the postmitotic epigenomic landscape, supporting longevity by regulating both pericentric and telomeric retrotransposons and by suppressing aberrant immune signaling.

Arsenic Impairs Differentiation of Human Induced Pluripotent Stem Cells into Cholinergic Motor Neurons.

Arsenic exposure during embryogenesis can lead to improper neurodevelopment and changes in locomotor activity. Additionally, in vitro studies have shown that arsenic inhibits the differentiation of sensory neurons and skeletal muscle. In the current study, human-induced pluripotent stem (iPS) cells were differentiated into motor neurons over 28 days, while being exposed to up to 0.5 µM arsenic. On day 6, neuroepithelial progenitor cells (NEPs) exposed to arsenic had reduced transcript levels of the neural progenitor/stem cell marker nestin (NES) and neuroepithelial progenitor marker SOX1, while levels of these transcripts were increased in motor neuron progenitors (MNPs) at day 12. In day 18 early motor neurons (MNs), choline acetyltransferase (CHAT) expression was reduced two-fold in cells exposed to 0.5 µM arsenic. RNA sequencing demonstrated that the cholinergic synapse pathway was impaired following exposure to 0.5 µM arsenic, and that transcript levels of genes involved in acetylcholine synthesis (CHAT), transport (solute carriers, SLC18A3 and SLC5A7) and degradation (acetylcholinesterase, ACHE) were all downregulated in day 18 early MNs. In day 28 mature motor neurons, arsenic significantly downregulated protein expression of microtubule-associated protein 2 (MAP2) and ChAT by 2.8- and 2.1-fold, respectively, concomitantly with a reduction in neurite length. These results show that exposure to environmentally relevant arsenic concentrations dysregulates the differentiation of human iPS cells into motor neurons and impairs the cholinergic synapse pathway, suggesting that exposure impairs cholinergic function in motor neurons.

Arsenic impairs stem cell differentiation via the Hippo signaling pathway.

Arsenic is a ubiquitous toxic metalloid, with over 150 million people exposed to arsenic concentrations above the current 10 ppb drinking water standard through contaminated food and water. Arsenic is a known developmental toxicant as neuronal and muscle development are disrupted following arsenic exposure during embryogenesis. In this study, murine embryonic stem cells were chronically exposed to 0.1 µM (7.5 ppb) arsenic for 32 weeks. RNA sequencing showed that the Hippo signaling pathway, which is involved in embryonic development and pluripotency maintenance, is impaired following arsenic exposure. Thus, temporal changes in the Hippo pathway's core components and its downstream target genes Ctgf and c-Myc were investigated. Protein expression of the pathway's main effector YAP in its active form was significantly upregulated by 3.7-fold in arsenic-exposed cells at week 8, while protein expression of inactive phosphorylated YAP was significantly downregulated by 2.5- and 2-fold at weeks 8 and 16. Exposure to arsenic significantly increased the ratio between nuclear and cytoplasmic YAP by 1.9-fold at weeks 16 and 28. The ratio between nuclear and cytoplasmic transcriptional enhancer factor domain was similarly increased in arsenic-treated samples by 3.4- and 1.6-fold at weeks 16 and 28, respectively. Levels of Ctgf and c-Myc were also upregulated following arsenic exposure. These results suggest that chronic exposure to an environmentally relevant arsenic concentration might hinder cellular differentiation and maintain pluripotency through the impairment of the Hippo signaling pathway resulting in increased YAP activation.

Distinct roles for canonical and variant histone H3 lysine-36 in Polycomb silencing.

Polycomb complexes regulate cell type-specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. We show that mutants of Drosophila replication-dependent (H3.2K36R) or replication-independent (H3.3K36R) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined (H3.3K36RH3.2K36R) mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that the H3.2K36R mutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected in H3.3K36R animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive Polycomb response elements located outside of silenced domains but relatively depleted from those inside. We conclude that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.

Thyroid hormone action controls multiple components of cell junctions at the ventricular zone in the newborn rat brain.

Thyroid hormone (TH) action controls brain development in a spatiotemporal manner. Previously, we demonstrated that perinatal hypothyroidism led to formation of a periventricular heterotopia in developing rats. This heterotopia occurs in the posterior telencephalon, and its formation was preceded by loss of radial glia cell polarity. As radial glia mediate cell migration and originate in a progenitor cell niche called the ventricular zone (VZ), we hypothesized that TH action may control cell signaling in this region. Here we addressed this hypothesis by employing laser capture microdissection and RNA-Seq to evaluate the VZ during a known period of TH sensitivity. Pregnant rats were exposed to a low dose of propylthiouracil (PTU, 0.0003%) through the drinking water during pregnancy and lactation. Dam and pup THs were quantified postnatally and RNA-Seq of the VZ performed in neonates. The PTU exposure resulted in a modest increase in maternal thyroid stimulating hormone and reduced thyroxine (T4). Exposed neonates exhibited hypothyroidism and T4 and triiodothyronine (T3) were also reduced in the telencephalon. RNA-Seq identified 358 differentially expressed genes in microdissected VZ cells of hypothyroid neonates as compared to controls (q-values ≤0.05). Pathway analyses showed processes like maintenance of the extracellular matrix and cytoskeleton, cell adhesion, and cell migration were significantly affected by hypothyroidism. Immunofluorescence also demonstrated that collagen IV, F-actin, radial glia, and adhesion proteins were reduced in the VZ. Immunohistochemistry of integrin αvß3 and isoforms of both thyroid receptors (TRα/TRß) showed highly overlapping expression patterns, including enrichment in the VZ. Taken together, our results show that TH action targets multiple components of cell junctions in the VZ, and this may be mediated by both genomic and nongenomic mechanisms. Surprisingly, this work also suggests that the blood-brain and blood-cerebrospinal fluid barriers may also be affected in hypothyroid newborns.

Lysine-36 of Drosophila histone H3.3 supports adult longevity.

Aging is a multifactorial process that disturbs homeostasis, increases disease susceptibility, and ultimately results in death. Although the definitive set of molecular mechanisms responsible for aging remain to be discovered, epigenetic change over time is proving to be a promising piece of the puzzle. Several posttranslational histone modifications (PTMs) have been linked to the maintenance of longevity. Here, we focus on lysine-36 of the replication-independent histone protein, H3.3 (H3.3K36). To interrogate the role of this residue in Drosophila developmental gene regulation, we generated a lysine to arginine mutant that blocks the activity of its cognate modifying enzymes. We found that an H3.3BK36R mutation causes a significant reduction in adult lifespan, accompanied by dysregulation of the genomic and transcriptomic architecture. Transgenic co-expression of wild-type H3.3B completely rescues the longevity defect. Because H3.3 is known to accumulate in non-dividing tissues, we carried out transcriptome profiling of young vs aged adult fly heads. The data show that loss of H3.3K36 results in age-dependent misexpression of NF-κB and other innate immune target genes, as well as defects in silencing of heterochromatin. We propose H3.3K36 maintains the postmitotic epigenomic landscape, supporting longevity by regulating both pericentric and telomeric retrotransposons and by suppressing aberrant immune signaling.

Long-term arsenic exposure impairs differentiation in mouse embryonal stem cells.

Arsenic is a contaminant found in many foods and drinking water. Exposure to arsenic during development can cause improper neuronal progenitor cell development, differentiation, and function, while in vitro studies have determined that acute arsenic exposure to stem and progenitor cells reduced their ability to differentiate. In the current study, P19 mouse embryonal stem cells were exposed continuously to 0.1-µM (7.5 ppb) arsenic for 32 weeks. A cell lineage array examining messenger RNA (mRNA) changes after 8 and 32 weeks of exposure showed that genes involved in pluripotency were increased, whereas those involved in differentiation were reduced. Therefore, temporal changes of select pluripotency and neuronal differentiation markers throughout the 32-week chronic arsenic exposure were investigated. Sox2 and Oct4 mRNA expression were increased by 1.9- to 2.5-fold in the arsenic-exposed cells, beginning at Week 12. Sox2 protein expression was similarly increased starting at Week 16 and remained elevated by 1.5-fold to sixfold. One target of Sox2 is N-cadherin, whose expression is a hallmark of epithelial-mesenchymal transitions (EMTs). Exposure to arsenic significantly increased N-cadherin protein levels beginning at Week 20, concurrent with increased grouping of N-cadherin positive cells at the perimeter of the embryoid body. Expression of Zeb1, which helps increase the expression of Sox2, was also increased started at Week 16. In contrast, Gdf3 mRNA expression was reduced by 3.4- to 7.2-fold beginning at Week 16, and expression of its target protein, phospho-Smad2/3, was also reduced. These results suggest that chronic, low-level arsenic exposure may delay neuronal differentiation and maintain pluripotency.