Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells.

Paternal environmental factors can epigenetically influence gene expressions in offspring. We demonstrate that restraint stress, an experimental model for strong psychological stress, to fathers affects the epigenome, transcriptome, and metabolome of offspring in a MEKK1-dATF2 pathway-dependent manner in Drosophila melanogaster. Genes involved in amino acid metabolism are upregulated by paternal restraint stress, while genes involved in glycolysis and the tricarboxylic acid (TCA) cycle are downregulated. The effects of paternal restraint stress are also confirmed by metabolome analysis. dATF-2 is highly expressed in testicular germ cells, and restraint stress also induces p38 activation in the testes. Restraint stress induces Unpaired 3 (Upd3), a Drosophila homolog of Interleukin 6 (IL-6). Moreover, paternal overexpression of upd3 in somatic cells disrupts heterochromatin in offspring but not in offspring from dATF-2 mutant fathers. These results indicate that paternal restraint stress affects metabolism in offspring via inheritance of dATF-2-dependent epigenetic changes.

Publisher Correction: Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A common molecular mechanism underlies the role of Mps1 in chromosome biorientation and the spindle assembly checkpoint.

The Mps1 kinase corrects improper kinetochore-microtubule attachments, thereby ensuring chromosome biorientation. Yet, its critical phosphorylation targets in this process remain largely elusive. Mps1 also controls the spindle assembly checkpoint (SAC), which halts chromosome segregation until biorientation is attained. Its role in SAC activation is antagonised by the PP1 phosphatase and involves phosphorylation of the kinetochore scaffold Knl1/Spc105, which in turn recruits the Bub1 kinase to promote assembly of SAC effector complexes. A crucial question is whether error correction and SAC activation are part of a single or separable pathways. Here, we isolate and characterise a new yeast mutant, mps1-3, that is severely defective in chromosome biorientation and SAC signalling. Through an unbiased screen for extragenic suppressors, we found that mutations lowering PP1 levels at Spc105 or forced association of Bub1 with Spc105 reinstate both chromosome biorientation and SAC signalling in mps1-3 cells. Our data argue that a common mechanism based on Knl1/Spc105 phosphorylation is critical for Mps1 function in error correction and SAC signalling, thus supporting the idea that a single sensory apparatus simultaneously elicits both pathways.

ATF7-Dependent Epigenetic Changes Are Required for the Intergenerational Effect of a Paternal Low-Protein Diet.

Paternal dietary conditions may contribute to metabolic disorders in offspring. We have analyzed the role of the stress-dependent epigenetic regulator cyclic AMP-dependent transcription factor 7 (ATF7) in paternal low-protein diet (pLPD)-induced gene expression changes in mouse liver. Atf7+/- mutations cause an offspring phenotype similar to that caused by pLPD, and the effect of pLPD almost vanished when paternal Atf7+/- mice were used. ATF7 binds to the promoter regions of ∼2,300 genes, including cholesterol biosynthesis-related and tRNA genes in testicular germ cells (TGCs). LPD induces ATF7 phosphorylation by p38 via reactive oxygen species (ROS) in TGCs. This leads to the release of ATF7 and a decrease in histone H3K9 dimethylation (H3K9me2) on its target genes. These epigenetic changes are maintained and induce expression of some tRNA fragments in spermatozoa. These results indicate that LPD-induced and ATF7-dependent epigenetic changes in TGCs play an important role in paternal diet-induced metabolic reprograming in offspring.

The Phosphatase PP1 Promotes Mitotic Slippage through Mad3 Dephosphorylation.

Accurate chromosome segregation requires bipolar attachment of kinetochores to spindle microtubules. A conserved surveillance mechanism, the spindle assembly checkpoint (SAC), responds to lack of kinetochore-microtubule connections and delays anaphase onset until all chromosomes are bipolarly attached [1]. SAC signaling fires at kinetochores and involves a soluble mitotic checkpoint complex (MCC) that inhibits the anaphase-promoting complex (APC) [2, 3]. The mitotic delay imposed by SAC, however, is not everlasting. If kinetochores fail to establish bipolar connections, cells can escape from the SAC-induced mitotic arrest through a process called mitotic slippage [4]. Mitotic slippage occurs in the presence of SAC signaling at kinetochores [5, 6], but whether and how MCC stability and APC inhibition are actively controlled during slippage is unknown. The PP1 phosphatase has emerged as a key factor in SAC silencing once all kinetochores are bipolarly attached [7, 8]. PP1 turns off SAC signaling through dephosphorylation of the SAC scaffold Knl1/Blinkin at kinetochores [9-11]. Here, we show that, in budding yeast, PP1 is also required for mitotic slippage. However, its involvement in this process is not linked to kinetochores but rather to MCC stability. We identify S268 of Mad3 as a critical target of PP1 in this process. Mad3 S268 dephosphorylation destabilizes the MCC without affecting the initial SAC-induced mitotic arrest. Conversely, it accelerates mitotic slippage and overcomes the slippage defect of PP1 mutants. Thus, slippage is not the mere consequence of incomplete APC inactivation that brings about mitotic exit, as originally proposed, but involves the exertive antagonism between kinases and phosphatases.

Chromosome-associated RNA-protein complexes promote pairing of homologous chromosomes during meiosis in Schizosaccharomyces pombe.

Pairing of homologous chromosomes in meiosis is essential for sexual reproduction. We have previously demonstrated that the fission yeast sme2 RNA, a meiosis-specific long noncoding RNA (lncRNA), accumulates at the sme2 chromosomal loci and mediates their robust pairing in meiosis. However, the mechanisms underlying lncRNA-mediated homologous pairing have remained elusive. In this study, we identify conserved RNA-binding proteins that are required for robust pairing of homologous chromosomes. These proteins accumulate mainly at the sme2 and two other chromosomal loci together with meiosis-specific lncRNAs transcribed from these loci. Remarkably, the chromosomal accumulation of these lncRNA-protein complexes is required for robust pairing. Moreover, the lncRNA-protein complexes exhibit phase separation properties, since 1,6-hexanediol treatment reversibly disassembled these complexes and disrupted the pairing of associated loci. We propose that lncRNA-protein complexes assembled at specific chromosomal loci mediate recognition and subsequent pairing of homologous chromosomes.

Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells.

BACKGROUND: Endothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood. RESULTS: To characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We developed a robust procedure for comparative epigenome analysis that circumvents variations at the level of the individual and technical noise derived from sample preparation under various conditions. Through this approach, we identified 3765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. We found that the nine EC types can be divided into two subgroups, corresponding to those with upper-body origins and lower-body origins, based on their epigenomic landscape. Epigenomic variations were highly correlated with gene expression patterns, but also provided unique information. Most of the deferentially expressed genes and enhancers were cooperatively enriched in more than one EC type, suggesting that the distinct combinations of multiple genes play key roles in the diverse phenotypes across EC types. Notably, many homeobox genes were differentially expressed across EC types, and their expression was correlated with the relative position of each organ in the body. This reflects the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing. CONCLUSIONS: This comprehensive analysis of epigenome characterization of EC types reveals diverse transcriptional regulation across human vascular systems. These datasets provide a valuable resource for understanding the vascular system and associated diseases.

Comparative analysis demonstrates cell type-specific conservation of SOX9 targets between mouse and chicken.

SRY (sex-determining region Y)-box 9 (SOX9) is a transcription factor regulating both chondrogenesis and sex determination. Among vertebrates, SOX9's functions in chondrogenesis are well conserved, while they vary in sex determination. To investigate the conservation of SOX9's regulatory functions in chondrogenesis and gonad development among species, we performed chromatin immunoprecipitation sequencing (ChIP-seq) using developing limb buds and male gonads from embryos of two vertebrates, mouse and chicken. In both mouse and chicken, SOX9 bound to intronic and distal regions of genes more frequently in limb buds than in male gonads, while SOX9 bound to the proximal upstream regions of genes more frequently in male gonads than in limb buds. In both species, SOX palindromic repeats were identified more frequently in SOX9 binding regions in limb bud genes compared with those in male gonad genes. The conservation of SOX9 binding regions was significantly higher in limb bud genes. In addition, we combined RNA expression analysis (RNA sequencing) with the ChIP-seq results at the same stage in developing chondrocytes and Sertoli cells and determined SOX9 target genes in these cells of the two species and disclosed that SOX9 targets showed high similarity of targets in chondrocytes, but not in Sertoli cells.

Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation.

Elg1, the major subunit of a Replication Factor C-like complex, is critical to ensure genomic stability during DNA replication, and is implicated in controlling chromatin structure. We investigated the consequences of Elg1 loss for the dynamics of chromatin re-formation following DNA replication. Measurement of Okazaki fragment length and the micrococcal nuclease sensitivity of newly replicated DNA revealed a defect in nucleosome organization in the absence of Elg1. Using a proteomic approach to identify Elg1 binding partners, we discovered that Elg1 interacts with Rtt106, a histone chaperone implicated in replication-coupled nucleosome assembly that also regulates transcription. A central role for Elg1 is the unloading of PCNA from chromatin following DNA replication, so we examined the relative importance of Rtt106 and PCNA unloading for chromatin reassembly following DNA replication. We find that the major cause of the chromatin organization defects of an ELG1 mutant is PCNA retention on DNA following replication, with Rtt106-Elg1 interaction potentially playing a contributory role.

Mapping of histone-binding sites in histone replacement-completed spermatozoa.

The majority of histones are replaced by protamines during spermatogenesis, but small amounts are retained in mammalian spermatozoa. Since nucleosomes in spermatozoa influence epigenetic inheritance, it is important to know how histones are distributed in the sperm genome. Conflicting data, which may result from different conditions used for micrococcal nuclease (MNase) digestion, have been reported: retention of nucleosomes at either gene promoter regions or within distal gene-poor regions. Here, we find that the swim-up sperm used in many studies contain about 10% population of sperm which have not yet completed the histone-to-protamine replacement. We develop a method to purify histone replacement-completed sperm (HRCS) and to completely solubilize histones from cross-linked HRCS without MNase digestion. Our results indicate that histones are retained at specific promoter regions in HRCS. This method allows the study of epigenetic status in mature sperm.

Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks.

Despite its evolutionarily conserved function in controlling DNA replication, the chromosomal binding sites of the budding yeast Rif1 protein are not well understood. Here, we analyse genome-wide binding of budding yeast Rif1 by chromatin immunoprecipitation, during G1 phase and in S phase with replication progressing normally or blocked by hydroxyurea. Rif1 associates strongly with telomeres through interaction with Rap1. By comparing genomic binding of wild-type Rif1 and truncated Rif1 lacking the Rap1-interaction domain, we identify hundreds of Rap1-dependent and Rap1-independent chromosome interaction sites. Rif1 binds to centromeres, highly transcribed genes and replication origins in a Rap1-independent manner, associating with both early and late-initiating origins. Interestingly, Rif1 also binds around activated origins when replication progression is blocked by hydroxyurea, suggesting association with blocked forks. Using nascent DNA labelling and DNA combing techniques, we find that in cells treated with hydroxyurea, yeast Rif1 stabilises recently synthesised DNA Our results indicate that, in addition to controlling DNA replication initiation, budding yeast Rif1 plays an ongoing role after initiation and controls events at blocked replication forks.

Re-evaluating the Localization of Sperm-Retained Histones Revealed the Modification-Dependent Accumulation in Specific Genome Regions.

The question of whether retained histones in the sperm genome localize to gene-coding regions or gene deserts has been debated for years. Previous contradictory observations are likely caused by the non-uniform sensitivity of sperm chromatin to micrococcal nuclease (MNase) digestion. Sperm chromatin has a highly condensed but heterogeneous structure and is composed of 90%∼99% protamines and 1%∼10% histones. In this study, we utilized nucleoplasmin (NPM) to improve the solubility of sperm chromatin by removing protamines in vitro. NPM treatment efficiently solubilized histones while maintaining quality and quantity. Chromatin immunoprecipitation sequencing (ChIP-seq) analyses using NPM-treated sperm demonstrated the predominant localization of H4 to distal intergenic regions, whereas modified histones exhibited a modification-dependent preferential enrichment in specific genomic elements, such as H3K4me3 at CpG-rich promoters and H3K9me3 in satellite repeats, respectively, implying the existence of machinery protecting modified histones from eviction.

Cohesin Ubiquitylation and Mobilization Facilitate Stalled Replication Fork Dynamics.

Replication fork integrity is challenged in conditions of stress and protected by the Mec1/ATR checkpoint to preserve genome stability. Still poorly understood in fork protection is the role played by the structural maintenance of chromosomes (SMC) cohesin complex. We uncovered a role for the Rsp5Bul2 ubiquitin ligase in promoting survival to replication stress by preserving stalled fork integrity. Rsp5Bul2 physically interacts with cohesin and the Mec1 kinase, thus promoting checkpoint-dependent cohesin ubiquitylation and cohesin-mediated fork protection. Ubiquitylation mediated by Rsp5Bul2 promotes cohesin mobilization from chromatin neighboring stalled forks, likely by stimulating the Cdc48/p97 ubiquitin-selective segregase, and its timely association to nascent chromatids. This Rsp5Bul2 fork protection mechanism requires the Wpl1 cohesin mobilizer as well as the function of the Eco1 acetyltransferase securing sister chromatid entrapment. Our data indicate that ubiquitylation facilitates cohesin dynamic interfacing with replication forks within a mechanism preserving stalled-fork functional architecture.

interleukin-11 induces and maintains progenitors of different cell lineages during Xenopus tadpole tail regeneration.

Unlike mammals, Xenopus laevis tadpoles possess high ability to regenerate their lost organs. In amphibians, the main source of regenerated tissues is lineage-restricted tissue stem cells, but the mechanisms underlying induction, maintenance and differentiation of these stem/progenitor cells in the regenerating organs are poorly understood. We previously reported that interleukin-11 (il-11) is highly expressed in the proliferating cells of regenerating Xenopus tadpole tails. Here, we show that il-11 knockdown (KD) shortens the regenerated tail length, and the phenotype is rescued by forced-il-11-expression in the KD tadpoles. Moreover, marker genes for undifferentiated notochord, muscle, and sensory neurons are downregulated in the KD tadpoles, and the forced-il-11-expression in intact tadpole tails induces expression of these marker genes. Our findings demonstrate that il-11 is necessary for organ regeneration, and suggest that IL-11 plays a key role in the induction and maintenance of undifferentiated progenitors across cell lineages during Xenopus tail regeneration. Xenopus laevis tadpoles have maintained their ability to regenerate various organs. Here, the authors show that interleukin-11 is necessary for organ regeneration, by inducing and maintaining undifferentiated progenitors across cell lineages during Xenopus tail regeneration.

An In Vitro Human Liver Model by iPSC-Derived Parenchymal and Non-parenchymal Cells.

During liver development, hepatoblasts and liver non-parenchymal cells (NPCs) such as liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs) constitute the liver bud where they proliferate and differentiate. Accordingly, we reasoned that liver NPCs would support the maturation of hepatocytes derived from human induced pluripotent stem cells (hiPSCs), which usually exhibit limited functions. We found that the transforming growth factor ß and Rho signaling pathways, respectively, regulated the proliferation and maturation of LSEC and HSC progenitors isolated from mouse fetal livers. Based on these results, we have established culture systems to generate LSECs and HSCs from hiPSCs. These hiPSC-derived NPCs exhibited distinctive phenotypes and promoted self-renewal of hiPSC-derived liver progenitor cells (LPCs) over the long term in the two-dimensional culture system without exogenous cytokines and hepatic maturation of hiPSC-derived LPCs. Thus, a functional human liver model can be constructed in vitro from the LPCs, LSECs, and HSCs derived from hiPSCs.

MRG15-mediated tethering of PALB2 to unperturbed chromatin protects active genes from genotoxic stress.

The partner and localiser of BRCA2 (PALB2) plays important roles in the maintenance of genome integrity and protection against cancer. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, recent studies provide evidence that PALB2 also associates with unperturbed chromatin. Here, we investigated the previously poorly described role of chromatin-associated PALB2 in undamaged cells. We found that PALB2 associates with active genes through its major binding partner, MRG15, which recognizes histone H3 trimethylated at lysine 36 (H3K36me3) by the SETD2 methyltransferase. Missense mutations that ablate PALB2 binding to MRG15 confer elevated sensitivity to the topoisomerase inhibitor camptothecin (CPT) and increased levels of aberrant metaphase chromosomes and DNA stress in gene bodies, which were suppressed by preventing DNA replication. Remarkably, the level of PALB2 at genic regions was frequently decreased, rather than increased, upon CPT treatment. We propose that the steady-state presence of PALB2 at active genes, mediated through the SETD2/H3K36me3/MRG15 axis, ensures an immediate response to DNA stress and therefore effective protection of these regions during DNA replication. This study provides a conceptual advance in demonstrating that the constitutive chromatin association of repair factors plays a key role in the maintenance of genome stability and furthers our understanding of why PALB2 defects lead to human genome instability syndromes.

Decreased cohesin in the brain leads to defective synapse development and anxiety-related behavior.

Abnormal epigenetic regulation can cause the nervous system to develop abnormally. Here, we sought to understand the mechanism by which this occurs by investigating the protein complex cohesin, which is considered to regulate gene expression and, when defective, is associated with higher-level brain dysfunction and the developmental disorder Cornelia de Lange syndrome (CdLS). We generated conditional Smc3-knockout mice and observed greater dendritic complexity and larger numbers of immature synapses in the cerebral cortex of Smc3+/- mice. Smc3+/- mice also exhibited more anxiety-related behavior, which is a symptom of CdLS. Further, a gene ontology analysis after RNA-sequencing suggested the enrichment of immune processes, particularly the response to interferons, in the Smc3+/- mice. Indeed, fewer synapses formed in their cortical neurons, and this phenotype was rescued by STAT1 knockdown. Thus, low levels of cohesin expression in the developing brain lead to changes in gene expression that in turn lead to a specific and abnormal neuronal and behavioral phenotype.

ASBEL-TCF3 complex is required for the tumorigenicity of colorectal cancer cells.

Wnt/ß-catenin signaling plays a key role in the tumorigenicity of colon cancer. Furthermore, it has been reported that lncRNAs are dysregulated in several steps of cancer development. Here we show that ß-catenin directly activates the transcription of the long noncoding RNA (lncRNA) ASBEL [antisense ncRNA in the ANA (Abundant in neuroepithelium area)/BTG3 (B-cell translocation gene 3) locus] and transcription factor 3 (TCF3), both of which are required for the survival and tumorigenicity of colorectal cancer cells. ASBEL interacts with and recruits TCF3 to the activating transcription factor 3 (ATF3) locus, where it represses the expression of ATF3. Furthermore, we demonstrate that ASBEL-TCF3-mediated down-regulation of ATF3 expression is required for the proliferation and tumorigenicity of colon tumor cells. ATF3, in turn, represses the expression of ASBEL Our results reveal a pathway involving an lncRNA and two transcription factors that plays a key role in Wnt/ß-catenin-mediated tumorigenesis. These results may provide insights into the variety of biological and pathological processes regulated by Wnt/ß-catenin signaling.

ARCN1 Mutations Cause a Recognizable Craniofacial Syndrome Due to COPI-Mediated Transport Defects.

Cellular homeostasis is maintained by the highly organized cooperation of intracellular trafficking systems, including COPI, COPII, and clathrin complexes. COPI is a coatomer protein complex responsible for intracellular protein transport between the endoplasmic reticulum and the Golgi apparatus. The importance of such intracellular transport mechanisms is underscored by the various disorders, including skeletal disorders such as cranio-lenticulo-sutural dysplasia and osteogenesis imperfect, caused by mutations in the COPII coatomer complex. In this article, we report a clinically recognizable craniofacial disorder characterized by facial dysmorphisms, severe micrognathia, rhizomelic shortening, microcephalic dwarfism, and mild developmental delay due to loss-of-function heterozygous mutations in ARCN1, which encodes the coatomer subunit delta of COPI. ARCN1 mutant cell lines were revealed to have endoplasmic reticulum stress, suggesting the involvement of ER stress response in the pathogenesis of this disorder. Given that ARCN1 deficiency causes defective type I collagen transport, reduction of collagen secretion represents the likely mechanism underlying the skeletal phenotype that characterizes this condition. Our findings demonstrate the importance of COPI-mediated transport in human development, including skeletogenesis and brain growth.