Inheritance of a Phenotypically Neutral Epimutation Evokes Gene Silencing in Later Generations.

Small RNAs trigger the formation of epialleles that are silenced across generations. Consequently, RNA-directed epimutagenesis is associated with persistent gene repression. Here, we demonstrate that small interfering RNA-induced epimutations in fission yeast are still inherited even when the silenced gene is reactivated, and descendants can reinstate the silencing phenotype that only occurred in their ancestors. This process is mediated by the deposition of a phenotypically neutral molecular mark composed of tri-methylated histone H3 lysine 9 (H3K9me3). Its stable propagation is coupled to RNAi and requires maximal binding affinity of the Clr4/Suvar39 chromodomain to H3K9me3. In wild-type cells, this mark has no visible impact on transcription but causes gene silencing if RNA polymerase-associated factor 1 complex (Paf1C) activity is impaired. In sum, our results reveal a distinct form of epigenetic memory in which cells acquire heritable, transcriptionally active epialleles that confer gene silencing upon modulation of Paf1C.

The Histone Acetyltransferase Mst2 Protects Active Chromatin from Epigenetic Silencing by Acetylating the Ubiquitin Ligase Brl1.

Faithful propagation of functionally distinct chromatin states is crucial for maintaining cellular identity, and its breakdown can lead to diseases such as cancer. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we report a positive feedback loop that preserves the transcription-competent state of RNA polymerase II-transcribed genes. We found that Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3-marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides acetylating histone H3K14, Mst2 also acetylates Brl1, a component of the histone H2B ubiquitin ligase complex. Brl1 acetylation increases histone H2B ubiquitination, which positively feeds back on transcription and prevents ectopic heterochromatin assembly. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.

An enhancer screen identifies new suppressors of small-RNA-mediated epigenetic gene silencing.

Small non-protein coding RNAs are involved in pathways that control the genome at the level of chromatin. In Schizosaccharomyces pombe, small interfering RNAs (siRNAs) are required for the faithful propagation of heterochromatin that is found at peri-centromeric repeats. In contrast to repetitive DNA, protein-coding genes are refractory to siRNA-mediated heterochromatin formation, unless siRNAs are expressed in mutant cells. Here we report the identification of 20 novel mutant alleles that enable de novo formation of heterochromatin at a euchromatic protein-coding gene by using trans-acting siRNAs as triggers. For example, a single amino acid substitution in the pre-mRNA cleavage factor Yth1 enables siRNAs to trigger silent chromatin formation with unparalleled efficiency. Our results are consistent with a kinetic nascent transcript processing model for the inhibition of small-RNA-directed de novo formation of heterochromatin and lay a foundation for further mechanistic dissection of cellular activities that counteract epigenetic gene silencing.

The RNA-induced transcriptional silencing complex targets chromatin exclusively via interacting with nascent transcripts.

Small RNAs regulate chromatin modification and transcriptional gene silencing across the eukaryotic kingdom. Although these processes have been well studied, fundamental mechanistic aspects remain obscure. Specifically, it is unclear exactly how small RNA-loaded Argonaute protein complexes target chromatin to mediate silencing. Here, using fission yeast, we demonstrate that transcription of the target locus is essential for RNA-directed formation of heterochromatin. However, high transcriptional activity is inhibitory; thus, a transcriptional window exists that is optimal for silencing. We further found that pre-mRNA splicing is compatible with RNA-directed heterochromatin formation. However, the kinetics of pre-mRNA processing is critical. Introns close to the 5' end of a transcript that are rapidly spliced result in a bistable response whereby the target either remains euchromatic or becomes fully silenced. Together, our results discount siRNA-DNA base pairing in RNA-mediated heterochromatin formation, and the mechanistic insights further reveal guiding paradigms for the design of small RNA-directed chromatin silencing studies in multicellular organisms.

The Paf1 complex represses small-RNA-mediated epigenetic gene silencing.

RNA interference (RNAi) refers to the ability of exogenously introduced double-stranded RNA to silence expression of homologous sequences. Silencing is initiated when the enzyme Dicer processes the double-stranded RNA into small interfering RNAs (siRNAs). Small RNA molecules are incorporated into Argonaute-protein-containing effector complexes, which they guide to complementary targets to mediate different types of gene silencing, specifically post-transcriptional gene silencing and chromatin-dependent gene silencing. Although endogenous small RNAs have crucial roles in chromatin-mediated processes across kingdoms, efforts to initiate chromatin modifications in trans by using siRNAs have been inherently difficult to achieve in all eukaryotic cells. Using fission yeast, here we show that RNAi-directed heterochromatin formation is negatively controlled by the highly conserved RNA polymerase-associated factor 1 complex (Paf1C). Temporary expression of a synthetic hairpin RNA in Paf1C mutants triggers stable heterochromatin formation at homologous loci, effectively silencing genes in trans. This repressed state is propagated across generations by the continual production of secondary siRNAs, independently of the synthetic hairpin RNA. Our data support a model in which Paf1C prevents targeting of nascent transcripts by the siRNA-containing RNA-induced transcriptional silencing complex and thereby epigenetic gene silencing, by promoting efficient transcription termination and rapid release of the RNA from the site of transcription. We show that although compromised transcription termination is sufficient to initiate the formation of bi-stable heterochromatin by trans-acting siRNAs, impairment of both transcription termination and nascent transcript release is imperative to confer stability to the repressed state. Our work uncovers a novel mechanism for small-RNA-mediated epigenome regulation and highlights fundamental roles for Paf1C and the RNAi machinery in building epigenetic memory.

A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

An extended dsRBD with a novel zinc-binding motif mediates nuclear retention of fission yeast Dicer.

Dicer proteins function in RNA interference (RNAi) pathways by generating small RNAs (sRNAs). Here, we report the solution structure of the C-terminal domain of Schizosaccharomyces pombe Dicer (Dcr1). The structure reveals an unusual double-stranded RNA binding domain (dsRBD) fold embedding a novel zinc-binding motif that is conserved among dicers in yeast. Although the C-terminal domain of Dcr1 still binds nucleic acids, this property is dispensable for proper functioning of Dcr1. In contrast, disruption of zinc coordination renders Dcr1 mainly cytoplasmic and leads to remarkable changes in gene expression and loss of heterochromatin assembly. In summary, our results reveal novel insights into the mechanism of nuclear retention of Dcr1 and raise the possibility that this new class of dsRBDs might generally function in nucleocytoplasmic trafficking and not substrate binding. The C-terminal domain of Dcr1 constitutes a novel regulatory module that might represent a potential target for therapeutic intervention with fungal diseases.

Machine learning trial to detect sex differences in simple sticker arts of 1606 preschool children.

BACKGROUND: Previous studies suggested that drawings made by preschool boys and girls show distinguishable differences. However, children's drawings on their own are too complexly determined and inherently ambiguous to be a reliable indicator. In the present study, we attempted to develop a machine learning algorithm for classification of sex of the subjects using children's artworks. METHODS: We studied three types of simple sticker artworks from 1606 Japanese preschool children aged 51-83 months (803 boys and 803 girls). Those artworks were processed into digitalized data. Simulated data based on the original data were also generated. Logistic regression approach was applied to each dataset to make a classifier, and run on each dataset in a stratified ten-fold cross-validation with hyperparameter tuning. A probability score was calculated in each sample and utilized for sex classification. Prediction performance was evaluated using accuracy, recall, and precision scores, as well as learning curves. RESULTS: Two models created from the original and simulated data showed comparably low metrics. The distributions of probability scores in the samples from boys and girls mostly overlapped and were indistinguishable. Learning curves of the models showed an extremely under-fitted pattern. CONCLUSIONS: Our machine learning algorithm was unable to distinguish simple sticker arts created by boys and girls. More complex tasks will enable to develop an accurate classifier.

Variations in gender identity and sexual orientation of university students.

Background: Previous studies have shown that a small percentage of people in the general population have atypical gender identity and/or sexual orientation. Aim: This study aimed to explore variations in gender identity and sexual orientation in university students and determine genetic factors associated with these variations. Methods: Deviations from complete gender congruence and exclusive heterosexual orientation in 736 Japanese university students were quantitatively assessed with self-assessment questionnaires. Next, we conducted genetic tests for 80 participants who showed relatively low gender identity scores and/or atypical sexual orientation. These genetic tests consisted of repeat number analysis of the androgen receptor gene (AR) and a SKAT-O: an optimal unified sequence kernel association test, which is an exome-based rare variant association study. The results of the genetic tests were compared with the Japanese reference data and the results of our 637 control samples. Outcomes: We calculated the gender identity and sexual orientation scores of all participants and analyzed the molecular data of 80 selected participants. Results: The gender identity scores of 736 participants were broadly distributed: only ~15% of natal males and ~5% of natal females had the maximum score that corresponds to complete gender congruence. The sexual orientation scores also varied: ~80% of natal males and ~60% of natal females showed exclusive heterosexual orientation. We found no association between gender characteristics and AR repeat numbers. The SKAT-O showed that rare damaging variants of TDRP and 3 other genes were more common in the 80 participants than in the control group. Clinical Implications: Our data support the view that gender is a phenotypic continuum rather than a binary trait. Strength and Limitations: This study quantitatively assessed the gender characteristics of a large cohort of university students. Moreover, we conducted systematic screening for genetic factors associated with gender variations. The weaknesses of the study were the limited analytic power of the questionnaires, the relatively small sample for molecular analyses, and incomplete clinical information and relatively advanced ages of the control group. Conclusion: This study revealed significant variations in gender identity and sexual orientation in university students, which may be partly associated with variants in TDRP or other genes.

Schizosaccharomyces pombe reporter strains for relative quantitative assessment of heterochromatin silencing.

The fission yeast Schizosaccharomyces pombe has been emerging as an important model organism for studying the formation and repression of heterochromatin. To enable simple and relative quantitative assessment of heterochromatin silencing, we have created bioluminescence-based reporter strains. A green-emitting click beetle luciferase (CBG68) gene was inserted within pericentromeric heterochromatin or at the silent mating-type locus via homologous recombination. In the same strains, a red-emitting click beetle luciferase (CBR) gene is expressed from the euchromatic leu1(+) locus and can be used as a reference in dual-colour assays. Our reporter strains are suitable for performing Chroma-Glo™ assays, which can be carried out directly in the culture medium without prior cell lysis and in a multiwell format. Our reporter system reliably reflects the state of chromatin and can be easily adapted for use in high-throughput screening approaches.

Physicochemical and Biochemical Evaluation of Amorphous Solid Dispersion of Naringenin Prepared Using Hot-Melt Extrusion.

Naringenin (NRG) is a plant-derived flavonoid. Due to its antioxidant, anti-inflammatory, and analgesic activities it is beneficial to human health and is often used as a functional food ingredient; however, it has poor water solubility and low in vivo bioavailability. Therefore, the efficacy of NRG can be improved by enhancing its water solubility to increase gastrointestinal absorption. Conventional methods for the formulation of NRG are very complex and use toxic organic solvents, making them impractical for the production of functional foods. The objective of this study was to develop a safe and effective NRG-based functional food material. Previously, we established a technology to prepare amorphous solid dispersions (SDs) from functional food ingredients with poor water solubility and used hot-melt extrusion technology that is comparatively simple and does not involve the use of organic solvents. In this study, we prepared NRG SD and evaluated them both physicochemically and biochemically. NRG SD had superior water solubility and gastrointestinal absorption relative to native NRG and showed higher analgesic efficacy in rats than crystalline NRG. NRG SD was administered to mice in a mixed diet for 28 days, and organ weights and hematological/clinical biochemical parameters were assessed. NRG SD did not demonstrate severe adverse effects. The results suggest that NRG SD is a safe and highly efficacious formulation that can be used as a functional food material in the future.

Regulation of plasma-membrane-associated sialidase NEU3 gene by Sp1/Sp3 transcription factors.

Gene expression of the human plasma membrane-associated sialidase (NEU3), a key enzyme for ganglioside degradation, is relatively high in brain and is modulated in response to many cellular processes, including neuronal cell differentiation and tumorigenesis. We demonstrated previously that NEU3 is markedly up-regulated in various human cancers and showed that NEU3 transgenic mice developed a diabetic phenotype and were susceptible to azoxymethane-induced aberrant crypt foci in their colon tissues. These results suggest that appropriate control of NEU3 gene expression is required for homoeostasis of cellular functions. To gain insights into regulation mechanisms, we determined the gene structure and assessed transcription factor involvement. Oligo-capping analysis indicated the existence of alternative promoters for the NEU3 gene. Transcription started from two clusters of multiple TSSs (transcription start sites); one cluster is preferentially utilized in brain and another in other tissues and cells. Luciferase reporter assays showed further that the region neighbouring the two clusters has promoter activity in the human cell lines analysed. The promoter lacks TATA, but contains CCAAT and CAAC, elements, whose deletions led to a decrease in promoter activity. Electrophoretic mobility-shift assays and chromatin immunoprecipitation demonstrated binding of transcription factors Sp (specificity protein) 1 and Sp3 to the promoter region. Down-regulation of the factors by siRNAs (short interfering RNAs) increased transcription from brain-type TSSs and decreased transcription from other TSSs, suggesting a role for Sp1 and Sp3 in selection of the TSSs. These results indicate that NEU3 expression is diversely regulated by Sp1/Sp3 transcription factors binding to alternative promoters, which might account for multiple modulation of gene expression.

Cholesterol-binding toxins and anti-cholesterol antibodies as structural probes for cholesterol localization.

Cholesterol is one of the major constituents of mammalian cell membranes. It plays an indispensable role in regulating the structure and function of cell membranes and affects the pathology of various diseases. In recent decades much attention has been paid to the existence of membrane microdomains, generally termed lipid "rafts", and cholesterol, along with sphingolipids, is thought to play a critical role in raft structural organization and function. Cholesterol-binding probes are likely to provide useful tools for analyzing the distribution and dynamics of membrane cholesterol, as a structural element of raft microdomains, and elsewhere within the cell. Among the probes, non-toxic derivatives of perfringolysin O, a cholesterol-binding cytolysin, bind cholesterol in a concentration-dependent fashion with a strict threshold. They selectively recognize cholesterol in cholesterol-enriched membranes, and have been used in many studies to detect microdomains in plasma and intracellular membranes. Anti-cholesterol antibodies that recognize cholesterol in domain structures have been developed in recent years. In this chapter, we describe the characteristics of these cholesterol-binding proteins and their applications to studies on membrane cholesterol localization.

Translocation of lysosomal cathepsin D caused by oxidative stress or proteasome inhibition in primary cultured neurons and astrocytes.

We reported previously that N-linked glycoproteins were accumulated in the cytosol of the normal aging rat brain, and that one protein had been identified as cathepsin D (Mech. Ageing Dev., 127, 771-778 (2006)). In this study, to elucidate the mechanism of cathepsin D accumulation in the cytosol, we examined the effects of oxidative stress and proteasome inhibition on the apoptosis and subcellular localization of cathepsin D in primary cultured neurons and astrocytes. Using 4'-6-diamidino-2-phenylindole (DAPI)- or Hoechst 33342-staining and annexin V detection, we found that oxidative stress caused by tert-butyl hydroperoxide and proteasome inhibition by lactacystin induced apoptosis in neurons and astrocytes. Furthermore, after cell fractionation, it was demonstrated that cathepsin D was translocated from lysosomes to cytosol under apoptosis-inducing conditions in both cells. These results suggested that oxidative stress and the suppression of proteasome activity triggered the translocation of cathepsin D from lysosomes to cytosol. The possible mechanism of age-related accumulation of cathepsin D in the cytosol of the normal rat brain will be discussed.

A fully automated deep learning pipeline for high-throughput colony segmentation and classification.

Adenine auxotrophy is a commonly used non-selective genetic marker in yeast research. It allows investigators to easily visualize and quantify various genetic and epigenetic events by simply reading out colony color. However, manual counting of large numbers of colonies is extremely time-consuming, difficult to reproduce and possibly inaccurate. Using cutting-edge neural networks, we have developed a fully automated pipeline for colony segmentation and classification, which speeds up white/red colony quantification 100-fold over manual counting by an experienced researcher. Our approach uses readily available training data and can be smoothly integrated into existing protocols, vastly speeding up screening assays and increasing the statistical power of experiments that employ adenine auxotrophy.

MOV10 as a novel telomerase-associated protein.

A novel telomerase-associated protein was isolated from porcine testis. The 115-kDa protein, purified with telomerase activity, was molecular cloned using human cDNA library, and identified as MOV10. The expression levels of both MOV10 mRNA and MOV10 protein in cancer cells were 2-3 times higher than that of the normal cells, and MOV10 mRNA was highly expressed in human testis and ovary. The anti-MOV10 antibody precipitated the telomerase activity from cancer cell extracts, and inhibited the telomerase activity in vitro. Sf9-expressed MOV10 protein bound to G-rich strand of both single- and double-stranded telomere-sequenced DNA, but not to single C-rich strand. ChIP assay showed the binding of MOV10 to telomere region in vivo. These data suggest that MOV10 is involved in the progression of telomerase-catalyzing reaction via the interaction of telomerase protein and telomere DNA.

Impairment in a negative regulatory system for TCR signaling in CD4+ T cells from old mice.

To examine the involvement of lipid rafts in an age-associated decline in T cell function, we analyzed the effect of aging on the constituents of lipid rafts in resting mouse CD4(+) T cells. We found a pronounced, age-dependent reduction in PAG/Cbp, which is involved in the regulation of Src family kinases (SFKs) by recruiting Csk (a negative regulator of SFKs) to lipid rafts. This reduction is specific for T cells and is attributed, at least in part, to the reduction in its mRNA level. The reduction of PAG accompanies marked impairment in recruiting Csk to lipid rafts and a concomitant decrease in the inactive forms of SFKs. These findings indicate that old mouse CD4(+) T cells have a defect in a negative SFK regulatory system.

Separation of a cholesterol-enriched microdomain involved in T-cell signal transduction.

We isolated a cholesterol-enriched membrane subpopulation from the so-called lipid raft fractions of Jurkat T-cells by taking advantage of its selective binding to a cholesterol-binding probe, BCtheta. The BCtheta-bound membrane subpopulation has a much higher cholesterol/phospholipid (C/P) molar ratio (approximately 1.0) than the BCtheta-unbound population in raft fractions (approximately 0.3). It contains not only the raft markers GM1 and flotillin, but also some T-cell receptor (TCR) signalling molecules, including Lck, Fyn and LAT. In addition, Csk and PAG, inhibitory molecules of the TCR signalling cascade, are also contained in the BCtheta-bound membranes. On the other hand, CD3epsilon, CD3zeta and Zap70 are localized in the BCtheta-unbound membranes, segregated from other TCR signalling molecules under nonstimulated conditions. However, upon stimulation of TCR, portions of CD3epsilon, CD3zeta and Zap70 are recruited to the BCtheta-bound membranes. The Triton X-100 concentration used for lipid raft preparation affects neither the C/P ratio nor protein composition of the BCtheta-bound membranes. These results show that our method is useful for isolating a particular cholesterol-rich membrane domain of T-cells, which could be a core domain controlling the TCR signalling cascade.

Control of cell polarity in response to intra- and extracellular signals in budding yeast.

Budding yeast serves as a powerful genetic model organism for studying the molecular mechanisms of cell polarity in single cells. Like other polarized eukaryotic cells, yeast cells possess polarity programs that regulate where they grow and divide. Establishment of a site of cell polarity may be conceptualized in several stages. First, cells mark a specific location at the cell surface for polarized cell growth and cell division. To define these sites, cells use intrinsic cues present in the cell or landmarks determined by extracellular signals such as morphogens. Second, these landmark proteins then recruit or activate polarity establishment proteins including small GTPases and their regulators. Positive and negative feedback mechanisms are required to transform these site-selection processes into a stable axis of polarity. Finally, these locally activated GTPase modules recruit and activate proteins that organize the actin cytoskeleton and cell growth. In this short review, we describe molecular pathways required to establish oriented cell polarity, and emphasize recent advances in defining positive and negative feedback mechanisms that together may translate an initially weak symmetry-breaking signal into a robust axis of polarity.

Dicer and Hsp104 function in a negative feedback loop to confer robustness to environmental stress.

Epigenetic mechanisms can be influenced by environmental cues and thus evoke phenotypic variation. This plasticity can be advantageous for adaptation but also detrimental if not tightly controlled. Although having attracted considerable interest, it remains largely unknown if and how environmental cues such as temperature trigger epigenetic alterations. Using fission yeast, we demonstrate that environmentally induced discontinuous phenotypic variation is buffered by a negative feedback loop that involves the RNase Dicer and the protein disaggregase Hsp104. In the absence of Hsp104, Dicer accumulates in cytoplasmic inclusions and heterochromatin becomes unstable at elevated temperatures, an epigenetic state inherited for many cell divisions after the heat stress. Loss of Dicer leads to toxic aggregation of an exogenous prionogenic protein. Our results highlight the importance of feedback regulation in building epigenetic memory and uncover Hsp104 and Dicer as homeostatic controllers that buffer environmentally induced stochastic epigenetic variation and toxic aggregation of prionogenic proteins.