Systematic identification of factors for provirus silencing in embryonic stem cells.

Embryonic stem cells (ESCs) repress the expression of exogenous proviruses and endogenous retroviruses (ERVs). Here, we systematically dissected the cellular factors involved in provirus repression in embryonic carcinomas (ECs) and ESCs by a genome-wide siRNA screen. Histone chaperones (Chaf1a/b), sumoylation factors (Sumo2/Ube2i/Sae1/Uba2/Senp6), and chromatin modifiers (Trim28/Eset/Atf7ip) are key determinants that establish provirus silencing. RNA-seq analysis uncovered the roles of Chaf1a/b and sumoylation modifiers in the repression of ERVs. ChIP-seq analysis demonstrates direct recruitment of Chaf1a and Sumo2 to ERVs. Chaf1a reinforces transcriptional repression via its interaction with members of the NuRD complex (Kdm1a, Hdac1/2) and Eset, while Sumo2 orchestrates the provirus repressive function of the canonical Zfp809/Trim28/Eset machinery by sumoylation of Trim28. Our study reports a genome-wide atlas of functional nodes that mediate proviral silencing in ESCs and illuminates the comprehensive, interconnected, and multi-layered genetic and epigenetic mechanisms by which ESCs repress retroviruses within the genome.

Interplay between chromatin marks in development and disease.

DNA methylation (DNAme) and histone post-translational modifications (PTMs) have important roles in transcriptional regulation. Although many reports have characterized the functions of such chromatin marks in isolation, recent genome-wide studies reveal surprisingly complex interactions between them. Here, we focus on the interplay between DNAme and methylation of specific lysine residues on the histone H3 tail. We describe the impact of genetic perturbation of the relevant methyltransferases in the mouse on the landscape of chromatin marks as well as the transcriptome. In addition, we discuss the specific neurodevelopmental growth syndromes and cancers resulting from pathogenic mutations in the human orthologues of these genes. Integrating these observations underscores the fundamental importance of crosstalk between DNA and histone H3 methylation in development and disease.

Paternal MTHFR deficiency leads to hypomethylation of young retrotransposons and reproductive decline across two successive generations.

5,10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in the folate metabolic pathway with a key role in generating methyl groups. As MTHFR deficiency impacts male fertility and sperm DNA methylation, there is the potential for epimutations to be passed to the next generation. Here, we assessed whether the impact of MTHFR deficiency on testis morphology and sperm DNA methylation is exacerbated across generations in mouse. Although MTHFR deficiency in F1 fathers has only minor effects on sperm counts and testis weights and histology, F2 generation sons show further deterioration in reproductive parameters. Extensive loss of DNA methylation is observed in both F1 and F2 sperm, with >80% of sites shared between generations, suggestive of regions consistently susceptible to MTHFR deficiency. These regions are generally methylated during late embryonic germ cell development and are enriched in young retrotransposons. As retrotransposons are resistant to reprogramming of DNA methylation in embryonic germ cells, their hypomethylated state in the sperm of F1 males could contribute to the worsening reproductive phenotype observed in F2 MTHFR-deficient males, compatible with the intergenerational passage of epimutations.

Long Terminal Repeats: From Parasitic Elements to Building Blocks of the Transcriptional Regulatory Repertoire.

The life cycle of endogenous retroviruses (ERVs), also called long terminal repeat (LTR) retrotransposons, begins with transcription by RNA polymerase II followed by reverse transcription and re-integration into the host genome. While most ERVs are relics of ancient integration events, "young" proviruses competent for retrotransposition-found in many mammals, but not humans-represent an ongoing threat to host fitness. As a consequence, several restriction pathways have evolved to suppress their activity at both transcriptional and post-transcriptional stages of the viral life cycle. Nevertheless, accumulating evidence has revealed that LTR sequences derived from distantly related ERVs have been exapted as regulatory sequences for many host genes in a wide range of cell types throughout mammalian evolution. Here, we focus on emerging themes from recent studies cataloging the diversity of ERV LTRs acting as important transcriptional regulatory elements in mammals and explore the molecular features that likely account for LTR exaptation in developmental and tissue-specific gene regulation.

Factor Structure for the Sport Concussion Assessment Tool Symptom Scale in Adolescents After Concussion.

OBJECTIVE: To examine the factor structure of the Sport Concussion Assessment Tool-5 (SCAT5) symptom scale in adolescents on their initial presentation to a concussion clinic within the typical recovery period after concussion (ie, <30 days). We hypothesize that the SCAT5 symptoms represent various clinically meaningful groups. A secondary purpose was to examine the effects of sex on the factor structure of the SCAT5 symptom scale. STUDY DESIGN: Retrospective cross-sectional analysis. SETTING: Tertiary, institutional. PATIENTS: Nine hundred eighty-one adolescents (45% women) aged between 13 and 18 years. INDEPENDENT VARIABLES: Adolescents completed the SCAT5 symptom scale. MAIN OUTCOME MEASURES: The factor structure of SCAT5 examined using a principal axis factor analysis. RESULTS: A 5-factor structure model explained 61% of the variance in symptoms. These 5 factors are identified as Energy (17%), Mental Health (13%), Migrainous (13%), Cognitive (9%), and Vestibulo-Ocular (9%). A similar 5-factor model emerged for each sex, and the proportion of variance in symptoms explained by the 5-factor model was comparable between the sexes. CONCLUSIONS: The findings of this report indicate that the SCAT5 symptoms aggregated into 5 delineated factors, and these factors were largely consistent across the sexes. The delineation of symptoms into 5 factors provides preliminary validation for the presence of different concussion phenotypes. Confirmatory factor analysis is warranted to examine the applicability and clinical utility of the use of the 5-factor structure in a clinical setting.

Setdb1 is required for germline development and silencing of H3K9me3-marked endogenous retroviruses in primordial germ cells.

Transcription of endogenous retroviruses (ERVs) is inhibited by de novo DNA methylation during gametogenesis, a process initiated after birth in oocytes and at approximately embryonic day 15.5 (E15.5) in prospermatogonia. Earlier in germline development, the genome, including most retrotransposons, is progressively demethylated. Young ERVK and ERV1 elements, however, retain intermediate methylation levels. As DNA methylation reaches a low point in E13.5 primordial germ cells (PGCs) of both sexes, we determined whether retrotransposons are marked by H3K9me3 and H3K27me3 using a recently developed low-input ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) method. Although these repressive histone modifications are found predominantly on distinct genomic regions in E13.5 PGCs, they concurrently mark partially methylated long terminal repeats (LTRs) and LINE1 elements. Germline-specific conditional knockout of the H3K9 methyltransferase SETDB1 yields a decrease of both marks and DNA methylation at H3K9me3-enriched retrotransposon families. Strikingly, Setdb1 knockout E13.5 PGCs show concomitant derepression of many marked ERVs, including intracisternal A particle (IAP), ETn, and ERVK10C elements, and ERV-proximal genes, a subset in a sex-dependent manner. Furthermore, Setdb1 deficiency is associated with a reduced number of male E13.5 PGCs and postnatal hypogonadism in both sexes. Taken together, these observations reveal that SETDB1 is an essential guardian against proviral expression prior to the onset of de novo DNA methylation in the germline.

H3S10ph broadly marks early-replicating domains in interphase ESCs and shows reciprocal antagonism with H3K9me2.

Phosphorylation of histone H3 at serine 10 (H3S10ph) by Aurora kinases plays an important role in mitosis; however, H3S10ph also marks regulatory regions of inducible genes in interphase mammalian cells, implicating mitosis-independent functions. Using the fluorescent ubiquitin-mediated cell cycle indicator (FUCCI), we found that 30% of the genome in interphase mouse embryonic stem cells (ESCs) is marked with H3S10ph. H3S10ph broadly demarcates gene-rich regions in G1 and is positively correlated with domains of early DNA replication timing (RT) but negatively correlated with H3K9me2 and lamin-associated domains (LADs). Consistent with mitosis-independent kinase activity, this pattern was preserved in ESCs treated with Hesperadin, a potent inhibitor of Aurora B/C kinases. Disruption of H3S10ph by expression of nonphosphorylatable H3.3S10A results in ectopic spreading of H3K9me2 into adjacent euchromatic regions, mimicking the phenotype observed in Drosophila JIL-1 kinase mutants. Conversely, interphase H3S10ph domains expand in Ehmt1 (also known as Glp) null ESCs, revealing that H3S10ph deposition is restricted by H3K9me2. Strikingly, spreading of H3S10ph at RT transition regions (TTRs) is accompanied by aberrant transcription initiation of genes co-oriented with the replication fork in Ehmt1-/- and Ehmt2-/- ESCs, indicating that establishment of repressive chromatin on the leading strand following DNA synthesis may depend upon these lysine methyltransferases. H3S10ph is also anti-correlated with H3K9me2 in interphase murine embryonic fibroblasts (MEFs) and is restricted to intragenic regions of actively transcribing genes by EHMT2. Taken together, these observations reveal that H3S10ph may play a general role in restricting the spreading of repressive chromatin in interphase mammalian cells.

Does Exercise Increase Vestibular and Ocular Motor Symptom Detection After Sport-Related Concussion?

BACKGROUND AND PURPOSE: Postconcussive vestibular and ocular motor symptoms are common and contribute to longer recovery. The Vestibular/Ocular Motor Screening (VOMS) is used to detect such symptoms, but a VOMS performed at rest may miss symptoms that are only provoked by exertion. Supervised exercise challenges (SECs) have been shown to detect concussion-related symptoms provoked by physical exertion. The purpose of this study was to determine whether athletes undergoing an SEC will exhibit greater symptom provocation with the VOMS compared to a VOMS performed at rest prior to an SEC. METHODS: Thirty-six athletes (58.3% male) between ages 10 and 18 years and within 30 days of concussion were included. All participants completed VOMS assessments at rest and immediately after an SEC. VOMS total symptom score increases were calculated for both pre- and post-SEC assessments compared using Wilcoxon ranked sum tests. The frequencies of positive assessments for each VOMS item were compared using McNemar's test. RESULTS: There were significant increases in post-SEC symptom provocation scores compared with pre-SEC scores for all VOMS items. The post-SEC VOMS identified 29 participants (80.6%) as positive in at least 1 VOMS item compared with 21 participants (58.3%) identified as positive pre-SEC (P = 0.008). For all VOMS items, the post-SEC VOMS identified participants who were previously negative on a pre-SEC VOMS but became positive after the SEC. DISCUSSION AND CONCLUSIONS: An SEC performed prior to a VOMS assessment may increase the detection of vestibular and ocular motor symptoms that may be missed if the VOMS was performed only at rest.Video Abstract available for more insight from the authors (see the Video Supplemental Digital Content 1, available at: http://links.lww.com/JNPT/A342).

Do male and female adolescents report symptoms differently after concussion?

We investigated sex-based differences in the presence and severity of aggregated symptom indicators as well as individual concussion symptoms.Materials and Methods: A cross-sectional examination of sex differences in symptoms reported by adolescents upon initial concussion evaluation at a concussion clinic. Nine hundred and eighty-six adolescents completed the Sport Concussion Assessment Tool (SCAT5) symptom checklist. Chi-square and Mann-Whitney U tests were used for differences in the presence and severity of symptoms, respectively. Sex differences in global indices of symptom distress were compared.Results: Females endorsed more symptoms (Female: Median (M)=15, Interquartile range (IQR):9-18 vs. Male: M=11, IQR: 6-15, p<0.001) and a greater total symptom score (Female: M=37, IQR:16-45 vs. Male: M=20, IQR:8-39). After False Discovery Rate (FDR) adjustment, females endorsed the presence of 21 of 22 individual symptoms more frequently than males (p≤ 0.039), with greater symptom severity for 20 of 22 individual symptoms (p≤0.036). Moderate ESs were observed for sex-based differences in the total symptom score and the global severity index. Small ESs was observed for differences in most individual symptoms.Conclusions: The greater frequency and severity of concussion symptoms reported by female adolescents highlights the importance of considering sex as a modifier for the management of concussion.

Use of Supervised Exercise During Recovery Following Sports-Related Concussion.

OBJECTIVE: To assess the safety of supervised exercise (SE) in acute sport-related concussion (SRC) and its influence on recovery. DESIGN: Retrospective cohort study. SETTING: University SRC clinic at a tertiary care center. PATIENTS: One hundred ninety-four consecutive new patient charts were reviewed. Patients were included if they were seen within 30 days of sustaining a SRC, and their medical records included all required data elements. One hundred twenty-six patients were included in the analysis. INTERVENTIONS: Symptomatic patients who initiated SE within 16 days of SRC (n = 24) were compared with those who did not undergo SE or initiated SE after postinjury day 16 (n = 84). Age, sex, history of previous concussions, injury severity, relevant comorbidities, and other treatments received were included in the analysis. MAIN OUTCOME MEASURES: The association between early SE and clearance for return to sport was determined using a hazard ratio (HR). The number of days from SRC until clearance for return to sport and the number of days symptomatic from concussion were also compared between early SE and nonearly SE cohorts. RESULTS: No serious adverse events occurred in the early SE group. Early SE was associated with earlier return to sport (HR = 2.35, P = 0.030). The early SE group had fewer days from SRC until clearance for return to sport (mean 26.5 ± 11.2 days vs 35.1 ± 26.5 days, P = 0.020). There was a trend toward fewer symptomatic days in the early SE group (P = 0.054). CONCLUSION: Early SE performed in the symptomatic stage of SRC was safe and associated with earlier return to sport.

Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells.

Naive embryonic stem cells hold great promise for research and therapeutics as they have broad and robust developmental potential. While such cells are readily derived from mouse blastocysts it has not been possible to isolate human equivalents easily, although human naive-like cells have been artificially generated (rather than extracted) by coercion of human primed embryonic stem cells by modifying culture conditions or through transgenic modification. Here we show that a sub-population within cultures of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) manifests key properties of naive state cells. These naive-like cells can be genetically tagged, and are associated with elevated transcription of HERVH, a primate-specific endogenous retrovirus. HERVH elements provide functional binding sites for a combination of naive pluripotency transcription factors, including LBP9, recently recognized as relevant to naivety in mice. LBP9-HERVH drives hESC-specific alternative and chimaeric transcripts, including pluripotency-modulating long non-coding RNAs. Disruption of LBP9, HERVH and HERVH-derived transcripts compromises self-renewal. These observations define HERVH expression as a hallmark of naive-like hESCs, and establish novel primate-specific transcriptional circuitry regulating pluripotency.

Changes in Vestibular/Ocular-Motor Screen Scores in Adolescents Treated With Vestibular Therapy After Concussion.

PURPOSE: To examine interrelationships among Vestibular/Ocular-Motor Screen (VOMS) items and to characterize the recovery of VOMS performance in a sample of adolescents treated with vestibular physical therapy (VPT) after concussion. METHODS: Seventy-seven patients with concussion and 77 participants without concussion completed the study. Adolescents with concussion received an individualized VPT intervention consisting of targeted exercises for gaze stability, postural stability, ocular-motor control, habituation, and aerobic activities. The exercises were performed during a weekly clinic visit and via a home exercise program. RESULTS: Except for near-point convergence distance, all VOMS items were significantly interrelated. Over the course of VPT, significant improvements in VOMS performance were observed, and discharge scores were similar to scores observed in adolescents without concussion. CONCLUSIONS: The VOMS measured moderately related functions and captured changes over the course of VPT. Clinicians should consider the contextual risk of "false positive" in their interpretation of VOMS.

Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells.

DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germ line, and may be mediated by Tet (ten eleven translocation) enzymes, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet enzymes have been studied extensively in mouse embryonic stem (ES) cells, which are generally cultured in the absence of vitamin C, a potential cofactor for Fe(II) 2-oxoglutarate dioxygenase enzymes such as Tet enzymes. Here we report that addition of vitamin C to mouse ES cells promotes Tet activity, leading to a rapid and global increase in 5hmC. This is followed by DNA demethylation of many gene promoters and upregulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site of genes affected by vitamin C treatment. Importantly, vitamin C, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay, and the vitamin-C-induced changes in 5hmC and 5mC are entirely suppressed in Tet1 and Tet2 double knockout ES cells. Vitamin C has a stronger effect on regions that gain methylation in cultured ES cells compared to blastocysts, and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A particle retroelements, which are resistant to demethylation in the early embryo, are resistant to vitamin-C-induced DNA demethylation. Collectively, the results of this study establish vitamin C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells.

Correction: hnRNP K Coordinates Transcriptional Silencing by SETDB1 in Embryonic Stem Cells.

[This corrects the article DOI: 10.1371/journal.pgen.1004933.].

Development and application of an integrated allele-specific pipeline for methylomic and epigenomic analysis (MEA).

BACKGROUND: Allele-specific transcriptional regulation, including of imprinted genes, is essential for normal mammalian development. While the regulatory regions controlling imprinted genes are associated with DNA methylation (DNAme) and specific histone modifications, the interplay between transcription and these epigenetic marks at allelic resolution is typically not investigated genome-wide due to a lack of bioinformatic packages that can process and integrate multiple epigenomic datasets with allelic resolution. In addition, existing ad-hoc software only consider SNVs for allele-specific read discovery. This limitation omits potentially informative INDELs, which constitute about one fifth of the number of SNVs in mice, and introduces a systematic reference bias in allele-specific analyses. RESULTS: Here, we describe MEA, an INDEL-aware Methylomic and Epigenomic Allele-specific analysis pipeline which enables user-friendly data exploration, visualization and interpretation of allelic imbalance. Applying MEA to mouse embryonic datasets yields robust allele-specific DNAme maps and low reference bias. We validate allele-specific DNAme at known differentially methylated regions and show that automated integration of such methylation data with RNA- and ChIP-seq datasets yields an intuitive, multidimensional view of allelic gene regulation. MEA uncovers numerous novel dynamically methylated loci, highlighting the sensitivity of our pipeline. Furthermore, processing and visualization of epigenomic datasets from human brain reveals the expected allele-specific enrichment of H3K27ac and DNAme at imprinted as well as novel monoallelically expressed genes, highlighting MEA's utility for integrating human datasets of distinct provenance for genome-wide analysis of allelic phenomena. CONCLUSIONS: Our novel pipeline for standardized allele-specific processing and visualization of disparate epigenomic and methylomic datasets enables rapid analysis and navigation with allelic resolution. MEA is freely available as a Docker container at https://github.com/julienrichardalbert/MEA .

hnRNP K coordinates transcriptional silencing by SETDB1 in embryonic stem cells.

Retrotransposition of endogenous retroviruses (ERVs) poses a substantial threat to genome stability. Transcriptional silencing of a subset of these parasitic elements in early mouse embryonic and germ cell development is dependent upon the lysine methyltransferase SETDB1, which deposits H3K9 trimethylation (H3K9me3) and the co-repressor KAP1, which binds SETDB1 when SUMOylated. Here we identified the transcription co-factor hnRNP K as a novel binding partner of the SETDB1/KAP1 complex in mouse embryonic stem cells (mESCs) and show that hnRNP K is required for ERV silencing. RNAi-mediated knockdown of hnRNP K led to depletion of H3K9me3 at ERVs, concomitant with de-repression of proviral reporter constructs and specific ERV subfamilies, as well as a cohort of germline-specific genes directly targeted by SETDB1. While hnRNP K recruitment to ERVs is dependent upon KAP1, SETDB1 binding at these elements requires hnRNP K. Furthermore, an intact SUMO conjugation pathway is necessary for SETDB1 recruitment to proviral chromatin and depletion of hnRNP K resulted in reduced SUMOylation at ERVs. Taken together, these findings reveal a novel regulatory hierarchy governing SETDB1 recruitment and in turn, transcriptional silencing in mESCs.

ChAsE: chromatin analysis and exploration tool.

: We present ChAsE, a cross-platform desktop application developed for interactive visualization, exploration and clustering of epigenomic data such as ChIP-seq experiments. ChAsE is designed and developed in close collaboration with several groups of biologists and bioinformaticians with a focus on usability and interactivity. Data can be analyzed through k-means clustering, specifying presence or absence of signal in epigenetic data and performing set operations between clusters. Results can be explored in an interactive heat map and profile plot interface and exported for downstream analysis or as high quality figures suitable for publications. AVAILABILITY AND IMPLEMENTATION: Software, source code (MIT License), data and video tutorials available at http://chase.cs.univie.ac.at CONTACT: : mkarimi@brc.ubc.ca or torsten.moeller@univie.ac.atSupplementary information: Supplementary data are available at Bioinformatics online.

Regulation of DNA methylation turnover at LTR retrotransposons and imprinted loci by the histone methyltransferase Setdb1.

During mammalian development, DNA methylation patterns need to be reset in primordial germ cells (PGCs) and preimplantation embryos. However, many LTR retrotransposons and imprinted genes are impervious to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that a subset of such genomic regions are resistant to widespread erasure of DNA methylation in mouse embryonic stem cells (mESCs) lacking the de novo DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b. Intriguingly, these loci are enriched for H3K9me3 in mESCs, implicating this mark in DNA methylation homeostasis. Indeed, deletion of the H3K9 methyltransferase SET domain bifurcated 1 (Setdb1) results in reduced H3K9me3 and DNA methylation levels at specific loci, concomitant with increased 5-hydroxymethylation (5hmC) and ten-eleven translocation 1 binding. Taken together, these data reveal that Setdb1 promotes the persistence of DNA methylation in mESCs, likely reflecting one mechanism by which DNA methylation is maintained at LTR retrotransposons and imprinted genes during developmental stages when DNA methylation is reprogrammed.