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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 translocat

Retrotransposons (RTEs) have been postulated to reactivate with age and contribute to aging through activated innate immune response and inflammation. Here, we analyzed the relationship between RTE expression and aging using published transcriptomic and methylomic datasets of human blood. Despite no observed correlation between RTE activity and chronological age, the expression of most RTE classes and families except short interspersed nuclear elements (SINEs) correlated with biological age-associated gene signature scores. Strikingly, we found that the expression of SINEs was linked to upregulated DNA repair pathways in multiple cohorts. We also observed DNA hypomethylation with aging and the significant increase in RTE expression level in hypomethylated RTEs except for SINEs. Additionally, our single-cell transcriptomic analysis suggested a role for plasma cells in aging mediated by RTEs. Altogether, our multi-omics analysis of large human cohorts highlights the role of RTEs in biological aging and suggests possible mechanisms and cell populations for future investigations., Competing Interests: YT, NS, IT, XZ, WM, SG, GM No competing interests declared, MK Reviewing editor, eLife, (© 2024, Tsai, Seymen et al.)

Mutational profiles of myelodysplastic syndromes (MDS) have established that a relatively small number of genetic aberrations, including SF3B1 and SRSF2 spliceosome mutations, lead to specific phenotypes and prognostic subgrouping. We performed a multi-omics factor analysis (MOFA) on two published MDS cohorts of bone marrow mononuclear cells (BMMNCs) and CD34 + cells with three data modalities (clinical, genotype, and transcriptomics). Seven different views, including immune profile, inflammation/aging, retrotransposon (RTE) expression, and cell-type composition, were derived from these modalities to identify the latent factors with significant impact on MDS prognosis. SF3B1 was the only mutation among 13 mutations in the BMMNC cohort, indicating a significant association with high inflammation. This trend was also observed to a lesser extent in the CD34 + cohort. Interestingly, the MOFA factor representing the inflammation shows a good prognosis for MDS patients with high inflammation. In contrast, SRSF2 mutant cases show a granulocyte-monocyte progenitor (GMP) pattern and high levels of senescence, immunosenescence, and malignant myeloid cells, consistent with their poor prognosis. Furthermore, MOFA identified RTE expression as a risk factor for MDS. This work elucidates the efficacy of our integrative approach to assess the MDS risk that goes beyond all the scoring systems described thus far for MDS., Competing Interests: SG, NS, SH, WM, IT, SJ, DK, AI, AP, JB, GN, GM No competing interests declared, MK Reviewing editor, eLife, (© 2024, Gerlevik, Seymen, Hama et al.)

Origin recognition complex (ORC)-dependent loading of the replicative helicase MCM2-7 onto replication origins in G1-phase forms the basis of replication fork establishment in S-phase. However, how ORC and MCM2-7 facilitate genome-wide DNA licensing is not fully understood. Mapping the molecular footprints of budding yeast ORC and MCM2-7 genome-wide, we discovered that MCM2-7 loading is associated with ORC release from origins and redistribution to non-origin sites. Our bioinformatic analysis revealed that origins are compact units, where a single MCM2-7 double hexamer blocks repetitive loading through steric ORC binding site occlusion. Analyses of A-elements and an improved B2-element consensus motif uncovered that DNA shape, DNA flexibility, and the correct, face-to-face spacing of the two DNA elements are hallmarks of ORC-binding and efficient helicase loading sites. Thus, our work identified fundamental principles for MCM2-7 helicase loading that explain how origin licensing is realised across the genome., (© 2024. The Author(s).)

The role and molecular mechanisms of intermittent fasting (IF) in non-alcoholic steatohepatitis (NASH) and its transition to hepatocellular carcinoma (HCC) are unknown. Here, we identified that an IF 5:2 regimen prevents NASH development as well as ameliorates established NASH and fibrosis without affecting total calorie intake. Furthermore, the IF 5:2 regimen blunted NASH-HCC transition when applied therapeutically. The timing, length, and number of fasting cycles as well as the type of NASH diet were critical parameters determining the benefits of fasting. Combined proteome, transcriptome, and metabolome analyses identified that peroxisome-proliferator-activated receptor alpha (PPARα) and glucocorticoid-signaling-induced PCK1 act co-operatively as hepatic executors of the fasting response. In line with this, PPARα targets and PCK1 were reduced in human NASH. Notably, only fasting initiated during the active phase of mice robustly induced glucocorticoid signaling and free-fatty-acid-induced PPARα signaling. However, hepatocyte-specific glucocorticoid receptor deletion only partially abrogated the hepatic fasting response. In contrast, the combined knockdown of Ppara and Pck1 in vivo abolished the beneficial outcomes of fasting against inflammation and fibrosis. Moreover, overexpression of Pck1 alone or together with Ppara in vivo lowered hepatic triglycerides and steatosis. Our data support the notion that the IF 5:2 regimen is a promising intervention against NASH and subsequent liver cancer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Telomeric repeat-containing RNAs are long non-coding RNAs generated from the telomeres. TERRAs are essential for the establishment of heterochromatin marks at telomeres, which serve for the binding of members of the heterochromatin protein 1 (HP1) protein family of epigenetic modifiers involved with chromatin compaction and gene silencing. While HP1γ is enriched on gene bodies of actively transcribed human and mouse genes, it is unclear if its transcriptional role is important for HP1γ function in telomere cohesion and telomere maintenance. We aimed to study the effect of mouse HP1γ on the transcription of telomere factors and molecules that can affect telomere maintenance., Main Methods: We investigated the telomere function of HP1γ by using HP1γ deficient mouse embryonic fibroblasts (MEFs). We used gene expression analysis of HP1γ deficient MEFs and validated the molecular and mechanistic consequences of HP1γ loss by telomere FISH, immunofluorescence, RT-qPCR and DNA-RNA immunoprecipitation (DRIP)., Key Findings: Loss of HP1γ in primary MEFs led to a downregulation of various telomere and telomere-accessory transcripts, including the shelterin protein TRF1. Its downregulation is associated with increased telomere replication stress and DNA damage (γH2AX), effects more profound in females. We suggest that the source for the impaired telomere maintenance is a consequence of increased telomeric DNA-RNA hybrids and TERRAs arising at and from mouse chromosomes 18 and X., Significance: Our results suggest an important transcriptional control by mouse HP1γ of various telomere factors including TRF1 protein and TERRAs that has profound consequences on telomere stability, with a potential sexually dimorphic nature., Competing Interests: Declaration of competing interest There are no conflicts of interest., (Copyright © 2023 Imperial College London. Published by Elsevier Inc. All rights reserved.)

We present a general purpose visual analysis system that can be used for exploring parameters of a variety of computer models. Our proposed system offers key components of a visual parameter analysis framework including parameter sampling, deriving output summaries, and an exploration interface. It also provides an API for rapid development of parameter space exploration solutions as well as the flexibility to support custom workflows for different application domains. We evaluate the effectiveness of our system by demonstrating it in three domains: data mining, machine learning and specific application in bioinformatics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Younesy, Pober, Möller and Karimi.)

Innate immune responses rely on inducible gene expression programmes which, in contrast to steady-state transcription, are highly dependent on cohesin. Here we address transcriptional parameters underlying this cohesin-dependence by single-molecule RNA-FISH and single-cell RNA-sequencing. We show that inducible innate immune genes are regulated predominantly by an increase in the probability of active transcription, and that probabilities of enhancer and promoter transcription are coordinated. Cohesin has no major impact on the fraction of transcribed inducible enhancers, or the number of mature mRNAs produced per transcribing cell. Cohesin is, however, required for coupling the probabilities of enhancer and promoter transcription. Enhancer-promoter coupling may not be explained by spatial proximity alone, and at the model locus Il12b can be disrupted by selective inhibition of the cohesinopathy-associated BET bromodomain BD2. Our data identify discrete steps in enhancer-mediated inducible gene expression that differ in cohesin-dependence, and suggest that cohesin and BD2 may act on shared pathways., (© 2022. The Author(s).)

RUNX1 is a transcription factor that plays key roles in hematopoietic development and in hematopoiesis and lymphopoiesis. In this article, we report that RUNX1 regulates a gene expression program in naive mouse B cells that affects the dynamics of cell cycle entry in response to stimulation of the BCR. Conditional knockout of Runx1 in mouse resting B cells resulted in accelerated entry into S-phase after BCR engagement. Our results indicate that Runx1 regulates the cyclin D2 ( Ccnd2 ) gene, the immediate early genes Fosl2 , Atf3 , and Egr2 , and the Notch pathway gene Rbpj in mouse B cells, reducing the rate at which transcription of these genes increases after BCR stimulation. RUNX1 interacts with the chromatin remodeler SNF-2-related CREB-binding protein activator protein (SRCAP), recruiting it to promoter and enhancer regions of the Ccnd2 gene. BCR-mediated activation triggers switching between binding of RUNX1 and its paralog RUNX3 and between SRCAP and the switch/SNF remodeling complex member BRG1. Binding of BRG1 is increased at the Ccnd2 and Rbpj promoters in the Runx1 knockout cells after BCR stimulation. We also find that RUNX1 exerts positive or negative effects on a number of genes that affect the activation response of mouse resting B cells. These include Cd22 and Bank1 , which act as negative regulators of the BCR, and the IFN receptor subunit gene Ifnar1 The hyperresponsiveness of the Runx1 knockout B cells to BCR stimulation and its role in regulating genes that are associated with immune regulation suggest that RUNX1 could be involved in regulating B cell tolerance., (Copyright © 2021 by The Authors.)

Motivation: Deciphering nucleosome-nucleosome interactions is an important step toward mesoscale description of chromatin organization but computational tools to perform such analyses are not publicly available., Results: We developed iNucs, a user-friendly and efficient Python-based bioinformatics tool to compute and visualize nucleosome-resolved interactions using standard pairs format input generated from pairtools., Availabilityand Implementation: https://github.com/Karimi-Lab/inucs/., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press.)

Cell competition is emerging as a quality-control mechanism that eliminates unfit cells in a wide range of settings from development to the adult. However, the nature of the cells normally eliminated by cell competition and what triggers their elimination remains poorly understood. In mice, 35% of epiblast cells are eliminated before gastrulation. Here we show that cells with mitochondrial defects are eliminated by cell competition during early mouse development. Using single-cell transcriptional profiling of eliminated mouse epiblast cells, we identify hallmarks of cell competition and mitochondrial defects. We demonstrate that mitochondrial defects are common to a range of different loser cell types and that manipulating mitochondrial function triggers cell competition. Moreover, we show that in the mouse embryo, cell competition eliminates cells with sequence changes in mt-Rnr1 and mt-Rnr2, and that even non-pathological changes in mitochondrial DNA sequences can induce cell competition. Our results suggest that cell competition is a purifying selection that optimizes mitochondrial performance before gastrulation., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Epigenetic modifications and nucleosome positioning play an important role in modulating gene expression. However, how the patterns of epigenetic modifications and nucleosome positioning are established around promoters is not well understood. Here, we have addressed these questions in a series of genome-wide experiments coupled to a novel bioinformatic analysis approach. Our data reveal a clear correlation between CpG density, promoter activity and accumulation of active or repressive histone marks. CGI boundaries define the chromatin promoter regions that will be epigenetically modified. CpG-rich promoters are targeted by histone modifications and histone variants, while CpG-poor promoters are regulated by DNA methylation. CGIs boundaries, but not transcriptional activity, are essential determinants of H2A.Z positioning in vicinity of the promoters, suggesting that the presence of H2A.Z is not related to transcriptional control. Accordingly, H2A.Z depletion has no impact on gene expression of arrested mouse embryonic fibroblasts. Therefore, the underlying DNA sequence, the promoter CpG density and, to a lesser extent, transcriptional activity, are key factors implicated in promoter chromatin architecture., Competing Interests: Declarations of Interest None., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

CD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling. Here we use single cell RNA sequencing (scRNA-seq) to classify mouse thymocyte selection intermediates by coreceptor gene expression. In the unperturbed thymus, Cd4 + Cd8a - selection intermediates appear before Cd4 - Cd8a + selection intermediates, but the timing of these subsets is flexible according to the strength of TCR signals. Our data show that selection intermediates discriminate MHC class prior to the loss of coreceptor expression and suggest a model where signal strength informs the timing of coreceptor gene activity and ultimately CD4/CD8 lineage choice.

Chromosome movements and programmed DNA double-strand breaks (DSBs) promote homologue pairing and initiate recombination at meiosis onset. Meiotic progression involves checkpoint-controlled termination of these events when all homologue pairs achieve synapsis and form crossover precursors. Exploiting the temporo-spatial organisation of the C. elegans germline and time-resolved methods of protein removal, we show that surveillance of the synaptonemal complex (SC) controls meiotic progression. In nuclei with fully synapsed homologues and crossover precursors, removing different meiosis-specific cohesin complexes, which are individually required for SC stability, or a SC central region component causes functional redeployment of the chromosome movement and DSB machinery, triggering whole-nucleus reorganisation. This apparent reversal of the meiotic programme requires CHK-2 kinase reactivation via signalling from chromosome axes containing HORMA proteins, but occurs in the absence of transcriptional changes. Our results uncover an unexpected plasticity of the meiotic programme and show how chromosome signalling orchestrates nuclear organisation and meiotic progression.

Genetic variation in the enzymes that catalyze posttranslational modification of proteins is a potentially important source of phenotypic variation during evolution. Ubiquitination is one such modification that affects turnover of virtually all of the proteins in the cell in addition to roles in signaling and epigenetic regulation. UBE2D3 is a promiscuous E2 enzyme, which acts as an ubiquitin donor for E3 ligases that catalyze ubiquitination of developmentally important proteins. We have used protein sequence comparison of UBE2D3 orthologs to identify a position in the C-terminal α-helical region of UBE2D3 that is occupied by a conserved serine in amniotes and by alanine in anamniote vertebrate and invertebrate lineages. Acquisition of the serine (S138) in the common ancestor to modern amniotes created a phosphorylation site for Aurora B. Phosphorylation of S138 disrupts the structure of UBE2D3 and reduces the level of the protein in mouse embryonic stem cells (ESCs). Substitution of S138 with the anamniote alanine (S138A) increases the level of UBE2D3 in ESCs as well as being a gain of function early embryonic lethal mutation in mice. When mutant S138A ESCs were differentiated into extraembryonic primitive endoderm, levels of the PDGFRα and FGFR1 receptor tyrosine kinases were reduced and primitive endoderm differentiation was compromised. Proximity ligation analysis showed increased interaction between UBE2D3 and the E3 ligase CBL and between CBL and the receptor tyrosine kinases. Our results identify a sequence change that altered the ubiquitination landscape at the base of the amniote lineage with potential effects on amniote biology and evolution., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Summary: Transposable elements (TEs) influence the evolution of novel transcriptional networks yet the specific and meaningful interpretation of how TE-derived transcriptional initiation contributes to the transcriptome has been marred by computational and methodological deficiencies. We developed LIONS for the analysis of RNA-seq data to specifically detect and quantify TE-initiated transcripts., Availability and Implementation: Source code, container, test data and instruction manual are freely available at www.github.com/ababaian/LIONS., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cell-fate determination is influenced by interactions between master transcription factors (TFs) and cis-regulatory elements. Hepatocyte nuclear factor 4 alpha (HNF4A), a liver-enriched TF, acts as a master controller in specification of hepatic progenitor cells by regulating a network of TFs to control onset of hepatocyte cell fate. Using analysis of genome-wide histone modifications, DNA methylation, and hydroxymethylation in mouse hepatocytes, we show that HNF4A occupies active enhancers in hepatocytes and is essential for active histone and DNA signatures, especially acetylation of lysine 27 of histone 3 (H3K27ac) and 5-hydroxymethylcytosine (5hmC). In mice lacking HNF4A protein in hepatocytes, we observed a decrease in both H3K27ac and hydroxymethylation at regions bound by HNF4A. Mechanistically, HNF4A-associated hydroxymethylation (5hmC) requires its interaction with ten-eleven translocation methylcytosine dioxygenase 3 (TET3), a protein responsible for oxidation from 5mC to 5hmC. Furthermore, HNF4A regulates TET3 expression in liver by directly binding to an enhancer region. Conclusion: In conclusion, we identified that HNF4A is required for the active epigenetic state at enhancers that amplifies transcription of genes in hepatocytes., (© 2019 by the American Association for the Study of Liver Diseases.)

Cohesin is a regulator of genome architecture with roles in sister chromatid cohesion and chromosome compaction. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes. Here, we show that the role of cohesin in chromosome organization requires the histone chaperone FACT ('facilitates chromatin transcription') in Saccharomyces cerevisiae. We find that FACT interacts directly with cohesin, and is dynamically required for its localization on chromatin. Depletion of FACT in metaphase cells prevents cohesin accumulation at pericentric regions and causes reduced binding on chromosome arms. Using the Hi-C technique, we show that cohesin-dependent TAD (topological associated domain)-like structures in G1 and metaphase chromosomes are reduced in the absence of FACT. Sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our data show that FACT contributes to the formation of cohesin-dependent TADs, thus uncovering a new role for this complex in nuclear organization during interphase and mitotic chromosome folding.

De novo DNA methylation (DNAme) during mouse oogenesis occurs within transcribed regions enriched for H3K36me3. As many oocyte transcripts originate in long terminal repeats (LTRs), which are heterogeneous even between closely related mammals, we examined whether species-specific LTR-initiated transcription units (LITs) shape the oocyte methylome. Here we identify thousands of syntenic regions in mouse, rat, and human that show divergent DNAme associated with private LITs, many of which initiate in lineage-specific LTR retrotransposons. Furthermore, CpG island (CGI) promoters methylated in mouse and/or rat, but not human oocytes, are embedded within rodent-specific LITs and vice versa. Notably, at a subset of such CGI promoters, DNAme persists on the maternal genome in fertilized and parthenogenetic mouse blastocysts or in human placenta, indicative of species-specific epigenetic inheritance. Polymorphic LITs are also responsible for disparate DNAme at promoter CGIs in distantly related mouse strains, revealing that LITs also promote intra-species divergence in CGI DNAme.

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 .

Transposable elements (TEs) comprise almost half of primate genomes and their aberrant regulation can result in deleterious effects. In pluripotent stem cells, rapidly evolving KRAB-ZNF genes target TEs for silencing by H3K9me3. To investigate the evolution of TE silencing, we performed H3K9me3 ChIP-seq experiments in induced pluripotent stem cells from 10 human and 7 chimpanzee individuals. We identified four million orthologous TEs and found the SVA and ERV families to be marked most frequently by H3K9me3. We found little evidence of inter-species differences in TE silencing, with as many as 82% of putatively silenced TEs marked at similar levels in humans and chimpanzees. TEs that are preferentially silenced in one species are a similar age to those silenced in both species and are not more likely to be associated with expression divergence of nearby orthologous genes. Our data suggest limited species-specificity of TE silencing across 6 million years of primate evolution., Competing Interests: MW, SZ, KL, BP, MK, MS, YG No competing interests declared, (© 2018, Ward et al.)

The development of cell therapy for repairing damaged or diseased skeletal muscle has been hindered by the inability to significantly expand immature, transplantable myogenic stem cells (MuSCs) in culture. To overcome this limitation, a deeper understanding of the mechanisms regulating the transition between activated, proliferating MuSCs and differentiation-primed, poorly engrafting progenitors is needed. Here, we show that methyltransferase Setd7 facilitates such transition by regulating the nuclear accumulation of β-catenin in proliferating MuSCs. Genetic or pharmacological inhibition of Setd7 promotes in vitro expansion of MuSCs and increases the yield of primary myogenic cell cultures. Upon transplantation, both mouse and human MuSCs expanded with a Setd7 small-molecule inhibitor are better able to repopulate the satellite cell niche, and treated mouse MuSCs show enhanced therapeutic potential in preclinical models of muscular dystrophy. Thus, Setd7 inhibition may help bypass a key obstacle in the translation of cell therapy for muscle disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (© 2018 Chen et al.; Published by Cold Spring Harbor Laboratory Press.)

Remnants of ancient transposable elements (TEs) are abundant in mammalian genomes. These sequences contain multiple regulatory motifs and hence are capable of influencing expression of host genes. TEs are known to be released from epigenetic repression and can become transcriptionally active in cancer. Such activation could also lead to lineage-inappropriate activation of oncogenes, as previously described in lymphomas. However, there are few reports of this mechanism occurring in non-blood cancers. Here, we re-analyzed whole transcriptome data from a large cohort of patients with colon cancer, compared to matched normal colon control samples, to detect genes or transcripts ectopically expressed through activation of TE promoters. Among many such transcripts, we identified six where the affected gene has described role in cancer and where the TE-driven gene mRNA is expressed in primary colon cancer, but not normal matched tissue, and confirmed expression in colon cancer-derived cell lines. We further characterized a TE-gene chimeric transcript involving the Interleukin 33 (IL-33) gene (termed LTR-IL-33), that is ectopically expressed in a subset of colon cancer samples through the use of an endogenous retroviral long terminal repeat (LTR) promoter of the MSTD family. The LTR-IL-33 chimeric transcript encodes a novel shorter isoform of the protein, which is missing the initial N-terminus (including many conserved residues) of Native IL-33. In vitro studies showed that LTR-IL-33 expression is required for optimal CRC cell line growth as 3D colonospheres. Taken together, these data demonstrate the significance of TEs as regulators of aberrant gene expression in colon cancer.

: 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., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Repression of endogenous retroviruses (ERVs) in mammals involves several epigenetic mechanisms. Acute loss of the maintenance methyltransferase Dnmt1 induces widespread DNA demethylation and transcriptional activation of ERVs, including CpG-rich IAP (intracisternal A particle) proviruses. Here, we show that this effect is not due simply to a loss of DNA methylation. Conditional deletions reveal that both Dnmt1 and Np95 are essential for maintenance DNA methylation. However, while IAPs are derepressed in Dnmt1-ablated embryos and embryonic stem cells (ESCs), these ERVs remain silenced when Np95 is deleted alone or in combination with Dnmt1. This paradoxical phenotype results from an ectopic interaction between NP95 and the H3K9 methyltransferase SETDB1. Normally, SETDB1 maintains silencing of IAPs, but in the absence of DNMT1, prolonged binding of NP95 to hemimethylated DNA transiently disrupts SETDB1-dependent H3K9me3 deposition. Thus, our observations reveal an unexpected antagonistic interplay between two repressive pathways involved in retroviral silencing in mammalian cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The transcription factor interferon regulatory factor 5 (IRF5) is upregulated in Hodgkin lymphoma (HL) and is a key regulator of the aberrant transcriptome characteristic of this disease. Here we show that IRF5 upregulation in HL is driven by transcriptional activation of a normally dormant endogenous retroviral LOR1a long terminal repeat (LTR) upstream of IRF5. Specifically, through screening of RNA-sequencing libraries, we detected LTR-IRF5 chimeric transcripts in multiple HL cell lines but not in normal B-cell controls. In HL, the LTR was in an open and hypomethylated epigenetic state, and we further show the LTR is the site of transcriptional initiation. Among HL cell lines, usage of the LTR promoter strongly correlates with overall levels of IRF5 mRNA and protein, indicating that LTR transcriptional awakening is a major contributor to IRF5 upregulation in HL. Taken together, oncogenic IRF5 overexpression in HL is the result of a specific LTR transcriptional activation. We propose that such LTR derepression is a distinct mechanism of oncogene activation ('onco-exaptation'), and that such a mechanism warrants further investigation in molecular and cancer research.

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.

Combined chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) has enabled genome-wide epigenetic profiling of numerous cell lines and tissue types. A major limitation of ChIP-seq, however, is the large number of cells required to generate high-quality data sets, precluding the study of rare cell populations. Here, we present an ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP) and sequencing method to generate genome-wide histone mark profiles with high resolution from as few as 10(3) cells. We demonstrate that ULI-NChIP-seq generates high-quality maps of covalent histone marks from 10(3) to 10(6) embryonic stem cells. Subsequently, we show that ULI-NChIP-seq H3K27me3 profiles generated from E13.5 primordial germ cells isolated from single male and female embryos show high similarity to recent data sets generated using 50-180 × more material. Finally, we identify sexually dimorphic H3K27me3 enrichment at specific genic promoters, thereby illustrating the utility of this method for generating high-quality and -complexity libraries from rare cell populations.

Background: Several tools have been developed to enable biologists to perform initial browsing and exploration of sequencing data. However the computational tool set for further analyses often requires significant computational expertise to use and many of the biologists with the knowledge needed to interpret these data must rely on programming experts., Results: We present VisRseq, a framework for analysis of sequencing datasets that provides a computationally rich and accessible framework for integrative and interactive analyses without requiring programming expertise. We achieve this aim by providing R apps, which offer a semi-auto generated and unified graphical user interface for computational packages in R and repositories such as Bioconductor. To address the interactivity limitation inherent in R libraries, our framework includes several native apps that provide exploration and brushing operations as well as an integrated genome browser. The apps can be chained together to create more powerful analysis workflows., Conclusions: To validate the usability of VisRseq for analysis of sequencing data, we present two case studies performed by our collaborators and report their workflow and insights.

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., (© 2014 Liu et al.; Published by Cold Spring Harbor Laboratory Press.)

Remnants of ancient transposable elements (TEs) are abundant in mammalian genomes. These sequences harbor multiple regulatory motifs and hence are capable of influencing expression of host genes. In response to environmental changes, TEs are known to be released from epigenetic repression and to become transcriptionally active. Such activation could also lead to lineage-inappropriate activation of oncogenes, as one study described in Hodgkin lymphoma. However, little further evidence for this mechanism in other cancers has been reported. Here, we reanalyzed whole transcriptome data from a large cohort of patients with diffuse large B-cell lymphoma (DLBCL) compared with normal B-cell centroblasts to detect genes ectopically expressed through activation of TE promoters. We have identified 98 such TE-gene chimeric transcripts that were exclusively expressed in primary DLBCL cases and confirmed several in DLBCL-derived cell lines. We further characterized a TE-gene chimeric transcript involving a fatty acid-binding protein gene (LTR2-FABP7), normally expressed in brain, that was ectopically expressed in a subset of DLBCL patients through the use of an endogenous retroviral LTR promoter of the LTR2 family. The LTR2-FABP7 chimeric transcript encodes a novel chimeric isoform of the protein with characteristics distinct from native FABP7. In vitro studies reveal a dependency for DLBCL cell line proliferation and growth on LTR2-FABP7 chimeric protein expression. Taken together, these data demonstrate the significance of TEs as regulators of aberrant gene expression in cancer and suggest that LTR2-FABP7 may contribute to the pathogenesis of DLBCL in a subgroup of patients.

The assessment of expression and epigenomic status using sequencing based methods provides an unprecedented opportunity to identify and correlate allelic differences with epigenomic status. We present ALEA, a computational toolbox for allele-specific epigenomics analysis, which incorporates allelic variation data within existing resources, allowing for the identification of significant associations between epigenetic modifications and specific allelic variants in human and mouse cells. ALEA provides a customizable pipeline of command line tools for allele-specific analysis of next-generation sequencing data (ChIP-seq, RNA-seq, etc.) that takes the raw sequencing data and produces separate allelic tracks ready to be viewed on genome browsers. The pipeline has been validated using human and hybrid mouse ChIP-seq and RNA-seq data., Availability: The package, test data and usage instructions are available online at http://www.bcgsc.ca/platform/bioinfo/software/alea CONTACT: : mkarimi1@interchange.ubc.ca or sjones@bcgsc.ca Supplementary information: Supplementary data are available at Bioinformatics online., (© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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.

Background: In mouse embryonic stem cells (mESCs), transcriptional silencing of numerous class I and II endogenous retroviruses (ERVs), including IAP, ETn and MMERVK10C, is dependent upon the H3K9 methyltransferase (KMTase) SETDB1/ESET and its binding partner KAP1/TRIM28. In contrast, the H3K9 KMTases G9a and GLP and HP1 proteins are dispensable for this process. Intriguingly, MERVL retroelements are actively transcribed exclusively in the two-cell (2C) embryo, but the molecular basis of silencing of these class III ERVs at later developmental stages has not been systematically addressed., Results: Here, we characterized the roles of these chromatin factors in MERVL silencing in mESCs. While MMERVK10C and IAP ERVs are bound by SETDB1 and KAP1 and are induced following their deletion, MERVL ERVs show relatively low levels of SETDB1 and KAP1 binding and are upregulated exclusively following KAP1 depletion, indicating that KAP1 influences MERVL expression independent of SETDB1. In contrast to class I and class II ERVs, MERVL and MERVL LTR-driven genic transcripts are also upregulated following depletion of G9a or GLP, and G9a binds directly to these ERVs. Consistent with a direct role for H3K9me2 in MERVL repression, these elements are highly enriched for G9a-dependent H3K9me2, and catalytically active G9a is required for silencing of MERVL LTR-driven transcripts. MERVL is also derepressed in HP1α and HP1β KO ESCs. However, like KAP1, HP1α and HP1β are only modestly enriched at MERVL relative to IAP LTRs. Intriguingly, as recently shown for KAP1, RYBP, LSD1 and G9a-deficient mESCs, many genes normally expressed in the 2C embryo are also induced in HP1 KO mESCs, revealing that aberrant expression of a subset of 2C-specific genes is a common feature in each of these KO lines., Conclusions: Our results indicate that G9a and GLP, which are not required for silencing of class I and II ERVs, are recruited to MERVL elements and play a direct role in silencing of these class III ERVs, dependent upon G9a catalytic activity. In contrast, induction of MERVL expression in KAP1, HP1α and HP1β KO ESCs may occur predominantly as a consequence of indirect effects, in association with activation of a subset of 2C-specific genes.

The "arms race" relationship between transposable elements (TEs) and their host has promoted a series of epigenetic silencing mechanisms directed against TEs. Retrotransposons, a class of TEs, are often located in repressed regions and are thought to induce heterochromatin formation and spreading. However, direct evidence for TE-induced local heterochromatin in mammals is surprisingly scarce. To examine this phenomenon, we chose two mouse embryonic stem (ES) cell lines that possess insertionally polymorphic retrotransposons (IAP, ETn/MusD, and LINE elements) at specific loci in one cell line but not the other. Employing ChIP-seq data for these cell lines, we show that IAP elements robustly induce H3K9me3 and H4K20me3 marks in flanking genomic DNA. In contrast, such heterochromatin is not induced by LINE copies and only by a minority of polymorphic ETn/MusD copies. DNA methylation is independent of the presence of IAP copies, since it is present in flanking regions of both full and empty sites. Finally, such spreading into genes appears to be rare, since the transcriptional start sites of very few genes are less than one Kb from an IAP. However, the B3galtl gene is subject to transcriptional silencing via IAP-induced heterochromatin. Hence, although rare, IAP-induced local heterochromatin spreading into nearby genes may influence expression and, in turn, host fitness., Competing Interests: The authors have declared that no competing interests exist.

DNA methylation and histone H3 lysine 9 trimethylation (H3K9me3) play important roles in silencing of genes and retroelements. However, a comprehensive comparison of genes and repetitive elements repressed by these pathways has not been reported. Here we show that in mouse embryonic stem cells (mESCs), the genes upregulated after deletion of the H3K9 methyltransferase Setdb1 are distinct from those derepressed in mESC deficient in the DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b, with the exception of a small number of primarily germline-specific genes. Numerous endogenous retroviruses (ERVs) lose H3K9me3 and are concomitantly derepressed exclusively in SETDB1 knockout mESCs. Strikingly, ~15% of upregulated genes are induced in association with derepression of promoter-proximal ERVs, half in the context of "chimeric" transcripts that initiate within these retroelements and splice to genic exons. Thus, SETDB1 plays a previously unappreciated yet critical role in inhibiting aberrant gene transcription by suppressing the expression of proximal ERVs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The problem of determining haplotypes from genotypes has gained considerable prominence in the research community since the beginning of the HapMap project. Here the focus is on determining the sets of SNP values of individual chromosomes (haplotypes), since such information better captures the genetic causes of diseases. One of the main algorithmic tools for haplotyping is based on the assumption that the evolutionary history for the original haplotypes satisfies perfect phylogeny. This tool can be applied only on individual blocks of chromosomes, in which it is assumed that recombinations do not happen. However, exact determination of blocks is usually not possible. It would be desirable to develop a method for haplotyping which can account for recombinations, and thus can be applied on multiblock sections of chromosomes. A natural candidate for such a method is haplotyping via phylogenetic networks (which model recombinations) or their simplified version: galled-tree networks. However, even haplotyping via galled-tree networks appears hard, as the efficient algorithms exist only for very special cases: the galled-tree network has either a single gall or only small galls with two mutations each. Building on our previous results, we show that, in general, haplotyping via galled-tree networks is NP-complete, and it remains NP-complete when galls are allowed to have at most k mutations, for any k ≥ 3.