RUNX1 Regulates a Transcription Program That Affects the Dynamics of Cell Cycle Entry of Naive Resting B Cells.

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.

iNucs: inter-nucleosome interactions.

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.

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.

Evolution of an Amniote-Specific Mechanism for Modulating Ubiquitin Signaling via Phosphoregulation of the E2 Enzyme UBE2D3.

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.

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.

LIONS: analysis suite for detecting and quantifying transposable element initiated transcription from RNA-seq.

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.

Hepatocyte Nuclear Factor 4-Alpha Is Essential for the Active Epigenetic State at Enhancers in Mouse Liver.

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.

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.

Distinct isoform of FABP7 revealed by screening for retroelement-activated genes in diffuse large B-cell lymphoma.

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.

VisRseq: R-based visual framework for analysis of sequencing data.

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.

ALEA: a toolbox for allele-specific epigenomics analysis.

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.

Retrotransposon-induced heterochromatin spreading in the mouse revealed by insertional polymorphisms.

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.

MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing.

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.

Identification of novel myelodysplastic syndromes prognostic subgroups by integration of inflammation, cell-type composition, and immune signatures in the bone marrow.

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.

A 5:2 intermittent fasting regimen ameliorates NASH and fibrosis and blunts HCC development via hepatic PPARα and PCK1.

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.