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.

ModEx: a general purpose computer model exploration system.

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.

Cohesin couples transcriptional bursting probabilities of inducible enhancers and promoters.

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.

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.

Cell competition acts as a purifying selection to eliminate cells with mitochondrial defects during early mouse development.

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.

The order and logic of CD4 versus CD8 lineage choice and differentiation in mouse thymus.

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.

Surveillance of cohesin-supported chromosome structure controls meiotic progression.

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.

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.

FACT mediates cohesin function on chromatin.

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.

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.

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.

LTR retrotransposons transcribed in oocytes drive species-specific and heritable changes in DNA methylation.

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.

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 .

Silencing of transposable elements may not be a major driver of regulatory evolution in primate iPSCs.

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.

Inhibition of Methyltransferase Setd7 Allows the In Vitro Expansion of Myogenic Stem Cells with Improved Therapeutic Potential.

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 ß-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.

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.

A novel isoform of IL-33 revealed by screening for transposable element promoted genes in human colorectal cancer.

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.