ChromGene: gene-based modeling of epigenomic data.

Various computational approaches have been developed to annotate epigenomes on a per-position basis by modeling combinatorial and spatial patterns within epigenomic data. However, such annotations are less suitable for gene-based analyses. We present ChromGene, a method based on a mixture of learned hidden Markov models, to annotate genes based on multiple epigenomic maps across the gene body and flanks. We provide ChromGene assignments for over 100 cell and tissue types. We characterize the mixture components in terms of gene expression, constraint, and other gene annotations. The ChromGene method and annotations will provide a useful resource for gene-based epigenomic analyses.

An integrative approach for fine-mapping chromatin interactions.

MOTIVATION: Chromatin interactions play an important role in genome architecture and gene regulation. The Hi-C assay generates such interactions maps genome-wide, but at relatively low resolutions (e.g. 5-25 kb), which is substantially coarser than the resolution of transcription factor binding sites or open chromatin sites that are potential sources of such interactions. RESULTS: To predict the sources of Hi-C-identified interactions at a high resolution (e.g. 100 bp), we developed a computational method that integrates data from DNase-seq and ChIP-seq of TFs and histone marks. Our method, χ-CNN, uses this data to first train a convolutional neural network (CNN) to discriminate between called Hi-C interactions and non-interactions. χ-CNN then predicts the high-resolution source of each Hi-C interaction using a feature attribution method. We show these predictions recover original Hi-C peaks after extending them to be coarser. We also show χ-CNN predictions enrich for evolutionarily conserved bases, eQTLs and CTCF motifs, supporting their biological significance. χ-CNN provides an approach for analyzing important aspects of genome architecture and gene regulation at a higher resolution than previously possible. AVAILABILITY AND IMPLEMENTATION: χ-CNN software is available on GitHub (https://github.com/ernstlab/X-CNN). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype.

BACKGROUND: Improvement in the performance of eukaryotic microalgae for biofuel and bioproduct production is largely dependent on characterization of metabolic mechanisms within the cell. The marine diatom Cyclotella cryptica, which was originally identified in the Aquatic Species Program, is a promising strain of microalgae for large-scale production of biofuel and bioproducts, such as omega-3 fatty acids. RESULTS: We sequenced the nuclear genome and methylome of this oleaginous diatom to identify the genetic traits that enable substantial accumulation of triacylglycerol. The genome is comprised of highly methylated repetitive sequence, which does not significantly change under silicon starved lipid induction, and data further suggests the primary role of DNA methylation is to suppress DNA transposition. Annotation of pivotal glycolytic, lipid metabolism, and carbohydrate degradation processes reveal an expanded enzyme repertoire in C. cryptica that would allow for an increased metabolic capacity toward triacylglycerol production. Identification of previously unidentified genes, including those involved in carbon transport and chitin metabolism, provide potential targets for genetic manipulation of carbon flux to further increase its lipid phenotype. New genetic tools were developed, bringing this organism on a par with other microalgae in terms of genetic manipulation and characterization approaches. CONCLUSIONS: Functional annotation and detailed cross-species comparison of key carbon rich processes in C. cryptica highlights the importance of enzymatic subcellular compartmentation for regulation of carbon flux, which is often overlooked in photosynthetic microeukaryotes. The availability of the genome sequence, as well as advanced genetic manipulation tools enable further development of this organism for deployment in large-scale production systems.

Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming.

Pioneer transcription factors (TFs) access silent chromatin and initiate cell-fate changes, using diverse types of DNA binding domains (DBDs). FoxA, the paradigm pioneer TF, has a winged helix DBD that resembles linker histone and thereby binds its target sites on nucleosomes and in compacted chromatin. Herein, we compare the nucleosome and chromatin targeting activities of Oct4 (POU DBD), Sox2 (HMG box DBD), Klf4 (zinc finger DBD), and c-Myc (bHLH DBD), which together reprogram somatic cells to pluripotency. Purified Oct4, Sox2, and Klf4 proteins can bind nucleosomes in vitro, and in vivo they preferentially target silent sites enriched for nucleosomes. Pioneer activity relates simply to the ability of a given DBD to target partial motifs displayed on the nucleosome surface. Such partial motif recognition can occur by coordinate binding between factors. Our findings provide insight into how pioneer factors can target naive chromatin sites.

A Genome-Wide Chronological Study of Gene Expression and Two Histone Modifications, H3K4me3 and H3K9ac, during Developmental Leaf Senescence.

The genome-wide abundance of two histone modifications, H3K4me3 and H3K9ac (both associated with actively expressed genes), was monitored in Arabidopsis (Arabidopsis thaliana) leaves at different time points during developmental senescence along with expression in the form of RNA sequencing data. H3K9ac and H3K4me3 marks were highly convergent at all stages of leaf aging, but H3K4me3 marks covered nearly 2 times the gene area as H3K9ac marks. Genes with the greatest fold change in expression displayed the largest positively correlated percentage change in coverage for both marks. Most senescence up-regulated genes were premarked by H3K4me3 and H3K9ac but at levels below the whole-genome average, and for these genes, gene expression increased without a significant increase in either histone mark. However, for a subset of genes showing increased or decreased expression, the respective gain or loss of H3K4me3 marks was found to closely match the temporal changes in mRNA abundance; 22% of genes that increased expression during senescence showed accompanying changes in H3K4me3 modification, and they include numerous regulatory genes, which may act as primary response genes.

Repression of Sox9 by Jag1 is continuously required to suppress the default chondrogenic fate of vascular smooth muscle cells.

Acquisition and maintenance of vascular smooth muscle fate are essential for the morphogenesis and function of the circulatory system. Loss of contractile properties or changes in the identity of vascular smooth muscle cells (vSMCs) can result in structural alterations associated with aneurysms and vascular wall calcification. Here we report that maturation of sclerotome-derived vSMCs depends on a transcriptional switch between mouse embryonic days 13 and 14.5. At this time, Notch/Jag1-mediated repression of sclerotome transcription factors Pax1, Scx, and Sox9 is necessary to fully enable vSMC maturation. Specifically, Notch signaling in vSMCs antagonizes sclerotome and cartilage transcription factors and promotes upregulation of contractile genes. In the absence of the Notch ligand Jag1, vSMCs acquire a chondrocytic transcriptional repertoire that can lead to ossification. Importantly, our findings suggest that sustained Notch signaling is essential throughout vSMC life to maintain contractile function, prevent vSMC reprogramming, and promote vascular wall integrity.

A comprehensive resource of genomic, epigenomic and transcriptomic sequencing data for the black truffle Tuber melanosporum.

BACKGROUND: Tuber melanosporum, also known in the gastronomic community as "truffle", features one of the largest fungal genomes (125 Mb) with an exceptionally high transposable element (TE) and repetitive DNA content (>58%). The main purpose of DNA methylation in fungi is TE silencing. As obligate outcrossing organisms, truffles are bound to a sexual mode of propagation, which together with TEs is thought to represent a major force driving the evolution of DNA methylation. Thus, it was of interest to examine if and how T. melanosporum exploits DNA methylation to maintain genome integrity. FINDINGS: We performed whole-genome DNA bisulfite sequencing and mRNA sequencing on different developmental stages of T. melanosporum; namely, fruitbody ("truffle"), free-living mycelium and ectomycorrhiza. The data revealed a high rate of cytosine methylation (>44%), selectively targeting TEs rather than genes with a strong preference for CpG sites. Whole genome DNA sequencing uncovered multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs, almost exclusively in free-living mycelium propagated in vitro. Treatment of mycelia with 5-azacytidine partially reduced DNA methylation and increased TE transcription. Our transcriptome assembly also resulted in the identification of a set of novel transcripts from 614 genes. CONCLUSIONS: The datasets presented here provide valuable and comprehensive (epi)genomic information that can be of interest for evolutionary genomics studies of multicellular (filamentous) fungi, in particular Ascomycetes belonging to the subphylum, Pezizomycotina. Evidence derived from comparative methylome and transcriptome analyses indicates that a non-exhaustive and partly reversible methylation process operates in truffles.

Non-exhaustive DNA methylation-mediated transposon silencing in the black truffle genome, a complex fungal genome with massive repeat element content.

BACKGROUND: We investigated how an extremely transposon element (TE)-rich organism such as the plant-symbiotic ascomycete truffle Tuber melanosporum exploits DNA methylation to cope with the more than 45,000 repeated elements that populate its genome. RESULTS: Whole-genome bisulfite sequencing performed on different developmental stages reveals a high fraction of methylated cytosines with a strong preference for CpG sites. The methylation pattern is highly similar among samples and selectively targets TEs rather than genes. A marked trend toward hypomethylation is observed for TEs located within a 1 kb distance from expressed genes, rather than segregated in TE-rich regions of the genome. Approximately 300 hypomethylated or unmethylated TEs are transcriptionally active, with higher expression levels in free-living mycelium compared to fruitbody. Indeed, multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs are found almost exclusively in free-living mycelium. A reduction of DNA methylation, restricted to non-CpG sites and accompanied by an increase in TE expression, is observed upon treatment of free-living mycelia with 5-azacytidine. CONCLUSIONS: Evidence derived from analysis of the T. melanosporum methylome indicates that a non-exhaustive, partly reversible, methylation process operates in truffles. This allows for the existence of hypomethylated, transcriptionally active TEs that are associated with copy number variant regions of the genome. Non-exhaustive TE methylation may reflect a role of active TEs in promoting genome plasticity and the ability to adapt to sudden environmental changes.

Characterization and analysis of the Burkholderia pseudomallei BsaN virulence regulon.

BACKGROUND: Burkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis. A conserved type III secretion system (T3SS3) and type VI secretion system (T6SS1) are critical for intracellular survival and growth. The T3SS3 and T6SS1 genes are coordinately and hierarchically regulated by a TetR-type regulator, BspR. A central transcriptional regulator of the BspR regulatory cascade, BsaN, activates a subset of T3SS3 and T6SS1 loci. RESULTS: To elucidate the scope of the BsaN regulon, we used RNAseq analysis to compare the transcriptomes of wild-type B. pseudomallei KHW and a bsaN deletion mutant. The 60 genes positively-regulated by BsaN include those that we had previously identified in addition to a polyketide biosynthesis locus and genes involved in amino acid biosynthesis. BsaN was also found to repress the transcription of 51 genes including flagellar motility loci and those encoding components of the T3SS3 apparatus. Using a promoter-lacZ fusion assay in E. coli, we show that BsaN together with the chaperone BicA directly control the expression of the T3SS3 translocon, effector and associated regulatory genes that are organized into at least five operons (BPSS1516-BPSS1552). Using a mutagenesis approach, a consensus regulatory motif in the promoter regions of BsaN-regulated genes was shown to be essential for transcriptional activation. CONCLUSIONS: BsaN/BicA functions as a central regulator of key virulence clusters in B. pseudomallei within a more extensive network of genetic regulation. We propose that BsaN/BicA controls a gene expression program that facilitates the adaption and intracellular survival of the pathogen within eukaryotic hosts.

Nkx2-5 suppresses the proliferation of atrial myocytes and conduction system.

RATIONALE: Tight control of cardiomyocyte proliferation is essential for the formation of four-chambered heart. Although human mutation of NKX2-5 is linked to septal defects and atrioventricular conduction abnormalities, early lethality and hemodynamic alteration in the mutant models have caused controversy as to whether Nkx2-5 regulates cardiomyocyte proliferation. OBJECTIVE: In this study, we circumvented these limitations by atrial-restricted deletion of Nkx2-5. METHOD AND RESULTS: Atrial-specific Nkx2-5 mutants died shortly after birth with hyperplastic working myocytes and conduction system including two nodes and internodal tracts. Multicolor reporter analysis revealed that Nkx2-5-null cardiomyocytes displayed clonal proliferative activity throughout the atria, indicating the suppressive role of Nkx2-5 in cardiomyocyte proliferation after chamber ballooning stages. Transcriptome analysis revealed that aberrant activation of Notch signaling underlies hyperproliferation of mutant cardiomyocytes, and forced activation of Notch signaling recapitulates hyperproliferation of working myocytes but not the conduction system. CONCLUSIONS: Collectively, these data suggest that Nkx2-5 regulates the proliferation of atrial working and conduction myocardium in coordination with Notch pathway.

Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells.

Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.

Intrauterine calorie restriction affects placental DNA methylation and gene expression.

Maternal nutrient restriction causes the development of adult onset chronic diseases in the intrauterine growth restricted (IUGR) fetus. Investigations in mice have shown that either protein or calorie restriction during pregnancy leads to glucose intolerance, increased fat mass, and hypercholesterolemia in adult male offspring. Some of these phenotypes are shown to persist in successive generations. The molecular mechanisms underlying IUGR remain unclear. The placenta is a critical organ for mediating changes in the environment and the development of embryos. To shed light on molecular mechanisms that might affect placental responses to differing environments we examined placentas from mice that had been exposed to different diets. We measured gene expression and whole genome DNA methylation in both male and female placentas of mice exposed to either caloric restriction or ad libitum diets. We observed several differentially expressed pathways associated with IUGR phenotypes and, most importantly, a significant decrease in the overall methylation between these groups as well as sex-specific effects that are more pronounced in males. In addition, a set of significantly differentially methylated genes that are enriched for known imprinted genes were identified, suggesting that imprinted loci may be particularly susceptible to diet effects. Lastly, we identified several differentially methylated microRNAs that target genes associated with immunological, metabolic, gastrointestinal, cardiovascular, and neurological chronic diseases, as well as genes responsible for transplacental nutrient transfer and fetal development.

Estrogen and progesterone together expand murine endometrial epithelial progenitor cells.

Synchronous with massive shifts in reproductive hormones, the uterus and its lining the endometrium expand to accommodate a growing fetus during pregnancy. In the absence of an embryo the endometrium, composed of epithelium and stroma, undergoes numerous hormonally regulated cycles of breakdown and regeneration. The hormonally mediated regenerative capacity of the endometrium suggests that signals that govern the growth of endometrial progenitors must be regulated by estrogen and progesterone. Here, we report an antigenic profile for isolation of mouse endometrial epithelial progenitors. These cells are EpCAM(+) CD44(+) ITGA6(hi) Thy1(-) PECAM1(-) PTPRC(-) Ter119(-), comprise a minor subpopulation of total endometrial epithelia and possess a gene expression profile that is unique and different from other cells of the endometrium. The epithelial progenitors of the endometrium could regenerate in vivo, undergo multilineage differentiation and proliferate. We show that the number of endometrial epithelial progenitors is regulated by reproductive hormones. Coadministration of estrogen and progesterone dramatically expanded the endometrial epithelial progenitor cell pool. This effect was not observed when estrogen or progesterone was administered alone. Despite the remarkable sensitivity to hormonal signals, endometrial epithelial progenitors do not express estrogen or progesterone receptors. Therefore, their hormonal regulation must be mediated through paracrine signals resulting from binding of steroid hormones to the progenitor cell niche. Discovery of signaling defects in endometrial epithelial progenitors or their niche can lead to development of better therapies in diseases of the endometrium.