We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.

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Chromatin immunoprecipitation identifies specific interactions between genomic DNA and proteins, advancing our understanding of gene-level and chromosome-level regulation. Based on chromatin immunoprecipitation experiments using validated antibodies, we define the genome-wide distributions of 19 histone modifications, one histone variant, and eight chromatin-associated proteins in Caenorhabditis elegans embryos and L3 larvae. Cluster analysis identified five groups of chromatin marks with shared features: Two groups correlate with gene repression, two with gene activation, and one with the X chromosome. The X chromosome displays numerous unique properties, including enrichment of monomethylated H4K20 and H3K27, which correlate with the different repressive mechanisms that operate in somatic tissues and germ cells, respectively. The data also revealed striking differences in chromatin composition between the autosomes and between chromosome arms and centers. Chromosomes I and III are globally enriched for marks of active genes, consistent with containing more highly expressed genes, compared to chromosomes II, IV, and especially V. Consistent with the absence of cytological heterochromatin and the holocentric nature of C. elegans chromosomes, markers of heterochromatin such as H3K9 methylation are not concentrated at a single region on each chromosome. Instead, H3K9 methylation is enriched on chromosome arms, coincident with zones of elevated meiotic recombination. Active genes in chromosome arms and centers have very similar histone mark distributions, suggesting that active domains in the arms are interspersed with heterochromatin-like structure. These data, which confirm and extend previous studies, allow for in-depth analysis of the organization and deployment of the C. elegans genome during development.

BackgroundAlthough Caenorhabditis elegans was the first multicellular organism with a completely sequenced genome, how this genome is arranged within the nucleus is not known.ResultsWe determined the genomic regions associated with the nuclear transmembrane protein LEM-2 in mixed-stage C. elegans embryos via chromatin immunoprecipitation. Large regions of several megabases on the arms of each autosome were associated with LEM-2. The center of each autosome was mostly free of such interactions, suggesting that they are largely looped out from the nuclear membrane. Only the left end of the X chromosome was associated with the nuclear membrane. At a finer scale, the large membrane-associated domains consisted of smaller subdomains of LEM-2 associations. These subdomains were characterized by high repeat density, low gene density, high levels of H3K27 trimethylation, and silent genes. The subdomains were punctuated by gaps harboring highly active genes. A chromosome arm translocated to a chromosome center retained its association with LEM-2, although there was a slight decrease in association near the fusion point.ConclusionsLocal DNA or chromatin properties are the main determinant of interaction with the nuclear membrane, with position along the chromosome making a minor contribution. Genes in small gaps between LEM-2 associated regions tend to be highly expressed, suggesting that these small gaps are especially amenable to highly efficient transcription. Although our data are derived from an amalgamation of cell types in mixed-stage embryos, the results suggest a model for the spatial arrangement of C. elegans chromosomes within the nucleus.

SUMMARY Defects in the Piwi/piRNA pathway lead to transposon desilencing and immediate sterility in many organisms. We found that the C. elegans Piwi mutant prg-1 became sterile after growth for many generations. This phenotype did not occur for RNA interference mutants with strong transposon silencing defects and was separable from the role of PRG-1 in transgene silencing. Brief periods of starvation extended the transgenerational lifespan of prg-1 mutants by stimulating the DAF-16/FOXO longevity transcription factor. Constitutive activation of DAF-16 via reduced daf-2 insulin/IGF-1 signaling immortalized prg-1 strains via RNA interference proteins and histone H3 lysine 4 demethylases. In late-generation prg-1 mutants, desilencing of repetitive segments of the genome occurred, and silencing of repetitive loci was restored in prg-1; daf-2 mutants. This study reveals an unexpected interface between aging and transgenerational maintenance of germ cells, where somatic longevity is coupled to a genome silencing pathway that promotes germ cell immortality in parallel to the Piwi/piRNA system.

Dimethyl sulfoxide (DMSO), an amphipathic molecule, is widely used not only as a solvent for water-insoluble substances but also as a cryopreservant for various types of cells. Exposure to DMSO sometimes causes unexpected changes in cell fates. Because mammalian development and cellular differentiation are controlled epigenetically by DNA methylation and histone modifications, DMSO likely affects the epigenetic system. The effects of DMSO on transcription of three major DNA methyltransferases (Dnmts) and five well-studied histone modification enzymes were examined in mouse embryonic stem cells and embryoid bodies (EBs) by reverse transcription-polymerase chain reaction. Addition of DMSO (0.02%-1.0%) to EBs in culture induced an increase in Dnmt3a mRNA levels with increasing dosage. Increased expression of two subtypes of Dnmt3a in protein levels was confirmed by Western blotting. Southern blot analysis revealed that DMSO caused hypermethylation of two kinds of repetitive sequences in EBs. Furthermore, restriction landmark genomic scanning, by which DNA methylation status can be analyzed on thousands of loci in genic regions, revealed that DMSO affected DNA methylation status at multiple loci, inducing hypomethylation as well as hypermethylation depending on the genomic loci. In conclusion, DMSO has an impact on the epigenetic profile: upregulation of Dnmt3a expression and alteration of genomewide DNA methylation profiles with phenotypic changes in EBs.

Codevelopment of the lungs and heart underlies key evolutionary innovations in the transition to terrestrial life. Cardiac specializations that support pulmonary circulation, including the atrial septum, are generated by second heart field (SHF) cardiopulmonary progenitors (CPPs). It has been presumed that transcription factors required in the SHF for cardiac septation, e.g., Tbx5, directly drive a cardiac morphogenesis gene-regulatory network. Here, we report instead that TBX5 directly drives Wnt ligands to initiate a bidirectional signaling loop between cardiopulmonary mesoderm and the foregut endoderm for endodermal pulmonary specification and, subsequently, atrial septation. We show that Tbx5 is required for pulmonary specification in mice and amphibians but not for swim bladder development in zebrafish. TBX5 is non-cell-autonomously required for pulmonary endoderm specification by directly driving Wnt2 and Wnt2b expression in cardiopulmonary mesoderm. TBX5 ChIPsequencing identified cis-regulatory elements at Wnt2 sufficient for endogenous Wnt2 expression domains in vivo and required for Wnt2 expression in precardiac mesoderm in vitro. Tbx5 cooperated with Shh signaling to drive Wnt2b expression for lung morphogenesis. Tbx5 haploinsufficiency in mice, a model of Holt-Oram syndrome, caused a quantitative decrement of mesodermal-to-endodermal Wnt signaling and subsequent endodermal-to-mesodermal Shh signaling required for cardiac morphogenesis. Thus, Tbx5 initiates a mesodermendoderm-mesoderm signaling loop in lunged vertebrates that provides a molecular basis for the coevolution of pulmonary and cardiac structures required for terrestrial life. lung development | heart development | TBX5 | Wnt signaling | Hedgehog signaling

LMNA encodes nuclear lamin A/C that tethers lamina-associated heterochromatin domains (LADs) to the nuclear periphery. Point mutations in LMNA cause degenerative disorders including the premature aging disorder Hutchinson-Gilford progeria, but the mechanisms are unknown. We report that Ser22phosphorylated Lamin A/C (pS22-Lamin A/C) was localized to the interior of the nucleus in human fibroblasts throughout the cell cycle. pS22-Lamin A/C interacted with a specific subset of putative active enhancers, not LADs, primarily at locations co-bound by the transcriptional activator c-Jun. In progeriapatient fibroblasts, a subset of pS22-Lamin A/C-binding sites were lost whereas new pS22-Lamin A/Cbinding sites emerged in normally quiescent loci. These new pS22-Lamin A/C-binding sites displayed increased histone acetylation and c-Jun binding, implying increased enhancer activity. The genes near these new binding sites, implicated in clinical components of progeria including carotid artery diseases, hypertension, and cardiomegaly, were upregulated in progeria. These results suggest that Lamin A/C regulates gene expression by direct enhancer binding in the nuclear interior. Disruption of the gene regulatory rather than LAD function of Lamin A/C presents a novel mechanism for disorders caused by LMNA mutations including progeria. HIGHLIGHTS• pS22-Lamin A/C is present in the nuclear interior throughout interphase.• pS22-Lamin A/C associates with active enhancers, not lamina-associated domains.• pS22-Lamin A/C-genomic binding sites are co-bound by the transcriptional activator c-Jun.• New pS22-Lamin A/C binding in progeria accompanies upregulation of disease-related genes.