ABSTRACT
Temporally and spatially controlled accumulation underlies the functions of microRNAs (miRNAs) in various developmental processes. In Caenorhabditis elegans, this is exemplified by the temporal patterning miRNAs lin-4 and let-7, but for most miRNAs, developmental expression patterns remain poorly resolved. Indeed, experimentally observed long half-lives may constrain possible dynamics. Here, we profile miRNA expression throughout C. elegans postembryonic development at high temporal resolution, which identifies dynamically expressed miRNAs. We use mathematical models to explore the underlying mechanisms. For let-7, we can explain, and experimentally confirm, a striking stepwise accumulation pattern through a combination of rhythmic transcription and stage-specific regulation of precursor processing by the RNA-binding protein LIN-28. By contrast, the dynamics of several other miRNAs cannot be explained by regulation of production rates alone. Specifically, we show that a combination of oscillatory transcription and rhythmic decay drive rhythmic accumulation of miR-235, orthologous to miR-92 in other animals. We demonstrate that decay of miR-235 and additional miRNAs depends on EBAX-1, previously implicated in target-directed miRNA degradation (TDMD). Taken together, our results provide insight into dynamic miRNA decay and establish a resource to studying both the developmental functions of, and the regulatory mechanisms acting on, miRNAs.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Gene Expression Regulation, Developmental , MicroRNAs , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Larva/genetics , Larva/growth & development , Larva/metabolism , MicroRNAs/metabolism , MicroRNAs/genetics , Repressor Proteins , RNA Stability/genetics , Transcription Factors/metabolism , Transcription Factors/geneticsABSTRACT
The developmental role of histone H3K9 methylation (H3K9me), which typifies heterochromatin, remains unclear. In Caenorhabditis elegans, loss of H3K9me leads to a highly divergent upregulation of genes with tissue and developmental-stage specificity. During development H3K9me is lost from differentiated cell type-specific genes and gained at genes expressed in earlier developmental stages or other tissues. The continuous deposition of H3K9me2 by the SETDB1 homolog MET-2 after terminal differentiation is necessary to maintain repression. In differentiated tissues, H3K9me ensures silencing by restricting the activity of a defined set of transcription factors at promoters and enhancers. Increased chromatin accessibility following the loss of H3K9me is neither sufficient nor necessary to drive transcription. Increased ATAC-seq signal and gene expression correlate at a subset of loci positioned away from the nuclear envelope, while derepressed genes at the nuclear periphery remain poorly accessible despite being transcribed. In conclusion, H3K9me deposition can confer tissue-specific gene expression and maintain the integrity of terminally differentiated muscle by restricting transcription factor activity.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Cell Differentiation , Chromatin Assembly and Disassembly , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Protein Processing, Post-Translational , Transcription, Genetic , Animals , Animals, Genetically Modified , Binding Sites , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Chromatin Immunoprecipitation Sequencing , Gene Expression Profiling , Histone-Lysine N-Methyltransferase/genetics , Histones/genetics , Methylation , Protein Binding , Time Factors , TranscriptomeABSTRACT
DNA methylation is considered a stable epigenetic mark, yet methylation patterns can vary during differentiation and in diseases such as cancer. Local levels of DNA methylation result from opposing enzymatic activities, the rates of which remain largely unknown. Here we developed a theoretical and experimental framework enabling us to infer methylation and demethylation rates at 860,404 CpGs in mouse embryonic stem cells. We find that enzymatic rates can vary as much as two orders of magnitude between CpGs with identical steady-state DNA methylation. Unexpectedly, de novo and maintenance methylation activity is reduced at transcription factor binding sites, while methylation turnover is elevated in transcribed gene bodies. Furthermore, we show that TET activity contributes substantially more than passive demethylation to establishing low methylation levels at distal enhancers. Taken together, our work unveils a genome-scale map of methylation kinetics, revealing highly variable and context-specific activity for the DNA methylation machinery.
Subject(s)
CpG Islands/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA Demethylation , DNA Methylation/genetics , DNA-Binding Proteins/metabolism , Mouse Embryonic Stem Cells/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Binding Sites/genetics , Cell Line , Chromosome Mapping , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methyltransferase 3A , DNA-Binding Proteins/genetics , Dioxygenases/genetics , Dioxygenases/metabolism , Epigenesis, Genetic/genetics , Genome/genetics , Histones/metabolism , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription Factors/metabolism , Transcription, Genetic/genetics , DNA Methyltransferase 3BABSTRACT
In fission yeast and plants, RNA processing and degradation contribute to heterochromatin silencing, alongside conserved pathways of transcriptional repression. It has not been known whether similar pathways exist in metazoans. Here, we describe a pathway of silencing in Caenorhabditis elegans somatic cells, in which the highly conserved RNA-binding complex LSM2-8 contributes selectively to the repression of heterochromatic reporters and endogenous genes bearing the Polycomb mark, histone H3K27me3. This acts by degrading selected transcripts through the XRN-2 exoribonuclease. Disruption of the LSM2-8 pathway leads to mRNA stabilization. Unlike previously described pathways of heterochromatic RNA degradation, LSM2-8-mediated RNA degradation does not target nor require H3K9 methylation. Intriguingly, loss of this pathway coincides with a localized reduction in H3K27me3 at lsm-8-sensitive loci. We have thus uncovered a mechanism of RNA degradation that selectively contributes to the silencing of a subset of H3K27me3-marked genes, revealing a previously unrecognized layer of post-transcriptional control in metazoan heterochromatin.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Exoribonucleases/genetics , Histones/genetics , RNA Stability/genetics , Ribonucleoproteins, Small Nuclear/genetics , Animals , Caenorhabditis elegans/genetics , Gene Silencing/physiology , Heterochromatin/genetics , Methylation , Polycomb-Group Proteins/genetics , RNA Interference/physiology , RNA, Messenger/genetics , RNA, Small Interfering/geneticsABSTRACT
The execution of developmental programs of gene expression requires an accurate partitioning of the genome into subnuclear compartments, with active euchromatin enriched centrally and silent heterochromatin at the nuclear periphery1. The existence of degenerative diseases linked to lamin A mutations suggests that perinuclear binding of chromatin contributes to cell-type integrity2,3. The methylation of lysine 9 of histone H3 (H3K9me) characterizes heterochromatin and mediates both transcriptional repression and chromatin anchoring at the inner nuclear membrane4. In Caenorhabditis elegans embryos, chromodomain protein CEC-4 bound to the inner nuclear membrane tethers heterochromatin through H3K9me3,5, whereas in differentiated tissues, a second heterochromatin-sequestering pathway is induced. Here we use an RNA interference screen in the cec-4 background and identify MRG-1 as a broadly expressed factor that is necessary for this second chromatin anchor in intestinal cells. However, MRG-1 is exclusively bound to euchromatin, suggesting that it acts indirectly. Heterochromatin detachment in double mrg-1; cec-4 mutants is rescued by depleting the histone acetyltransferase CBP-1/p300 or the transcription factor ATF-8, a member of the bZIP family (which is known to recruit CBP/p300). Overexpression of CBP-1 in cec-4 mutants is sufficient to delocalize heterochromatin in an ATF-8-dependent manner. CBP-1 and H3K27ac levels increase in heterochromatin upon mrg-1 knockdown, coincident with delocalization. This suggests that the spatial organization of chromatin in C. elegans is regulated both by the direct perinuclear attachment of silent chromatin, and by an active retention of CBP-1/p300 in euchromatin. The two pathways contribute differentially in embryos and larval tissues, with CBP-1 sequestration by MRG-1 having a major role in differentiated cells.
Subject(s)
Caenorhabditis elegans/cytology , Caenorhabditis elegans/genetics , Chromatin/genetics , Chromatin/metabolism , Heterochromatin/genetics , Heterochromatin/metabolism , Animals , Caenorhabditis elegans/anatomy & histology , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Chromosomal Proteins, Non-Histone/deficiency , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Euchromatin/genetics , Euchromatin/metabolism , Gain of Function Mutation , Genes, Reporter/genetics , Histone Acetyltransferases/deficiency , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histones/chemistry , Histones/metabolism , Intestines/cytology , Transcription Factors/deficiency , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
In fission yeast and plants, RNA-processing pathways contribute to heterochromatin silencing, complementing well-characterized pathways of transcriptional repression. However, it was unclear whether this additional level of regulation occurs in metazoans. In a genetic screen, we uncovered a pathway of silencing in Caenorhabditis elegans somatic cells, whereby the highly conserved, RNA-binding complex LSM2-8 contributes to the repression of heterochromatic reporters and endogenous genes bearing the Polycomb mark H3K27me3. Importantly, the LSM2-8 complex works cooperatively with a 5'-3' exoribonuclease, XRN-2, and disruption of the pathway leads to selective mRNA stabilization. LSM2-8 complex-mediated RNA degradation does not target nor depend on H3K9me2/me3, unlike previously described pathways of heterochromatic RNA degradation. Up-regulation of lsm-8-sensitive loci coincides with a localized drop in H3K27me3 levels in the lsm-8 mutant. Put into the context of epigenetic control of gene expression, it appears that targeted RNA degradation helps repress a subset of H3K27me3-marked genes, revealing an unappreciated layer of regulation for facultative heterochromatin in animals.
ABSTRACT
RNA-binding proteins regulate all aspects of RNA metabolism. Their association with RNA is mediated by RNA-binding domains, of which many remain uncharacterized. A recently reported example is the NHL domain, found in prominent regulators of cellular plasticity like the C. elegans LIN-41. Here we employ an integrative approach to dissect the RNA specificity of LIN-41. Using computational analysis, structural biology, and in vivo studies in worms and human cells, we find that a positively charged pocket, specific to the NHL domain of LIN-41 and its homologs (collectively LIN41), recognizes a stem-loop RNA element, whose shape determines the binding specificity. Surprisingly, the mechanism of RNA recognition by LIN41 is drastically different from that of its more distant relative, the fly Brat. Our phylogenetic analysis suggests that this reflects a rapid evolution of the domain, presenting an interesting example of a conserved protein fold that acquired completely different solutions to RNA recognition.
Subject(s)
Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Evolution, Molecular , RNA, Helminth/genetics , Transcription Factors/chemistry , Transcription Factors/metabolism , Animals , Caenorhabditis elegans/classification , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Drosophila/classification , Drosophila/genetics , Drosophila/metabolism , Inverted Repeat Sequences , Nucleic Acid Conformation , Phylogeny , Protein Domains , RNA, Helminth/chemistry , RNA, Helminth/metabolism , Transcription Factors/geneticsABSTRACT
Endogenous RNAi (endoRNAi) is a conserved mechanism for fine-tuning gene expression. In the nematode Caenorhabditis elegans, several endoRNAi pathways are required for the successful development of reproductive cells. The CSR-1 endoRNAi pathway promotes germ cell development, primarily by facilitating the expression of germline genes. In this study, we report a novel function for the CSR-1 pathway in preventing premature activation of embryonic transcription in the developing oocytes, which is accompanied by a general Pol II activation. This CSR-1 function requires its RNase activity, suggesting that, by controlling the levels of maternal mRNAs, CSR-1-dependent endoRNAi contributes to an orderly reprogramming of transcription during the oocyte-to-embryo transition.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/embryology , Caenorhabditis elegans/genetics , Oocytes/physiology , RNA Interference , Animals , Animals, Genetically Modified , Caenorhabditis elegans Proteins/metabolism , Embryo, Nonmammalian , Female , Gene Expression Regulation, Developmental , Mutation , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Transcription Factors/geneticsABSTRACT
Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters-an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing.
Subject(s)
DNA/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Promoter Regions, Genetic , RNA Polymerase II/metabolism , RNA/biosynthesis , Single Molecule Imaging , Transcription, Genetic , Animals , Binding Sites , DNA/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Genome-Wide Association Study , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Half-Life , Kinetics , Protein Binding , Protein Stability , Proteolysis , RNA/genetics , RNA Polymerase II/genetics , TATA Box , Transcription Initiation Site , Transcription Initiation, Genetic , Transcription Termination, GeneticABSTRACT
The RNA-binding protein (RBP) LIN41, also known as LIN-41 or TRIM71, is a key regulator of animal development, but its physiological targets and molecular mechanism of action are largely elusive. Here we find that this RBP has two distinct mRNA-silencing activities. Using genome-wide ribosome profiling, RNA immunoprecipitation, and in vitro-binding experiments, we identify four mRNAs, each encoding a transcription factor or cofactor, as direct physiological targets of C. elegans LIN41. LIN41 silences three of these targets through their 3' UTRs, but it achieves isoform-specific silencing of one target, lin-29A, through its unique 5' UTR. Whereas the 3' UTR targets mab-10, mab-3, and dmd-3 undergo transcript degradation, lin-29A experiences translational repression. Through binding site transplantation experiments, we demonstrate that it is the location of the LIN41-binding site that specifies the silencing mechanism. Such position-dependent dual activity may, when studied more systematically, emerge as a feature shared by other RBPs.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/genetics , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Transcription Factors/metabolism , 3' Untranslated Regions , 5' Untranslated Regions , Animals , Binding Sites , RNA Interference , RNA Stability , RNA, Helminth/chemistry , RNA, Helminth/metabolismABSTRACT
Obesity is a global health issue, arousing interest in molecular mechanisms controlling fat. Transcriptional regulation of fat has received much attention, and key transcription factors involved in lipid metabolism, such as SBP-1/SREBP, LPD-2/C/EBP, and MDT-15, are conserved from nematodes to mammals. However, there is a growing awareness that lipid metabolism can also be controlled by post-transcriptional mechanisms. Here, we show that the Caenorhabditis elegans RNase, REGE-1, related to MCPIP1/Zc3h12a/Regnase-1, a key regulator of mammalian innate immunity, promotes accumulation of body fat. Using exon-intron split analysis, we find that REGE-1 promotes fat by degrading the mRNA encoding ETS-4, a fat-loss-promoting transcription factor. Because ETS-4, in turn, induces rege-1 transcription, REGE-1 and ETS-4 appear to form an auto-regulatory module. We propose that this type of fat regulation may be of key importance when, if faced with an environmental change, an animal must rapidly but precisely remodel its metabolism.
Subject(s)
Adipose Tissue/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Endoribonucleases/metabolism , Ribonucleases/metabolism , 3' Untranslated Regions/genetics , Animals , Cold Temperature , Gene Expression Regulation , Genome, Helminth , Intestines/enzymology , Models, Molecular , RNA Interference , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Interphase chromatin is organized in distinct nuclear sub-compartments, reflecting its degree of compaction and transcriptional status. In Caenorhabditis elegans embryos, H3K9 methylation is necessary to silence and to anchor repeat-rich heterochromatin at the nuclear periphery. In a screen for perinuclear anchors of heterochromatin, we identified a previously uncharacterized C. elegans chromodomain protein, CEC-4. CEC-4 binds preferentially mono-, di-, or tri-methylated H3K9 and localizes at the nuclear envelope independently of H3K9 methylation and nuclear lamin. CEC-4 is necessary for endogenous heterochromatin anchoring, but not for transcriptional repression, in contrast to other known H3K9 methyl-binders in worms, which mediate gene repression but not perinuclear anchoring. When we ectopically induce a muscle differentiation program in embryos, cec-4 mutants fail to commit fully to muscle cell fate. This suggests that perinuclear sequestration of chromatin during development helps restrict cell differentiation programs by stabilizing commitment to a specific cell fate. PAPERCLIP.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/embryology , Caenorhabditis elegans/genetics , Chromosomal Proteins, Non-Histone/metabolism , Embryo, Nonmammalian/cytology , Heterochromatin , Histone Code , Amino Acid Sequence , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , Cell Differentiation , Cell Nucleus/genetics , Cell Nucleus/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Embryo, Nonmammalian/metabolism , Molecular Sequence Data , Sequence AlignmentABSTRACT
RNA-seq experiments generate reads derived not only from mature RNA transcripts but also from pre-mRNA. Here we present a computational approach called exon-intron split analysis (EISA) that measures changes in mature RNA and pre-mRNA reads across different experimental conditions to quantify transcriptional and post-transcriptional regulation of gene expression. We apply EISA to 17 diverse data sets to show that most intronic reads arise from nuclear RNA and changes in intronic read counts accurately predict changes in transcriptional activity. Furthermore, changes in post-transcriptional regulation can be predicted from differences between exonic and intronic changes. EISA reveals both transcriptional and post-transcriptional contributions to expression changes, increasing the amount of information that can be gained from RNA-seq data sets.
Subject(s)
Exons/genetics , Gene Expression Regulation/genetics , Introns/genetics , RNA , Sequence Analysis, RNA/methods , Animals , Cell Line , Computational Biology , Gene Expression Profiling , Humans , Mice , Organ Specificity , RNA/genetics , RNA/metabolism , Transcription, Genetic/geneticsABSTRACT
MicroRNAs (miRNAs) regulate target mRNAs by silencing them. Reciprocally, however, target mRNAs can also modulate miRNA stability. Here, we uncover a remarkable efficacy of target RNA-directed miRNA degradation (TDMD) in rodent primary neurons. Coincident with degradation, and while still bound to Argonaute, targeted miRNAs are 3' terminally tailed and trimmed. Absolute quantification of both miRNAs and their decay-inducing targets suggests that neuronal TDMD is multiple turnover and does not involve co-degradation of the target but rather competes with miRNA-mediated decay of the target. Moreover, mRNA silencing, but not TDMD, relies on cooperativity among multiple target sites to reach high efficacy. This knowledge can be harnessed for effective depletion of abundant miRNAs. Our findings bring insight into a potent miRNA degradation pathway in primary neurons, whose TDMD activity greatly surpasses that of non-neuronal cells and established cell lines. Thus, TDMD may be particularly relevant for miRNA regulation in the nervous system.
Subject(s)
Argonaute Proteins/metabolism , Cerebellum/metabolism , Hippocampus/metabolism , MicroRNAs/metabolism , Neurons/metabolism , RNA, Messenger/metabolism , Animals , Argonaute Proteins/genetics , Base Pairing , Base Sequence , Cerebellum/cytology , Gene Expression Regulation , Genetic Vectors , Hippocampus/cytology , Lentivirus/genetics , Mice , MicroRNAs/genetics , Molecular Sequence Data , Neurons/cytology , Primary Cell Culture , RNA Stability , RNA, Messenger/genetics , Rats , Signal TransductionABSTRACT
UNLABELLED: QuasR is a package for the integrated analysis of high-throughput sequencing data in R, covering all steps from read preprocessing, alignment and quality control to quantification. QuasR supports different experiment types (including RNA-seq, ChIP-seq and Bis-seq) and analysis variants (e.g. paired-end, stranded, spliced and allele-specific), and is integrated in Bioconductor so that its output can be directly processed for statistical analysis and visualization. AVAILABILITY AND IMPLEMENTATION: QuasR is implemented in R and C/C++. Source code and binaries for major platforms (Linux, OS X and MS Windows) are available from Bioconductor (www.bioconductor.org/packages/release/bioc/html/QuasR.html). The package includes a 'vignette' with step-by-step examples for typical work ï¬ows. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
Computational Biology , Contig Mapping , Genomics/methods , High-Throughput Nucleotide Sequencing , Sequence Analysis, DNA/methods , Software , Algorithms , HumansABSTRACT
The cold shock domain is one of the most highly conserved motifs between bacteria and higher eukaryotes. Y-box-binding proteins represent a subfamily of cold shock domain proteins with pleiotropic functions, ranging from transcription in the nucleus to translation in the cytoplasm. These proteins have been investigated in all major model organisms except Caenorhabditis elegans. In this study, we set out to fill this gap and present a functional characterization of CEYs, the C. elegans Y-box-binding proteins. We find that, similar to other organisms, CEYs are essential for proper gametogenesis. However, we also report a novel function of these proteins in the formation of large polysomes in the soma. In the absence of the somatic CEYs, polysomes are dramatically reduced with a simultaneous increase in monosomes and disomes, which, unexpectedly, has no obvious impact on animal biology. Because transcripts that are enriched in polysomes in wild-type animals tend to be less abundant in the absence of CEYs, our findings suggest that large polysomes might depend on transcript stabilization mediated by CEY proteins.
Subject(s)
Caenorhabditis elegans Proteins/physiology , Polyribosomes/metabolism , RNA-Binding Proteins/physiology , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/analysis , Cytoplasm/chemistry , Protein Biosynthesis , RNA, Messenger/metabolism , RNA-Binding Proteins/analysis , Ribonucleoproteins/chemistryABSTRACT
For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs), recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%-40%) of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS). Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R(2)) of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.
Subject(s)
CpG Islands/genetics , DNA Methylation/genetics , Genome , Animals , Cell Line , DNA/genetics , Mammals/genetics , Sequence Analysis, DNAABSTRACT
Oscillations are a key to achieving dynamic behavior and thus occur in biological systems as diverse as the beating heart, defecating worms, and nascent somites. Here we report pervasive, large-amplitude, and phase-locked oscillations of gene expression in developing C. elegans larvae, caused by periodic transcription. Nearly one fifth of detectably expressed transcripts oscillate with an 8 hr period, and hundreds change >10-fold. Oscillations are important for molting but occur in all phases, implying additional functions. Ribosome profiling reveals that periodic mRNA accumulation causes rhythmic translation, potentially facilitating transient protein accumulation as well as coordinated production of stable, complex structures such as the cuticle. Finally, large-amplitude oscillations in RNA sampled from whole worms indicate robust synchronization of gene expression programs across cells and tissues, suggesting that these oscillations will be a powerful new model to study coordinated gene expression in an animal.
Subject(s)
Caenorhabditis elegans Proteins/biosynthesis , Caenorhabditis elegans/genetics , Gene Expression Regulation, Developmental , Models, Genetic , Animals , Caenorhabditis elegans/growth & development , Caenorhabditis elegans Proteins/genetics , Circadian Clocks , Gene Expression Profiling , Larva/genetics , Larva/growth & development , Protein Biosynthesis/physiology , RNA, Helminth/metabolism , Time Factors , Transcription, GeneticABSTRACT
Although XRN2 proteins are highly conserved eukaryotic 5'â3' exonucleases, little is known about their function in animals. Here, we characterize Caenorhabditis elegans XRN2, which we find to be a broadly and constitutively expressed nuclear protein. An xrn-2 null mutation or loss of XRN2 catalytic activity causes a molting defect and early larval arrest. However, by generating a conditionally mutant xrn-2ts strain de novo through an approach that may be also applicable to other genes of interest, we reveal further functions in fertility, during embryogenesis and during additional larval stages. Consistent with the known role of XRN2 in controlling microRNA (miRNA) levels, we can demonstrate that loss of XRN2 activity stabilizes some rapidly decaying miRNAs. Surprisingly, however, other miRNAs continue to decay rapidly in xrn-2ts animals. Thus, XRN2 has unanticipated miRNA specificity in vivo, and its diverse developmental functions may relate to distinct substrates. Finally, our global analysis of miRNA stability during larval stage 1 reveals that miRNA passenger strands (miR*s) are substantially less stable than guide strands (miRs), supporting the notion that the former are mostly byproducts of biogenesis rather than a less abundant functional species.