Project description:Technology for crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) has identified the transcriptomic targets of hundreds of RNA-binding proteins in cells. To improve the power of existing and future CLIP-seq datasets, we introduce Skipper, an end-to-end workflow that converts unprocessed reads into annotated binding sites using an improved statistical framework. Compared to existing methods, Skipper on average calls 3.1-4.2 times more transcriptomic binding sites and sometimes >10 times more sites, providing deeper insight into post-transcriptional gene regulation. Skipper also calls binding to annotated repetitive elements and identifies bound elements for 99% of enhanced CLIP experiments. We perform nine translation factor enhanced CLIPs and apply Skipper to learn determinants of translation factor occupancy including transcript region, sequence, and subcellular localization. Furthermore, we observe depletion of genetic variation in occupied sites and nominate transcripts subject to selective constraint because of translation factor occupancy. Skipper offers fast, easy, customizable analysis of CLIP-seq data.
Project description:Heterosis is a fundamental biological phenomenon characterized by the superior performance of a hybrid over its parents in many traits, but the underlying molecular basis remains elusive. To investigate whether DNA methylation plays a role in heterosis, we compared at single base-pair resolution the DNA methylomes of Arabidopsis Ler and C24 parental lines and their reciprocal F1 hybrids that exhibited heterosis for many quantitative traits. Both hybrids displayed increased DNA methylation across their entire genomes, especially in transposable elements. Interestingly, we found that increased methylation of the hybrid genomes predominantly occurred in regions that were differentially methylated in the two parents and covered by small RNAs (sRNAs), implying that the RNA-directed DNA methylation (RdDM) pathway may direct DNA methylation in hybrids. In addition, we found that 77 genes sensitive to remodeling of DNA methylation were transcriptionally repressed in both reciprocal hybrids, including genes involved in flavonoid biosynthesis and two circadian oscillator genes, CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL. Moreover, growth vigor of F1 hybrids was compromised by treatment with an agent that demethylates DNA, and by abolishing production of functional small RNAs due to mutations in Arabidopsis RNA methyltransferase HUA ENHANCER1. Together, our data suggest that genome-wide remodeling of DNA methylation directed by the RdDM pathway may play a role in hybrid vigor. Examination of DNA methylation by Bisulfite sequencing in 2 Arabidopsis ecotypes and their reciprocal hybrids.
Project description:MicroRNAs (miRNAs) are involved in post-transcriptional regulation of gene expression. Since several miRNAs are known to affect the stability or translation of developmental regulatory genes, the origin of novel miRNAs may have contributed to the evolution of developmental processes and morphology. Lepidoptera (butterflies and moths) is a species-rich clade with a well-established phylogeny and abundant genomic resources, thereby representing an ideal system in which to study miRNA evolution. We sequenced small RNA libraries from developmental stages of two divergent lepidopterans, Cameraria ohridella (Horse chestnut Leafminer) and Pararge aegeria (Speckled Wood butterfly), discovering 90 and 81 conserved miRNAs respectively, and many species-specific miRNA sequences. Mapping miRNAs onto the lepidopteran phylogeny reveals rapid miRNA turnover and an episode of miRNA fixation early in lepidopteran evolution, implying that miRNA acquisition accompanied the early radiation of the Lepidoptera. One lepidopteran-specific miRNA gene, miR-2768, is located within an intron of the homeobox gene invected, involved in insect segmental and wing patterning. We identified cubitus interruptus (ci) as a likely direct target of miR-2768, and validated this suppression using a luciferase assay system. We propose a model by which miR-2768 modulates expression of ci in the segmentation pathway and in patterning of lepidopteran wing primordia. Examination of the small RNA complements pooled across life cycle stages in each of Cameraria ohridella and Pararge aegeria.
Project description:MicroRNAs (miRNAs) are involved in post-transcriptional regulation of gene expression. Since several miRNAs are known to affect the stability or translation of developmental regulatory genes, the origin of novel miRNAs may have contributed to the evolution of developmental processes and morphology. Lepidoptera (butterflies and moths) is a species-rich clade with a well-established phylogeny and abundant genomic resources, thereby representing an ideal system in which to study miRNA evolution. We sequenced small RNA libraries from developmental stages of two divergent lepidopterans, Cameraria ohridella (Horse chestnut Leafminer) and Pararge aegeria (Speckled Wood butterfly), discovering 90 and 81 conserved miRNAs respectively, and many species-specific miRNA sequences. Mapping miRNAs onto the lepidopteran phylogeny reveals rapid miRNA turnover and an episode of miRNA fixation early in lepidopteran evolution, implying that miRNA acquisition accompanied the early radiation of the Lepidoptera. One lepidopteran-specific miRNA gene, miR-2768, is located within an intron of the homeobox gene invected, involved in insect segmental and wing patterning. We identified cubitus interruptus (ci) as a likely direct target of miR-2768, and validated this suppression using a luciferase assay system. We propose a model by which miR-2768 modulates expression of ci in the segmentation pathway and in patterning of lepidopteran wing primordia.
Project description:we determine genome-wide binding profiles of a maize CCA1 homolog, ZmCCA1b, in maize inbreds and F1 hybrids at different times of the day. ZmCCA1b is characterized as a central clock regulator gene with evolutionarily conserved molecular and circadian functions and nonadditively expressed in F1 hybrid seedlings. ZmCCA1b binds to over 4,300 target genes in the maize genomes, of which annotation confirms energy metabolic pathways as the main output. We report that an altered temporal binding activity of ZmCCA1b in the hybrid seedlings, which increases expression of carbon fixation genes, increases carbon fixation rates and biomass, demonstrating a novel example of how circadian-regulatory networks directly contribute to growth vigor in maize hybrids. These results collectively offer new insights into clock-mediated regulation of growth vigor in hybrid plants and crops. Profiling genome-wide binding events of ZmCCA1b in the maize inbreds and F1 hybrids at ZT3, ZT9 and ZT15 using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). 2 biological replicates for each sample were used. Input DNA sample corresponding to each ChIP sample was also sequenced in parallel. We have developed a native antibody for the protein (GRMZM2G014902; epitope: residues 11-77) for the ChIP-seq study.