Nascent transcriptomic landscape in bread wheat detected Pol II-dependent enhancer transcription
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PROVIDER: PRJNA738183 | ENA |
REPOSITORIES: ENA
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Project description:Bread wheat is the major staple food of the world with a complex hexaploidy genome. The precise spatiotemporal gene expression is orchestrated by enhancers, which lack general sequence features and thus are difficult to be located, especially in large genomes. Epigenomic architecture, including chromatin openness and active chromatin marks, has been widely used to characterize enhancers. However, an active chromatin environment does not necessarily mean an active enhancer. Recently, enhancer RNAs (eRNAs), the hallmark for active enhancers, have been detected by nascent RNA sequencing in both Drosophila and mammalian. In order to answer whether plant enhancers could be transcribed, we investigated the transcriptome of bread wheat via two nascent RNA sequencing methods, GRO-seq and pNET-seq combining with epigenome profiling. Our study demonstrates the presence and wide distribution of transcription at intergenic enhancers, which accurately reflects high enhancer activity, shedding light on the complex gene expression regulation across subgenomes in bread wheat.
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Project description:Bread wheat (Triticum aestivum) has a large, complex and hexaploid genome consisting of A, B and D homoeologous chromosome sets. Therefore each wheat gene potentially exists as a trio of A, B and D homoeoalleles, each of which may contribute differentially to wheat phenotypes. We describe a novel approach combining wheat cytogenetic resources (chromosome substitution ânullisomic-tetrasomicâ lines) with next generation deep sequencing of gene transcripts (RNA-seq), to directly and accurately identify homoeologue-specific single nucleotide variants and quantify the relative homoeoallelic contribution to gene expression. We obtained mRNA-Seq datasets from non-normalized cDNA libraries created from shoot and root tissues of the euploid bread wheat cultivar Chinese Spring, from which the nullitetra lines are derived, from complete sets of chromosome 1 and 5 nullitetras, and from extant relatives of the diploid A (Triticum urartu) and D (Aegilops tauschii) genome donors, herein referred to as A and D genome diploids
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Project description:The goal of this study was to test the effect of metal cations (CaCl2 and MnCl2) during Pol II NET-seq. These metals are readily used in the majority of studies that use NET-seq to analyze Pol II occupancy, but their effect on nascent transcript capture has not been analyzed. Our results suggest that the inclusion of these metals in Pol II NET-seq experiments does not cause a significant change in Pol II occupancy in the untreated control vs. metal-treated (CaCl2 or MnCl2) samples.
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Project description:We have generated single-nucleotide resolution, nascent transcription profiles from HeLa cells by developing Native Elongation Transcript sequencing technology for mammalian chromatin (mNET-seq). Our extensive data sets provide a substantial resource to study mammalian nascent transcript profiles. We reveal unanticipated phosphorylation states for RNA polymerase II C-terminal domain (Pol II CTD) at both gene ends. We also observe that following 5’ splice site cleavage by the spliceosome, upstream exon transcripts are tethered to Pol II CTD phosphorylated on the serine 5 position (S5P) which is accumulated over downstream exons. We further show that depletion of termination factors substantially reduces Pol II pausing at gene ends leading to termination defects. Remarkably termination factors play an additional promoter role by restricting non-productive RNA synthesis and redistributing Pol II CTD S2P to promoters. These data demonstrate that CTD phosphorylation is more dynamic and variably distributed across mammalian transcription units than previously envisaged. To monitor nascent RNA within the mammalian Pol II complex, and its association with different CTD phosphorylation states, we employed mNET-seq methodology on HeLa cells, complemented with direct sequencing of chromatin-bound RNA (ChrRNA-seq). mNET-seq was preformed using the antibodies 8WG16, CMA602, CMA603 and CMA601, which are specific for unphosphorylated CTD, Ser2 phosphorylation, Ser5 phosphorylation and all CTD isoforms, respectively. In another experiment, to evaluate the effect of transcription termination factors in nascent RNA production by Pol II, mNET-seq and complemented with ChrRNA-seq was preformed on HeLa cells transfected with siRNA against PTBP1, CPSF73, CstF64+CstF64tau or Xrn2, and the gene profiles were compared with profiles from HeLa transfected with siRNA for Luciferase generated by the same protocol.
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