Pol II docking and pausing at growth and stress genes in C. elegans
ABSTRACT: Fluctuations in nutrient availability profoundly impact gene expression. Previous work revealed post-recruitment regulation of RNA Polymerase II (Pol II) during starvation and recovery in Caenorhabitis elegans, suggesting promoter-proximal pausing promotes rapid response to feeding. To test this hypothesis, we measured Pol II elongation genome-wide by two complementary approaches and analyzed elongation in conjunction with Pol II binding and expression. We confirmed bona fide pausing during starvation and also discovered Pol II docking. Pausing occurs at active stress-response genes that become down-regulated in response to feeding. In contrast “docked” Pol II accumulates without initiating upstream of inactive growth genes that become rapidly up-regulated upon feeding. Beyond differences in function and expression, these two sets of genes have different core promoter motifs, suggesting alternative transcriptional machinery. Our work suggests that growth and stress genes are both regulated post-recruitment during starvation, but at initiation and elongation, respectively, coordinating gene expression with nutrient availability. We sequenced short, capped RNA (scRNA-seq) from N2 starved L1 C. elegans as well as a TFIIS mutant (RB2083). We also prepared scRNA-seq libraries from L1 larvae using one, two, or three of the enymes used to prepare the scRNA sequencing libraries as well as scRNA-seq libraries from Drosophila S2 cells using one or two of the enzymes. We further sequenced the 5' end of mRNA in starved C. elegans L1 larvae using the same enzymes as scRNA-seq.
Project description:Our understanding of transcription by RNA polymerase II (Pol II) is limited by our knowledge of the factors that mediate this critically important process. Here we describe the identification of NDF, a nucleosome destabilizing factor that facilitates Pol II transcription in chromatin. NDF has a PWWP motif, interacts with nucleosomes near the dyad, partially disassembles nucleosomes in an ATP-independent manner, and enhances transcription by Pol II through nucleosomes in vitro and in cell nuclei. NDF affects gene expression and co-localizes with H3K36me3 over transcribed regions of a subset of active genes with a preference for longer genes relative to shorter genes. Moreover, induction of transcription leads to the recruitment of NDF over gene bodies. In humans, NDF is present in all tested tissue types, is essential in stem cells, and is frequently overexpressed in breast cancer. Thus, NDF is a factor of primary importance for Pol II transcription in chromatin.
Project description:We report scRNA-seq data captured from 9,410 cells obtained from the skin of K14E7 transgenic and wildtype C57/BL6 mice. The K14E7 mouse model harbors the HPV16 E7 oncogene driven from a Keratin 14 promoter for keratinocyte-specific expression. We used scRNA-seq to detect and measure E7 transcription with unprecedented accuracy and resolution. With these data, we uncovered transcriptional differences between the individual cells; demonstrated that increased HPV16 E7 copy number is associated with increased expression of E7-induced genes; and showed that E7 expression is predominantly associated with basal keratinocytes.
Project description:Metazoan gene expression is often regulated after the recruitment of RNA polymerase II (Pol II) to promoters, through the controlled release of promoter-proximally paused Pol II into productive RNA synthesis. Despite the prevalence of paused Pol II, very little is known about the dynamics of these early elongation complexes or the fate of short transcription start site-associated (tss) RNAs they produce. Here, we demonstrate that paused elongation complexes can be remarkably stable, with half-lives exceeding 15 minutes at genes with inefficient pause release. Promoter-proximal termination by Pol II is infrequent and released tssRNAs are targeted for rapid degradation. Further, we provide evidence that the predominant tssRNA species observed are nascent RNAs held within early elongation complexes. We propose that stable pausing of polymerase provides a temporal window of opportunity for recruitment of factors to modulate gene expression and that the nascent tssRNA represents an appealing target for these interactions. This submission includes 13 raw data files. Four samples (chromatin, soluble, mock-treated, and Rrp40-depleted) are represented by two biological replicate raw data files, while five samples (cells, DMSO, FP, mock-treated + FP, and Rrp-40depleted + FP) are represented by single raw data files.
Project description:Non-lymphoid tissues (NLTs) harbour a pool of adaptive immune cells distinct from their counterparts in lymphoid tissues, and their development and phenotype remains largely unexplored. We used scRNA-seq to survey CD4+ T regulatory (Treg) and memory T (Tmem) cells in spleen, lymph nodes, skin and colon in an unbiased way, in mouse. This cross-tissues comparison allows us to obtain marker genes for immune populations in specific locations, as well as examine each population's heterogeneity. Additionally, a continuous phenotype of Treg migration can be modelled from the mouse data, unravelling the transcriptional stages through which these cells transition between tissues.
Project description:We have employed short-capped RNA sequencing (sc-RNA-seq) in order to identify genes whose expression is regulated by promoter proximal pausing of RNA Polymerase II (RNAPI) in response to stress stimulation. We used serum-deprived mouse Swiss 3T3 fibroblasts, either untreated (control) or treated with anisomycin to induce the p38/MAP kinase pathway. Serum starved (72 h 0.2% FCS) mouse 3T3 cells were treated with anisomycin (188.5 nM) for 1 h (in duplicates). Untreated, serum-starved cells were used as a control. We isolated nuclear RNA, performed size fractionation followed by isolation of short-capped RNAs (scRNA). scRNAs were subsequently converted into DNA library and sequenced.
Project description:Poised RNA polymerase II is predominantly found at developmental control genes and is thought to allow their rapid and synchronous induction in response to extracellular signals. How the recruitment of poised RNA Pol II is regulated during development is not known. By isolating muscle tissue from Drosophila embryos at five stages of differentiation, we show that the recruitment of poised Pol II occurs at many genes de novo and this makes them permissive for future gene expression. When compared to other tissues, these changes are stage-specific and not tissue-specific. In contrast, Polycomb group repression is tissue-specific and in combination with Pol II (the balanced state) marks genes with highly dynamic expression. This suggests that poised Pol II is temporally regulated and is held in check in a tissue-specific fashion. We compare our data to mammalian embryonic stem cells and discuss a framework for predicting developmental programs based on chromatin state. mRNA-seq of Drosophila tissues during development
Project description:Human pluripotent stem cells (hPSCs) offer a unique cellular model to study lineage specifications of the primary germ layers during human development. We profiled single-cell RNA-seq (scRNA-seq) on four lineage-specific progenitor cells derived from hESCs. Our scRNA-seq analyses revealed each type of progenitors display various extend of heterogeneity. Specifically, definitive endoderm cells (DECs) not only show a greater degree of heterogeneity, but are also enriched in metabolic signatures. Followed by detailed temporal scRNA-seq profiling along DEC differentiation, we reconstructed a differentiation trajectory using a novel statistical pipeline named Wave-Crest. Wave-Crest further identifies candidate regulators during the transitioning phase from Brachyury (T)+ mesendoderm towards CXCR4+ DEC state. To functionally test identified novel regulators; we generated a live cell monitoring system, a T-2A-EGFP knock-in reporter cell line via CRISPR/CAS9. We demonstrated that, among the top candidate genes, KLF8 plays a pivotal role modulating mesendoderm to DEC differentiation. In this submission, 1810 raw fastq files are provided; 212 are re-analysis from GSE64016. Four expected count matrices are provided - 1) 1018 single cells from snapshot progenitors; 2) 758 single cells from time couse profiling; 3) 19 bulk RNA-seq sample from snapshot progenitors; 4) 15 bulk RNA-seq sample from time course profiling. Total 1018 single cells from snapshot progenitors and 758 single cells from time couse profiling. Matchd population bulk RNA-seq samples for both the progenitors snapshot (19 samples) and time course profiling (15 samples) also included in this submission. These data set are used to detect the transitioning phase from mesendoderm to definitive endoderm.
Project description:Endothelial cell (EC) metabolism regulates angiogenesis and is an emerging target for anti-angiogenic therapy in tumor and choroidal neovascularization (CNV). In contrast to tumor ECs (TECs), CNV-ECs cannot be isolated for unbiased metabolic target discovery. Here we used scRNA-sequencing to profile 28,337 choroidal ECs (CECs) from mice to in silico distinguish healthy CECs from CNV-ECs. Trajectory inference suggested that CNV-ECs plastically upregulate genes in central carbon metabolism and collagen biosynthesis during differentiation from quiescent postcapillary venous ECs. CEC-tailored genome scale metabolic modeling predicted essentiality of SQLE and ALDH18A1 for proliferation and collagen production, respectively. Comparative analysis in TECs revealed more outspoken metabolic transcriptome heterogeneity in subtypes and consistent upregulation of SQLE and ALDH18A1 across tumor types. Inhibition of SQLE and ALDH18A1 reduced sprouting angiogenesis in vitro. These findings demonstrate the potential of integrated scRNA-seq analysis to identify angiogenic metabolic targets in disease ECs.
Project description:Barcode swapping results in the mislabeling of sequencing reads between multiplexed samples on the new patterned flow cell Illumina sequencing machines. This may compromise the validity of numerous genomic assays, especially for single-cell studies where many samples are routinely multiplexed together. The severity and consequences of barcode swapping for single-cell transcriptomic studies remain poorly understood. We have used two statistical approaches to robustly quantify the fraction of swapped reads in each of two plate-based single-cell RNA sequencing datasets. We found that approximately 2.5% of reads were mislabeled between samples on the HiSeq 4000 machine, which is lower than previous reports. We observed no correlation between the swapped fraction of reads and the concentration of free barcode across plates. Further- more, we have demonstrated that barcode swapping may generate complex but artefactual cell libraries in droplet-based single-cell RNA sequencing studies. To eliminate these artefacts, we have developed an algorithm to exclude individual molecules that have swapped between samples in 10X Genomics experiments, exploiting the combinatorial complexity present in the data. This permits the continued use of cutting-edge sequencing machines for droplet-based experiments while avoiding the confounding effects of barcode swapping. This data repository contains the sequencing files associated with the droplet based scRNA-seq dataset in Griffiths et al. (2018). The data presented here should purely used for technical analysis, the biological motivation is nonetheless briefly described in the following: The mammary gland is a unique organ as it undergoes most of its development during puberty and adulthood. Characterising the hierarchy of the various mammary epithelial cells and how they are regulated in response to gestation, lactation and involution is important for understanding how breast cancer develops. Recent studies have used numerous markers to enrich, isolate and characterise the different epithelial cell compartments within the adult mammary gland. However, in all of these studies only a handful of markers were used to define and trace cell populations. Therefore, there is a need for an unbiased and comprehensive description of mammary epithelial cells within the gland at different developmental stages. To this end we used single cell RNA sequencing (scRNAseq) to determine the gene expression profile of individual mammary epithelial cells across four adult developmental stages; nulliparous, mid gestation, lactation and post weaning (full natural involution).
Project description:The small nuclear RNA (snRNA) genes have been widely used as a model system for understanding transcriptional regulation due to unique aspects of their promoter structure, selectivity for either RNA Polymerase (Pol) II or III and a unique mechanism of termination that is tightly linked with the promoter. Recently, we identified the Little Elongation Complex (LEC) in Drosophila that is required for the expression of Pol II-transcribed snRNA genes. Here, we identify the molecular mechanism by which LEC specifically regulates Pol II-dependent snRNA gene transcription. We present genetic and molecular evidence from both Drosophila and mammals that LEC regulates both initiation and elongation stages of transcription of Pol II-transcribed snRNA genes. In human HCT116 cells we performed: ChIP-seq of ICE1, ICE2, ZC3H8, ELL, and AFF4; total RNA-seq following ICE1 knock-down and non-targeting (GFP) knock-down; ChIP-seq of ICE1 and Pol II following non-targetting (shGFP) and ICE1 knock-down (shICE1). In fly S2 cells we performed: Ice1 ChIP-seq following small hairpin knock-down of GFP (shGFP/non-targeting control) and Ice1 (knock-down of Ice1); ChIP-seq of Pol II following small hairpin knock-down of GFP (shGFP/non-targeting control), Ice1 (knock-down of Ice1), and Ell (knock-down of Ell).