Small RNA profiling of strains and lifecycle of Chlamydomonas reinhardtii
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ABSTRACT:
ORGANISM(S): Chlamydomonas reinhardtii
SUBMITTER: Krys Kelly
PROVIDER: E-GEOD-23379 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:To interrogate single-base resolution 6mA sites in the genome-wide, we develop DA-6mA-seq (DpnI-Assisted N6-methylAdenine sequencing), an optimized sequencing method taking advantage of restriction enzyme DpnI, which exclusively cleaves methylated adenine sites. We find DpnI also recognizes other sequence motifs besides the canonical GATC restriction sites, largely expanding the application range of this method. DA-6mA-seq requires less starting material and lower sequencing depth than previous methods, but achieves higher sensitivity, providing a good strategy to identify 6mA in large genome with a low abundance of 6mA. We rebuild the 6mA maps of Chlamydomonas by DA-6mA-seq and then apply this method to another two eukaryotic organisms, Plasmodium and Penicillium. Further analysis reveals most 6mA sites are symmetric at various sequence contexts, suggesting 6mA may function as a new heritable epigenetic mark in eukaryotes. A new sequencing method is developed to detect 6mA in eukaryotes
2016-04-12 | E-GEOD-74430 | biostudies-arrayexpress
Project description:Phosphorus (P) is an essential nutrient that is limiting in many environments. When P is scarce organisms employ strategies for conservation of internal stores, and to efficiently scavenge P from their external surroundings. In this study we investigated the acclimation response of Chlamydomonas reinhardtii to P deficiency, comparing the transcriptional profiles of P starved wild-type cells to the P replete condition. RNA was prepared from P-containing or P-deprived logarithmic growth phase cells and subjected to RNA-Seq analysis. During the 24 hours after the imposition of P starvation we observed that from the 407 significantly changing genes (> 2 fold change, corrected p-value < 0.05) in the wild-type 317 genes were up-regulated, in average 8.36-fold, and 90 genes were down-regulated by 3.43-fold, in average. Many of the upregulated genes encoded enzymes involved in specific responses to P starvation, including PHOX, encoding the major secreted alkaline phosphatase, and multiple putative, high-efficiency phosphate transporter genes. More general responses included the up-regulation of genes involved in photoprotective processes (LHCSR3, LHCSR1, LHCBM9, PTOX1) and genes involved in protein modification and degradation. Down-regulated mRNAs indicated an early stage of the reduction of chloroplast ribosomal proteins, which are considered to be a reservoir for P in the cell. Chlamydomonas reinhardtii strain 21 gr (CC1690, wild-type) grown in TAP medium (Harris 1989) in a rotary incubator (200 rpm) at 25 M-BM-0C in continuous light (70 M-BM-5mol m-2 s-1). For 24 hours, either 1.1 mM phosphate or 0 mM were provided with the growth media. P deprivation was achieved by washing cells twice in midlogarithmic growth phase with liquid TAP medium without P (TAP-P) and cells were resuspended at a density of 2.5 mg/ml Chlorophyll in TAP or TAP-P. Cell aliquots were collected for mRNA isolation 24 h after being transferred either to TAP or TAP-P medium.
2014-04-05 | E-GEOD-56505 | biostudies-arrayexpress
Project description:Alga-derived lipids represent an attractive potential source of biofuels. However, lipid accumulation in algae is a stress response tightly coupled to growth arrest, thereby imposing a major limitation on productivity. To identify master regulators of lipid accumulation and decipher the regulation of lipid biosynthetic pathway, we performed an integrative chromatin signature and transcriptomic analysis in the alga Chlamydomonas reinhardtii. Genome-wide histone modification profiling revealed remarkable differences in functional chromatin states between algae and higher eukaryotes and uncovered regulatory components at the core of lipid accumulation pathways. We identified the transcription factor PSR1 as a pivotal master switch that triggers cytosolic lipid hyper-accumulation an order of magnitude higher than stress regimens have achieved. Dissection of the PSR1 target network corroborates its central role in coordinating multiple stress responses. The comprehensive maps of functional chromatin signatures in a major clade of eukaryotic life and the discovery of a central regulator of algal lipid metabolism will facilitate targeted engineering strategies in microalgae. 1. Genome-wide H3K4me3 time series profiling (at 0 hr, 10 min, 30 min, 1 hr, 2hr, 6 hr, 8 hr, 24 hr and 48 hr after nitrogen starvation) was performed to determine time point to capture maximal chromatin changes. 2. Genome-wide H3K4me3, H3K27ac, H3K9me3, H3K27me3, H3K36me3 and Pol II profiling were performed at 0 hr, 1 hr after nitrogen starvation and 1 hr after sulfur starvation to determine chromatin signatures. Genome-wide H3K4me2 profiling was performed at 0 hr before starvation. 3. Transcriptome time series profiling (at 0 hr, 10 min, 30 min, 1 hr, 2hr, 6 hr, 8 hr, 24 hr and 48 hr after nitrogen and sulfur starvation separately) for chromatin signature characterization and integrative analysis. 4. Genome-wide PSR1 binding profiling was performed with polyclonal antibody against PSR1 peptide A region and PSR1 peptide B region individually. (At 30 min and 1 hr after nitrogen starvation, and 1 hr, 2 hr and 6 hr after sulfur starvation.) Please note that the following reference genome and gene models used in these experiments are linked below; C.reinhardtii_v5.3_genomic_scaffold_plastids.fasta.gz reference_gene_model.gtf.gz These are based off Phytozome (http://www.phytozome.net/) which does not provide access to earlier version data.
2015-07-28 | E-GEOD-59629 | biostudies-arrayexpress
Project description:N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. Multiple sequencing methods are developed to profile the distribution of 6mA in Chlamydomonas including MeDIP-Seq, enzyme-treated DNA-Seq, MNase-Seq and RNA-Seq.
2015-04-30 | E-GEOD-62690 | biostudies-arrayexpress
Project description:Like many microalgae unicellular green alga, Chlamydomonas reinhardtii also accumulates energy rich storage lipid and starch under nutrient-limited conditions, such as phosphorus (P) and nitrogen (N) limitation. To dissect the transcriptional regulation of lipid and starch biosynthesis in response to nutrient limitation, we have characterized the biological role of a candidate regulator: the MYB family transcription factor, PSR1 (phosphorus starvation response 1). Genetically modified (psr1 compromised) and wild-type lines were grown under P limiting and sufficient conditions and assayed at two time points by SOLiD RNA-seq.
2016-01-08 | E-MTAB-2556 | biostudies-arrayexpress
Project description:We analysed global gene expression changes in Chlamydomonas reinhardtii in response to 1h UV-B, applied at the same low level that was seen to promote subsequent UV-B stress tolerance, in order to elucidate the transcriptional reprogramming that leads to UV-B acclimation. mRNA profiles generated by deep sequencing from triplicate replicate Chlamydomonas reinhardtii samples sourced from independent cultures either protected from UV-B or exposed to 1h acclimation-level UV-B.
2015-11-11 | E-GEOD-68739 | biostudies-arrayexpress
Project description:Three acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. Each gene also shows increased mRNA abundance in other TAG-accumulating conditions (-S, -P, -Zn, -Fe). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared to the parent strain, D66+, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analysis suggest than an SBP domain transcription factor protein, whose mRNA increases precede that of other genes like DGAT1, is a candidate regulator of the N-deficiency response. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared to the parental strain in N-starvation conditions and is unaffected by other nutrient stresses, suggesting the operation of multiple signaling pathways leading to stress-induced TAG accumulation. Sampling of Chlamydomonas 2137 cultivated in TAP, N-Free TAP or TAP with varying amounts of N.
2013-01-10 | E-GEOD-34585 | biostudies-arrayexpress
Project description:High throughput sequencing was used to investigate the production of small RNAs from cultivated tomato cultivar M82 and its wild relative Solanum pennellii. In order to understand the pattern of inheritance of the samll RNAs, interspecific hybrids (F1 and F2) along with series of introgressed lines comprising precise short genomic regions from S. pennellii in M82 background were used. Examination of small RNA production in several tomato lines.
2012-05-29 | E-GEOD-23562 | biostudies-arrayexpress
Project description:This project aims to investigate the metabolic pathways expressed by the active microbial community occurring at the deep continental subsurface. Subsurface chemoLithoautotrophic Microbial Ecosystems (SLiMEs) under oligotrophic conditions are supported by H2; however, the overall ecological trophic structures of these communities are poorly understood. Some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa appear to support inverted trophic pyramids wherein methanogens contributing <5% of the total DNA apparently produce CH4 that supports the rest of the community. Here we show the active metabolic relationships of one such trophic structure by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Four autotrophic β-proteobacteria genera that are capable of oxidizing sulfur by denitrification dominate. They co-occur with sulfate reducers, anaerobic methane oxidizers and methanogens, which each comprises <5% of the total community. Defining trophic levels of microbial chemolithoautotrophs by the number of transfers from the initial abiotic H2-driven CO2 fixation, we propose a top-down cascade influence of the metabolic consumers that enhances the fitness of the metabolic producers to explain the inverted biomass pyramid of a multitrophic SLiME. Symbiotic partnerships are pivotal in the deep biosphere on and potentially beyond the Earth.
2018-10-27 | PXD004634 | Pride
Project description:We used RNA-Seq to query the Chlamydomonas reinhardtii transcriptome for regulation by CO2 concentration and by the transcription regulator CIA5(CCM1). Both CO2 concentration and CIA5 are known to play roles in induction of an essential CO2-concentrating mechanism (CCM), but the degree of interaction and the extent of global regulation beyond the CCM were not previously understood. We compared the transcriptome of a wild type strain vs. a cia5 strain under 3 CO2 supply conditions: high CO2 (H-CO2; 5%); low CO2 (L-CO2; 0.03 to 0.05%); and very-low CO2 (VL-CO2; <0.02%). Our goals were to: 1) reveal candidate genes that, through changes in their expression, distinguish multiple acclimation states induced by H-CO2, L-CO2, and VL-CO2; 2) reveal genes regulated directly or indirectly by CIA5; and 3) reveal genes responding to the interaction between CIA5 and changes in CO2 concentration. Our results revealed a small group of genes as encoding putative Ci transporters based on their expression patterns. The results also showed a massive and much broader impact on global gene regulation by CIA5/CCM1, which directly or indirectly affected 15% of the Chlamydomonas genome. The transcriptomes under L-CO2 and VL-CO2 conditions were not significantly different, suggesting that these two acclimation states must be controlled by mechanisms operating beyond transcript abundance. Both 137c wild type (cc125) and cia5 mutant were induced for 4 hours induction under H-CO2 (5%), L-CO2 (300~500 ppm), and VL-CO2 (100~200 ppm) conditions. 2 biological replicates were done for each strain -CO2 combination.
2012-08-10 | E-GEOD-33927 | biostudies-arrayexpress