Arabidopsis thaliana
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA369530 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5222790.fastq.gz | Fastqsanger.gz | |||
| SRR5222790_1.fastq.gz | Fastqsanger.gz | |||
| SRR5222790_2.fastq.gz | Fastqsanger.gz | |||
| SRR5222800.fastq.gz | Fastqsanger.gz | |||
| SRR5222800_1.fastq.gz | Fastqsanger.gz |
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Project description:Background: Analysis of the effect that chromatin structure has on the expression patterns of eukaryotic genes has recently expanded knowledge of the complex influence genome accessibility has on genome function. Interlaced with regular nucleosomal patterning are other mobile and labile sub-nucleosomal-sized protein structures bound to the genome such as transcription factors (TF), initiation complexes, and modified nucleosomes. Results: We present chromatin structure maps of the A. thaliana Col-0 wild-type (in vitro cell culture) combining differential micrococcal nuclease (MNase) digestion and analysis by paired-end next-generation sequencing with RNASeq data to provide insight into the sensitive genomic regions and variable DNA-bound particle size related to transcriptional activity. The landscape of sub-nucleosomal sized particles (subNSPs) has been investigated, revealing the DNA-binding complex positioning genome-wide. We observe the dynamic changes in the distribution of these complexes surrounding genomic features, particularly at transcription start sites (TSS). Differential digestion reveals the presence of MNase-sensitive smaller particles and a labile -1 nucleosome upstream of the TSS of active genes. These changes correlate with gene expression differences resulting from different environmental conditions of light- or dark growth both globally and locally, and a complex profile of bound particles is directly visualised. Conclusions: Analysis of the A. thaliana genome reveals that resolution of chromatin particle size by differential MNase digestion allows detection of the sensitive features in chromatin structure that have hereto been obfuscated. The concomitant analysis of transcript levels reveals the impact of transient extrinsic factors in modifying the sub- nucleosomal landscape in association with transcriptional changes, giving insight into the binding of transcription-associated factors.
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Project description:Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that nucleosomes at 5% of budding yeast nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequence-based profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling. We have analyzed the chromatin landscape of the yeast genome using paired-end MNase-seq and the chromatin binding of yeast remodelers Swr1, Ino80 and RSC at base-pair resolution using native chromatin immunoprecipitation followed by sequencing (N-ChIP-seq).
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Project description:Here we use bisulfite conversion of RNA combined with high-throughput IIlumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites in ribosomal RNAs of all three sub-cellular transcriptomes in Arabidopsis thaliana. m5C sites in rRNAs were also anlyzed in Arabidopsis T-DNA knockouts for the RNA methyltransferases TRM4A, TRM4B, TRDMT1, NSUN5, NOP2A, NOP2B and NOP2C.
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Project description:Chromosome conformation capture (3C)-derived methods have become an indispensable tool in the study of gene regulation. The three-dimensional contacts can probe depend strongly on the properties of the enzyme used to fragment chromatin prior to proximity-driven ligation. Micrococcal nuclease (MNase), used in Micro-C, increases resolution at the expense of low ligation efficiency and the need for extensive enzyme titration. To overcome these limitations, we engineered a highly active, TEV protease-activatable caspase-activated DNase (CAD) to enable an efficient, low-sequence-bias, and high-resolution proximity ligation assay we call CAD-C. CAD-C was successful on the first attempt for each human cell line tested and the resulting datasets capture loops, TADs, compartments, and stripes similarly to Micro-C. However, compared to Micro-C and Hi-C, CAD-C shows enhanced sensitivity for promoter-enhancer loops. Leveraging the ligation-competent DNA ends produced by CAD cleavage, we show that CAD-C is compatible with a highly streamlined, repair-free protocol and produces multi-step CADwalks, consecutive ligations between nucleosomal or sub-nucleosomal fragments. With these walks, we probe local chromatin fiber folding contacts, nucleosomal and sub-nucleosomal footprints, and long-range nuclear organization regimes in human cell lines. CAD-C is an efficient, robust chromatin structure assay that can span sub-nucleosomal to chromosomal length scales in a single experiment.
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