Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1257452 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR33395656_1.fastq.gz | Fastqsanger.gz | |||
| SRR33395656_2.fastq.gz | Fastqsanger.gz | |||
| SRR33395657_1.fastq.gz | Fastqsanger.gz | |||
| SRR33395657_2.fastq.gz | Fastqsanger.gz | |||
| SRR33395658_1.fastq.gz | Fastqsanger.gz |
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Project description:Analysis of NAC53 and NAC78 interactome in normal condition or upon Pseudomonas syringea pv. tomato DC3000 ∆HopQ infection. Constructs promUBQ10::eGFP-NAC53/eGFP-NAC78 are used as baits and promUBQ10::eGFP is used as control. Analysis of ubiquitination sites on NAC53 and NAC78 upon HRD1a/b ubiquitination. Analysis of NAC53, NAC78 and MDP25 subcellular interactome. For NAC53 and NAC78, dual tagged constructs pLjUBQ1::mCerrulean-NAC53/78-mCherry were used to pull-down N-ter moieties (cytosolic/nuclear localization) or C-ter moieties (endoplasmic reticulum localization). pUBQ10::eGFP and p35S::mCherry-HDEL are used as controls. For MDP25, protein variants were used, MDP25FL (plasma membrane localization), MDP25∆N25 (cytosolic localization) or N25MDP25 (plasma membrane localization). pUBQ10::eGFP is used as control.
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Project description:Full genome replication in eukaryotes depends on the function of thousands of DNA replication origins (ORIs). The genome-wide identification of ORI location, achieved for animal and plant cells in culture, has been important to define their DNA and chromatin features. A major challenge in the field is to approach the biology of ORIs in whole organisms to understand their developmental plasticity. Here, we have determined the ORI location, activity and chromatin landscape in two developmental stages of Arabidopsis thaliana. We found that ORIs associate with multiple chromatin signatures including the most frequent at TSS but also at proximal and distal gene regulatory regions or repressed Polycomb domains. In constitutive heterochromatin, a high fraction of ORIs colocalize with GC-rich retrotransposons. Quantitative analysis of ORI activity led us to conclude that strong ORIs possess high scores of local GC content and clusters of GGN trinucleotides that may form G quadruplexes and other G-rich structures. We also found that development primarily influences ORI firing strength rather than the location of ORIs in different genomic loci. Moreover, ORIs that preferentially fire at early vegetative stages colocalize with GC-rich heterochromatin whereas those at later stages associate with transcribed genes. Our results provide the first identification of ORI features in a whole organism, opening new ways of studying DNA replication in different cell types in the context of development under normal and mutant conditions.
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Project description:Genomic mapping of DNA replication origins (ORIs) in mammals provides a powerful means for understanding the regulatory complexity of our genome. Here we combine a genome-wide approach to identify preferential sites of DNA replication initiation at 0.4% of the mouse genome with detailed molecular analysis at distinct classes of ORIs according to their location relative to the genes. Our study reveals that 85% of the replication initiation sites in mouse embryonic stem (ES) cells are associated with transcriptional units. Nearly half of the identified ORIs map at promoter regions and, interestingly, ORI density strongly correlates with promoter density, reflecting the coordinated organisation of replication and transcription in the mouse genome. Detailed analysis of ORI activity showed that CpG island promoter-ORIs are the most efficient ORIs in ES cells and both ORI specification and firing efficiency are maintained across cell types. Remarkably, the distribution of replication initiation sites at promoter-ORIs exactly parallels that of transcription start sites (TSS) suggesting a co-evolution of the regulatory regions driving replication and transcription. Moreover, we found that promoter-ORIs are significantly enriched in CAGE tags derived from early embryos relative to all promoters. This association implies that transcription initiation early in development sets the probability of ORI activation unveiling a new hallmark in ORI efficiency regulation in mammalian cells. Two biological replicates of lambda-exonuclease treated short nascent strands (100-600 or 300-800 nt in length) were co-hybridised with genomic DNA from the same cells to tiled genomic array covering 10.1 Mb of the mouse genome (Agilent Technologies)
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