Determination of local chromatin interactions using a combined CRISPR and peroxidase APEX2 system
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ABSTRACT:
PROVIDER: PRJNA464393 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7128240_1.fastq.gz | Fastqsanger.gz | |||
| SRR7128240_2.fastq.gz | Fastqsanger.gz | |||
| SRR7128241_1.fastq.gz | Fastqsanger.gz | |||
| SRR7128241_2.fastq.gz | Fastqsanger.gz | |||
| SRR7128242_1.fastq.gz | Fastqsanger.gz |
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Project description:The architecture and function of chromatin are largely regulated by local interacting molecules, such as transcription factors and noncoding RNAs. However, our understanding of these regulatory molecules at a given locus is limited because of technical difficulties. Here, we describe the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and an engineered ascorbate peroxidase 2 (APEX2) system to investigate local chromatin interactions (CAPLOCUS). We showed that with specific small-guide RNA targets, CAPLOCUS could efficiently identify both repetitive genomic regions and single-copy genomic locus with high resolution. Genome-wide sequencing revealed known and potential long-range chromatin interactions for a specific single-copy locus. CAPLOCUS also identified telomere-associated RNA. CAPLOCUS, followed by mass spectrometry, identified both known and novel telomere-associated proteins in their native states. Thus, CAPLOCUS may be a useful approach for studying local interacting molecules at any given chromosomal location.
2019-02-18 | GSE114133 | GEO
Project description:The architecture and function of chromatin are largely regulated by local interacting molecules, such as transcription factors and noncoding RNAs. However, our understanding of these regulatory molecules at a given locus is limited because of technical difficulties. Here, we describe the use of Clustered Regularly Interspaced Short Palindromic Repeats and an engineered ascorbate peroxidase 2 (APEX2) system to investigate local chromatin interactions (CAPLOCUS). We showed that with specific small-guide RNA targets, CAPLOCUS could efficiently identify both repetitive genomic regions and single-copy genomic locus with high resolution. Genome-wide sequencing revealed known and potential long-range chromatin interactions for a specific single-copy locus. CAPLOCUS also identified telomere-associated RNAs. CAPLOCUS, followed by mass spectrometry, identified both known and novel telomere-associated proteins in their native states. Thus, CAPLOCUS may be a useful approach for studying local interacting molecules at any given chromosomal location.
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Project description:We sought to map RBM6 interactome using ascorbate peroxidase (APEX2)-based proximity labelling combined with mass spectrometry. Toward this end, we used CRISPR-cas9 methodology to establish MCF10A cell line expressing APEX2 fused to the N-terminal of RBM6, hereafter called MCF10AAPEX2-RBM6. The peroxidase activity of APEX2-RBM6 fusion was confirmed. Next, RBM6 proximal proteins that were biotinylated by APEX2 activation using hydrogen peroxidase (H2O2) in the presence of biotin-phenol were isolated and subjected to mass spectrometry. We identified 173 (P-value<0.05) RBM6 proximity-interaction partners.
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Project description:We used the flu mutant of Arabidopsis and a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX) to address the interactions between different reactive oxygen species (ROS) signaling pathways. The conditional flu mutant of Arabidopsis accumulates excess protochlorophyllide in the dark within chloroplast membranes that upon illumination acts as a photosensitizer and generates singlet oxygen (1O2). Immediately after the release of singlet oxygen rapid changes in nuclear gene expression occur. Distinct sets of genes were activated that were different from those induced by other reactive oxygen species, superoxide or hydrogen peroxide (H2O2), suggesting that different types of active oxygen species activate distinct signaling pathways. It was not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H2O2 in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX, line 14/2 PMID: 15165186). In the flu mutant overexpressing tAPX, the expression of most of the nuclear genes that were rapidly activated after the release of 1O2 was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX had only a very minor impact on nuclear gene expression. The results suggest that H2O2 antagonizes the 1O2-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H2O2- and 1O2-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions. Experiment Overall Design: Arabidopsis thaliana rosette leaves were harvested after 2 h of reillumination following a 8h dark period for RNA extraction and hybridization on Affymetrix ATH1 microarrays. The entire experiment was performed six times, providing independent biological replicates. For each of the six experiments, all four lines, wild-type, thylakoidal ascorbate peroxidase overexpressor (over tAPX, line 14/2), flu mutant and flu plants overexpressing thylakoidal ascorbate peroxidase were grown for 3 weeks under continuous light at 90 mmol. m-2 . s-1, transferred to the dark for 8 h, and reilluminated for 120 min before the rosette leaves of at least 10 plants per line were harvested. Total RNAs from three separate biological experiments were pooled (= 1 biological rep.) for the preparation of cDNA and the subsequent synthesis of biotin-labeled complementary RNA as recommended by Affymetrix.
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