Single step cross-linking ChIP-seq
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA794167 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17431300_1.fastq.gz | Fastqsanger.gz | |||
| SRR17431300_2.fastq.gz | Fastqsanger.gz | |||
| SRR17431301_1.fastq.gz | Fastqsanger.gz | |||
| SRR17431301_2.fastq.gz | Fastqsanger.gz | |||
| SRR17431302_1.fastq.gz | Fastqsanger.gz |
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Project description:The chromatin remodeller ATRX interacts with the histone chaperone DAXX, to deposit the histone variant H3.3 at sites of nucleosome turnover. ATRX is known to bind repetitive, heterochromatic regions of the genome including telomeres, ribosomal DNA and pericentric repeats many of which are putative G-quadruplex forming sequences (PQS). At these sites ATRX plays an ancillary role in a wide range of nuclear processes facilitating replication, chromatin modification and transcription. Here, using an improved protocol for chromatin immunoprecipitation, we show that ATRX also binds active regulatory elements in euchromatin. Mutations in ATRX lead to perturbation of gene expression associated with a reduction in chromatin accessibility, histone modification, transcription factor binding and deposition of H3.3 at the sequences to which it normally binds. In erythroid cells where down regulation of a-globin expression is a hallmark of ATR-X syndrome, perturbation of chromatin accessibility and gene expression occurs in only a subset of cells. The stochastic nature of this process suggests that ATRX acts as a general facilitator of cell specific transcriptional and epigenetic programmes, both in heterochromatin and euchromatin.
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Project description:The chromatin remodeller ATRX interacts with the histone chaperone DAXX, to deposit the histone variant H3.3 at sites of nucleosome turnover. ATRX is known to bind repetitive, heterochromatic regions of the genome including telomeres, ribosomal DNA and pericentric repeats many of which are putative G-quadruplex forming sequences (PQS). At these sites ATRX plays an ancillary role in a wide range of nuclear processes facilitating replication, chromatin modification and transcription. Here, using an improved protocol for chromatin immunoprecipitation, we show that ATRX also binds active regulatory elements in euchromatin. Mutations in ATRX lead to perturbation of gene expression associated with a reduction in chromatin accessibility, histone modification, transcription factor binding and deposition of H3.3 at the sequences to which it normally binds. In erythroid cells where down regulation of a-globin expression is a hallmark of ATR-X syndrome, perturbation of chromatin accessibility and gene expression occurs in only a subset of cells. The stochastic nature of this process suggests that ATRX acts as a general facilitator of cell specific transcriptional and epigenetic programmes, both in heterochromatin and euchromatin.
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Project description:RNA-protein interactions mediate a vast number of intracellular processes. CLIR-MS (cross-linking of isotope labeled RNA and tandem mass spectrometry) is a mass spectrometric technique that allows the identification of RNA-protein interaction sites at single nucleotide/amino acid resolution in a single experiment. The use of isotopically labeled RNA segments for UV light induced cross-linking generates characteristic isotope patterns that constrain the sequence database searches, thus increasing resolution. Whereas the use of segmentally isotopically labeled RNA is effective, it is technically involved and not applicable in some settings, e.g. in cell or tissue samples. A straightforward approach that maintains the advantages of isotopic labeling but obviates the need for segmental RNA labeling would therefore advance the field. Here we introduce an extension of the CLIR-MS workflow that uses unlabeled RNA during the cross-linking reaction and subsequently adds an isotopic label during sample preparation for MS analysis. The approach uses commercially available reagents and can be performed without specialized equipment in any lab. After RNase and protease digests of a cross-linked complex, an RNA-peptide adduct consists of a single peptide and a short nucleic acid adduct. We label the nucleic acid part of these adducts using the enzyme T4 polynucleotide kinase (T4-PNK) and a 1:1 mixture of heavy (18O4-gamma-ATP) and light ATP. In this simple, one-step reaction three of the four heavy oxygen atoms are transferred from the gamma-phosphate to the 5'-end of the RNA adduct. The isotopic difference of light and heavy cross-linked peptides (6.01 Da) can be detected using tandem mass spectrometry after enrichment of the cross-linked peptides. We applied this approach to the RNA recognition motif (RRM) of the protein FOX1 in complex with its cognate binding substrate, FOX-binding element RNA (FBE-RNA). Using a variation of the approach, we were able to label a single phosphate within an RNA and unambiguously determine the cross-linking site of the FOX1-RRM to the FBE at single residue resolution on RNA- and protein level. Specifically introducing isotopic labels improves identification of cross-linked species and enables relative quantification based on isotope dilution.
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Project description:eIF4G2 cross-linking immunoprecipitation (CLIP) in primary cortical neurons
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Project description:Chemical cross-linking mass spectrometry (CXMS) has emerged as a powerful technology to analyze protein complex structure and interaction. However, the spectral fragmentation behavior and spectral data retrieval of cross-linked peptides are more complex than single peptides. In this study, we designed and synthesized a trehalose-based MS-cleavable cross-linker, Trehalose Disuccinimidyl Ester (TDS), which possesses a CID/HCD-cleavable glycosidic bond and has good bioorthogonality and amphipathicity. Using TDS, the cross-linked peptides were simplified into conventional single peptides via the selective cleavage between glycosidic and peptide bonds under individual MS collision energy, which enhances the matching degree and retrieval throughput of spectral identification. The deep coverage of the TDS method facilitated the accurate resolution of the structural dynamics of purified proteins with different physicochemical properties and yeast 26S proteasome complex. Additionally, the bioorthogonality and amphipathicity of TDS enabled the cross-linking reaction to occur in vivo without the introduction of any organic solvent. Through coinciding with this feature and MS-cleavable capacity, TDS provided us a high throughput snapshot of the structural architecture of protein complex in live cells. These results provide a promising TDS toolkit to study CXMS and decipher the protein conformations and interactions with high accuracy and easy portability for cross-linker design.
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