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.
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.
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.
Project description:Some molecular chaperones are involved not only in assisting the folding of proteins but also, given appropriate conditions, in their degradation. This is the case of Hsp70 and Hsp90, which in concert with the cochaperone CHIP –an E3 ligase–, direct their bound substrate to degradation through ubiquitination. We have generated complexes between the chaperone (Hsp70 or Hsp90), the cochaperone CHIP and, as substrate, a p53 variant containing the GST protein (p53-TMGST). The two ternary complexes (Hsp70:p53-TMGST:CHIP and Hsp90:p53-TMGST:CHIP) ubiquitinate the substrate, and this is done with a higher efficiency than in the absence of the chaperones. The 3D structures of the two complexes, obtained using a combination of cryoelectron microscopy and crosslinking mass spectrometry, show the substrate located between the chaperone and the cochaperone, which suggests an ubiquitination mechanism. Both complexes are extremely flexible, which is crucial for the ubiquitination process.
Project description:Acidic residues (Asp and Glu) have a high prevalence on protein surfaces, but cross-linking reactions targeting these residues are limited. Existing methods either require high-concentration coupling reagents or have low structural compatibility. Here we extended our previously reported “plant-and-cast” strategy to develop two heterobifunctional cross-linkers DE1 and OPAAZ. These cross-linking reaction proceeds at neutral pH and room temperature without coupling reagents.
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.
Project description:Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue- resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. Wefound two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible. Biological significance Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue- resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so.We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Ourwork lays the foundations of studying themolecular details of biological processes at greater ease than this could be done so far.