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Crosslinking experiment of the RAGE oligomeric complexes

ABSTRACT: XL-MS of RAGE oligomeric complexes. The Crosslinking agent is BS3. Samples were subjected to sequential digestion. 91212_AM_1_3_1_HCDFT, 91212_AM_1_3_2_HCDFT, 91212_AM_4_3_1_HCDFT, 91212_AM_4_3_2_HCDFT - Tryp-Gluc; 91212_AM_1_2_1_HCDFT, 91212_AM_1_2_2_HCDFT, 91212_AM_4_2_1_HCDFT, 91212_AM_4_2_2_HCDFT - Tryp-Chym; 91212_AM_1_1_1_HCDFT, 91212_AM_1_1_2_HCDFT, 91212_AM_4_1_1_HCDFT, 91212_AM_4_1_2_HCDFT - Tryp-Tryp.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Human

SUBMITTER: Alexander Moysa

PROVIDER: MSV000086438 | MassIVE | Wed Nov 11 12:09:00 GMT 2020

REPOSITORIES: MassIVE

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Project description:Each protein within a regulatory complex associates with the genome by either binding DNA directly or by forming protein-protein interactions with DNA-bound proteins. In the chromatin immunoprecipitation (ChIP) assay, each protein’s unique mode of genomic association may be reflected by their patterns of formaldehyde-induced crosslinks to the DNA sequences that are in very close proximity. The ChIP-exo protocol precisely delineates protein-DNA crosslinking patterns by combining ChIP with 5' to 3' exonuclease digestion. Within a regulatory complex, the physical distance of a regulatory protein to the DNA affects crosslinking efficiencies. Therefore, the spatial organization of a protein-DNA complex could potentially be inferred by analyzing how crosslinking signatures vary between the subunits of a regulatory complex, and how they remain consistent over a set of coordinately regulated regions. Here, we present a computational framework that aligns ChIP-exo crosslinking patterns from multiple proteins across a set of regulatory regions, and which detects and quantifies protein-DNA crosslinking events within the aligned profiles. Our gapped multiple profile alignment approach does not rely on sequence motif features, but rather operates directly on the multi-protein, strand separated ChIP-exo tag patterns. The output of the alignment approach is a set of composite profiles that represent the crosslinking signatures of the complex across analyzed regulatory regions. We then use a probabilistic mixture model to deconvolve individual crosslinking events within the aligned ChIP-exo profiles, enabling consistent measurements of protein-DNA crosslinking strengths across multiple proteins. Lastly, we apply dimensionality reduction to visualize the relative organization of proteins within the regulatory complex. We demonstrate our approach by applying it to characterize regulatory complex organization in three biological settings. Firstly, we demonstrate that our alignment approach can recover the known organization of regulatory proteins at yeast ribosomal protein genes, without relying on any DNA sequence features. Secondly, we apply our gapped alignment and crosslinking quantification approaches to a novel set of ChIP-exo data to characterize the spatial organization of Pol III transcriptional machinery assembly at yeast tRNA genes. Finally, we demonstrate that our approach can be used to quantify changes in protein-DNA complex organization when applied to ChIP-nexus data from Drosophila Pol II transcriptional components in two experimental conditions. Our results suggest that principled analyses of ChIP-exo crosslinking patterns enable inference of spatial organization within protein-DNA complexes.

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Crosslinking experiment of the RAGE oligomeric complexes

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