Suborganelle-specific protein complexes analysis enabled by in-vivo crosslinking coupled with proximal labeling
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: Lihua Zhang
PROVIDER: PXD034768 | JPOST Repository | Tue Jun 21 00:00:00 BST 2022
REPOSITORIES: jPOST
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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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Project description: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;
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Project description:Stress granules are dynamic non-membrane bound organelles made up of untranslating messenger ribonucleoproteins (mRNPs) that form when cells integrate stressful environmental cues resulting in stalled translation initiation complexes. Although stress granules dramatically alter mRNA and protein localization, understanding these complexes has proven to be challenging through conventional imaging, purification, and crosslinking approaches. We therefore developed an RNA proximity labeling technique, APEX-Seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-Seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching both the spatial transcriptome using APEX-seq, and the spatial proteome using APEX-mass spectrometry (APEX-MS) provide new insights into the organization of translation initiation complexes on active mRNAs, as well as revealing unanticipated complexity in stress granule contents, and provides a powerful approach to explore the spatial environment of macromolecules.
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Project description:Alignment and quantification of ChIP-exo crosslinking patterns reveal the spatial organization of protein-DNA complexes
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Project description:Mammalian chromosomes are folded into an intricate hierarchy of structural domains, within which topologically associating domains (TADs) and CTCF-associated loops partition the physical interactions between regulatory sequences. Current understanding of chromosome folding largely relies on chromosome conformation capture (3C)-based experiments, where chromosomal interactions are detected as ligation products after crosslinking of chromatin. To measure chromosome structure in vivo, quantitatively and without relying on crosslinking and ligation, we have implemented a new method named damC. DamC combines DNA-methylation based detection of chromosomal interactions with next-generation sequencing and a biophysical model of methylation kinetics. DamC performed in mouse embryonic stem cells provides the first in vivo validation of the existence of TADs and CTCF loops, confirms 3C-based measurements of the scaling of contact probabilities within TADs, and provides evidence that mammalian chromatin in vivo is essentially rigid below 5 kilobases. Combining damC with transposon-mediated genomic engineering shows that new loops can be formed between ectopically introduced and endogenous CTCF sites, which alters the partitioning of physical interactions within TADs. This establishes damC as a crosslinking- and ligation-free framework to measure and modify chromosome interactions combined with a solid theoretical background for rigorous data interpretation. This orthogonal approach to 3C validates the existence of key structural features of mammalian chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated.
2019-05-16 | GSE128015 | GEO
Project description:Mammalian chromosomes are folded into an intricate hierarchy of structural domains, within which topologically associating domains (TADs) and CTCF-associated loops partition the physical interactions between regulatory sequences. Current understanding of chromosome folding largely relies on chromosome conformation capture (3C)-based experiments, where chromosomal interactions are detected as ligation products after crosslinking of chromatin. To measure chromosome structure in vivo, quantitatively and without relying on crosslinking and ligation, we have implemented a new method named damC. DamC combines DNA-methylation based detection of chromosomal interactions with next-generation sequencing and a biophysical model of methylation kinetics. DamC performed in mouse embryonic stem cells provides the first in vivo validation of the existence of TADs and CTCF loops, confirms 3C-based measurements of the scaling of contact probabilities within TADs, and provides evidence that mammalian chromatin in vivo is essentially rigid below 5 kilobases. Combining damC with transposon-mediated genomic engineering shows that new loops can be formed between ectopically introduced and endogenous CTCF sites, which alters the partitioning of physical interactions within TADs. This establishes damC as a crosslinking- and ligation-free framework to measure and modify chromosome interactions combined with a solid theoretical background for rigorous data interpretation. This orthogonal approach to 3C validates the existence of key structural features of mammalian chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated.
2019-05-16 | GSE128013 | GEO
Project description:Mammalian chromosomes are folded into an intricate hierarchy of structural domains, within which topologically associating domains (TADs) and CTCF-associated loops partition the physical interactions between regulatory sequences. Current understanding of chromosome folding largely relies on chromosome conformation capture (3C)-based experiments, where chromosomal interactions are detected as ligation products after crosslinking of chromatin. To measure chromosome structure in vivo, quantitatively and without relying on crosslinking and ligation, we have implemented a new method named damC. DamC combines DNA-methylation based detection of chromosomal interactions with next-generation sequencing and a biophysical model of methylation kinetics. DamC performed in mouse embryonic stem cells provides the first in vivo validation of the existence of TADs and CTCF loops, confirms 3C-based measurements of the scaling of contact probabilities within TADs, and provides evidence that mammalian chromatin in vivo is essentially rigid below 5 kilobases. Combining damC with transposon-mediated genomic engineering shows that new loops can be formed between ectopically introduced and endogenous CTCF sites, which alters the partitioning of physical interactions within TADs. This establishes damC as a crosslinking- and ligation-free framework to measure and modify chromosome interactions combined with a solid theoretical background for rigorous data interpretation. This orthogonal approach to 3C validates the existence of key structural features of mammalian chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated.
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