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Systematic evaluation of chromosome conformation capture assays [1D]

ABSTRACT: Chromosome conformation capture (3C)-based assays are used to map chromatin interactions genome-wide. Quantitative analyses of chromatin interaction maps can lead to insights into the spatial organization of chromosomes and the mechanisms by which they fold. A number of protocols such as in situ Hi-C and Micro-C are now widely used and these differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of experimental parameters of 3C-based protocols. We find that different protocols capture different 3D genome features with different efficiencies. First, the use of crosslinkers such as DSG in addition to formaldehyde improves signal-to-noise allowing detection of thousands of additional loops and strengthening compartment signal. Second, fragmenting chromatin to the level of nucleosomes using MNase allows detection of more loops. On the other hand, protocols that generate larger multi-kb fragments produce stronger compartmentalization signals. We confirmed our results in multiple cell states such as pluripotent and differentiated cells as well as cell cycle stages; Mitosis and G1. Based on these insights we developed Hi-C 3.0, a single protocol that can be used to both efficiently detect chromatin loops and to quantify compartmentalization. Finally, this study produced ultra-deeply sequenced reference interaction maps using in situ Hi-C, Micro-C and Hi-C 3.0 for commonly cell lines in the 4D Nucleome Project.

ORGANISM(S): Homo sapiens

PROVIDER: GSE165895 | GEO | 2021/03/08

REPOSITORIES: GEO

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Systematic evaluation of chromosome conformation capture assays

Project description:Chromosome conformation capture (3C)-based assays are used to map chromatin interactions genome-wide. Quantitative analyses of chromatin interaction maps can lead to insights into the spatial organization of chromosomes and the mechanisms by which they fold. A number of protocols such as in situ Hi-C and Micro-C are now widely used and these differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of experimental parameters of 3C-based protocols. We find that different protocols capture different 3D genome features with different efficiencies. First, the use of crosslinkers such as DSG in addition to formaldehyde improves signal-to-noise allowing detection of thousands of additional loops and strengthening compartment signal. Second, fragmenting chromatin to the level of nucleosomes using MNase allows detection of more loops. On the other hand, protocols that generate larger multi-kb fragments produce stronger compartmentalization signals. We confirmed our results in multiple cell states such as pluripotent and differentiated cells as well as cell cycle stages; Mitosis and G1. Based on these insights we developed Hi-C 3.0, a single protocol that can be used to both efficiently detect chromatin loops and to quantify compartmentalization. Finally, this study produced ultra-deeply sequenced reference interaction maps using in situ Hi-C, Micro-C and Hi-C 3.0 for commonly cell lines in the 4D Nucleome Project.

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Comparison of Hi-C results using in-solution versus in-nucleus ligation

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Comparison of Hi-C results using in-solution versus in-nucleus ligation

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Project description:Mammalian chromosomes are folded into intricate hierarchies of interaction 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 modified version of damID 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 and confirms 3C-based measurements of the scaling of contact probabilities. 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 orthogonal approach to 3C provides the first crosslinking- and ligation-free validation of the existence of key structural features of mammalian chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated.

2019-06-03 | PXD013507 | Pride

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