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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 [Hi-C]

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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Systematic evaluation of chromosome conformation capture assays

2021-03-08 | GSE163666 | GEO

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

2015-08-12 | GSE70181 | GEO

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

Project description:Chromosome conformation capture (3C) and derivative (4C, 5C and Hi-C) methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. We previously described an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing "in-nucleus ligation" and the standard "in-solution ligation". The results show that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. Thus in-nucleus ligation not only simplifies the experimental procedures, but also produces higher quality data with benefits for the entire range of 3C applications. We created Hi-C libraries by two different methods, in-solution ligation and in-nucleus ligation, from two biological replicates each of mouse foetal liver cells (mouse-1 and mouse-2) and human ES cells (human-1 and human-2) or the mixture of these two species. We also sequenced a random ligation library prepared by reversal of the cross-links and purification of the DNA prior to ligation.

2015-08-12 | E-GEOD-70181 | biostudies-arrayexpress

Micro-C XL: assaying chromosome conformation at length scales from the nucleosome to the entire genome

Project description:Structural analysis of chromosome folding in vivo has been revolutionized by Chromosome Conformation Capture (3C) and related methods, which use proximity ligation to identify chromosomal loci in physical contact. We recently described a variant 3C technique, Micro-C, in which chromatin is fragmented to mononucleosomes using micrococcal nuclease, enabling nucleosome-resolution folding maps of the genome. Here, we describe an improved Micro-C protocol using long crosslinkers, termed Micro-C XL, which exhibits greatly increased signal to noise, and provides further insight into the folding of the yeast genome. We also find that signal to noise is much improved in Micro-C XL libraries generated from relatively insoluble chromatin as opposed to soluble material, providing a simple method to physically enrich for bona-fide long-range interactions. Micro-C XL maps of the budding and fission yeast genomes reveal both short-range chromosome fiber features such as chromosomally-interacting domains (CIDs), as well as higher-order features such as clustering of centromeres and telomeres, thereby addressing the primary discrepancy between prior Micro-C data and reported 3C and Hi-C analyses. Interestingly, comparison of chromosome folding maps of S. cerevisiae and S. pombe revealed widespread qualitative similarities, yet quantitative differences, between these distantly-related species. Micro-C XL thus provides a single assay suitable for interrogation of chromosome folding at length scales from the nucleosome to the full genome.

2016-09-13 | GSE85220 | GEO

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2020-12-11 | GSE134590 | GEO

Modeling of DNA methylation in cis reveals principles of chromatin folding in vivo in the absence of crosslinking and ligation

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

Distinct 3D Genome Organization in the Two Nuclei of Tetrahymena thermophila

Project description:Somatic macronucleus (MAC) and germline micronucleus (MIC) of Tetrahymena thermophila are different in chromosome numbers, sizes, functions and cohesin complex locations. Loss of cohesin complex resulted in genome-wide disappearance of topologically associating domains (TADs) and chromatin loops in mammalian cells. However, the higher-level chromatin organization in Tetrahymena thermophila which contains both cohesin free MAC and cohesin located MIC are largely unknown. Here, using the Hi-C and HiChIP methods, we reveal that, these two nuclei possess distinct three-dimensional genome structures. In the MAC, each chromosome occupies its own territory and there are no chromatin compartmentalization or chromatin domains. The chromatin loops in MAC are mainly related to chromatin structures rather than transcriptional regulation. The MIC also without chromatin compartmentalization, but with chromatin domains and the domain boundaries are consistent with chromatin breakage sites (CBSs) which indicates that each MIC chromatin domain developed to one MAC chromosome during conjugation. Besides, we found the MIC exhibits unique intra-arm and inter-chromosome interactions at the crescent stage of conjugation, when the MIC undergoes meiotic recombination.

2020-10-01 | GSE125628 | GEO

Cohesin depleted cells rebuild active and inactive nuclear compartments after mitosis

Project description:Nuclear functions are essentially linked to nuclear compartmentalization. This study demonstrates that cohesin rings as anchors of chromatin loops are dispensable to rebuild a functional nuclear compartmentalization in cohesin depleted cells after passing through mitosis and formation of one daughter cell with a multilobulated nucleus (MLN). Super-resolved microscopy reveals co-aligned active and inactive nuclear compartments (ANC/INC) in these postmitotic nuclei, likely corresponding to A and B compartments, detected with Hi-C. MLN carry chromosome territories, built from chromatin domain clusters, pervaded by a system of interchromatin channels harboring splicing speckles. Channels are lined by transcriptionally competent chromatin, whereas repressed chromatin with higher compaction locates in the interior of chromatin clusters. MLN pass through S-phase with typical early and mid-to-late replication patterns. Sites of DNA synthesis become physically larger, consistent with a model where cohesin dependent loop extrusion tends to compact intervals of replicating chromatin, whereas their genomic boundaries are associated with compartmentalization.

2020-10-05 | GSE145099 | GEO

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Project description:Hi-C technique is widely used to study 3-dimensional chromatin architecture and assemble genomes. Conventional in situ Hi-C protocol employs restriction enzymes to digest chromatin, which results in non-uniform genomic coverage. Using sequence-agnostic restriction enzymes such as DNAse I could overcome this limitation. Here we compared different DNAse Hi-C protocols and identified several critical steps which significantly impact protocol efficiency. We proposed a new robust protocol for preparation of DNAse Hi-C libraries, supplemented with experimental controls and computational pipeline for evaluation of libraries quality and data analysis.

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