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\n In situ Hi-C is a method to detect and quantify the pairwise interactions between chromosome regions across the entire genome. It was developed in 2014 as an improvement over the dilution Hi-C method. Compared to standard dilution Hi-C, this technique reduces the frequency of random ligation because the ligation is performed in situ inside the nucleus, a constrained space, instead of in solution, where DNA fragments are floating freely. In addition, this protocol can be done more quickly in the lab and was the first to introduce the use of 4-cutter restriction enzymes as opposed to the previous 6-cutters, providing higher resolution.\n

\n

\nThe protocol involves cross-linking the cells with formaldehyde to form links between physically adjacent DNA regions. The cells are then permeabilized with their nuclei intact. A 4-cutter restriction enzyme is used to digest the chromatin into multiple DNA fragments. The resulting fragments are biotinylated by end filling of the fragments ends. The fragments are then ligated and the DNA is purified and sheared. The biotinylated fragments are pulled down from the solution with streptavidin beads and a library is constructed and sequenced. Analysis of the resulting paired-end short read sequences produces a matrix that shows the number of interactions between different DNA regions.\n

\n

\nSee Rao et al., 2014 for more details.\n

\n\n
\n\n

\n Image source: Rao et al., 2014, Figure 1A\n
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\n In situ Hi-C is a method to detect and quantify the pairwise interactions between chromosome regions across the entire genome. It was developed in 2014 as an improvement over the dilution Hi-C method. Compared to standard dilution Hi-C, this technique reduces the frequency of random ligation because the ligation is performed in situ inside the nucleus, a constrained space, instead of in solution, where DNA fragments are floating freely. In addition, this protocol can be done more quickly in the lab and was the first to introduce the use of 4-cutter restriction enzymes as opposed to the previous 6-cutters, providing higher resolution.\n

\n

\nThe protocol involves cross-linking the cells with formaldehyde to form links between physically adjacent DNA regions. The cells are then permeabilized with their nuclei intact. A 4-cutter restriction enzyme is used to digest the chromatin into multiple DNA fragments. The resulting fragments are biotinylated by end filling of the fragments ends. The fragments are then ligated and the DNA is purified and sheared. The biotinylated fragments are pulled down from the solution with streptavidin beads and a library is constructed and sequenced. Analysis of the resulting paired-end short read sequences produces a matrix that shows the number of interactions between different DNA regions.\n

\n

\nSee Rao et al., 2014 for more details.\n

\n\n
\n\n

\n Image source: Rao et al., 2014, Figure 1A\n
", "filetype": "html", "content_as_html": "
In Situ Hi-C\n

\n In situ Hi-C is a method to detect and quantify the pairwise interactions between chromosome regions across the entire genome. It was developed in 2014 as an improvement over the dilution Hi-C method. Compared to standard dilution Hi-C, this technique reduces the frequency of random ligation because the ligation is performed in situ inside the nucleus, a constrained space, instead of in solution, where DNA fragments are floating freely. In addition, this protocol can be done more quickly in the lab and was the first to introduce the use of 4-cutter restriction enzymes as opposed to the previous 6-cutters, providing higher resolution.\n

\n

\nThe protocol involves cross-linking the cells with formaldehyde to form links between physically adjacent DNA regions. The cells are then permeabilized with their nuclei intact. A 4-cutter restriction enzyme is used to digest the chromatin into multiple DNA fragments. The resulting fragments are biotinylated by end filling of the fragments ends. The fragments are then ligated and the DNA is purified and sheared. The biotinylated fragments are pulled down from the solution with streptavidin beads and a library is constructed and sequenced. Analysis of the resulting paired-end short read sequences produces a matrix that shows the number of interactions between different DNA regions.\n

\n

\nSee Rao et al., 2014 for more details.\n

\n
\n\n

\nImage source: Rao et al., 2014, Figure 1A\n
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We demonstrate that in differentiating mouse embryonic stem cells, MLL3/4-dependent deposition of H3K4me1 at enhancers correlates with increased levels of chromatin interactions, whereas loss of this histone modification leads to reduced levels of chromatin interactions and defects in gene activation during differentiation. H3K4me1 facilitates recruitment of the Cohesin complex, a known regulator of chromatin organization, to chromatin in vitro and in vivo, providing a potential mechanism for MLL3/4 to promote chromatin interactions between enhancers and promoters. Taken together, our results support a role for MLL3/4-dependent H3K4me1 in orchestrating long-range chromatin interactions at enhancers in mammalian cells.", "url": "https://www.ncbi.nlm.nih.gov/pubmed/29313530", "authors": ["Yan J", "Chen SA", "Local A", "Liu T", "Qiu Y", "Dorighi KM", "Preissl S", "Rivera CM", "Wang C", "Ye Z", "Ge K", "Hu M", "Wysocka J", "Ren B"], "@type": ["Publication", "Item"], "status": "current", "uuid": "daea24a9-b600-40f2-aa43-dae13b63bffb", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "pubs_using": [], "publications_of_set": [{"abstract": "Long-range chromatin interactions between enhancers and promoters are essential for transcription of many developmentally controlled genes in mammals and other metazoans. Currently, the exact mechanisms that connect distal enhancers to their specific target promoters remain to be fully elucidated. Here, we show that the enhancer-specific histone H3 lysine 4 monomethylation (H3K4me1) and the histone methyltransferases MLL3 and MLL4 (MLL3/4) play an active role in this process. We demonstrate that in differentiating mouse embryonic stem cells, MLL3/4-dependent deposition of H3K4me1 at enhancers correlates with increased levels of chromatin interactions, whereas loss of this histone modification leads to reduced levels of chromatin interactions and defects in gene activation during differentiation. H3K4me1 facilitates recruitment of the Cohesin complex, a known regulator of chromatin organization, to chromatin in vitro and in vivo, providing a potential mechanism for MLL3/4 to promote chromatin interactions between enhancers and promoters. Taken together, our results support a role for MLL3/4-dependent H3K4me1 in orchestrating long-range chromatin interactions at enhancers in mammalian cells.", "title": "Histone H3 lysine 4 monomethylation modulates long-range chromatin interactions at enhancers.", "journal": "Cell research", "ID": "PMID:29313530", "@type": ["Publication", "Item"], "date_published": "2018-02", "display_title": "Yan J et al. (2018) PMID:29313530", "status": "current", "uuid": "daea24a9-b600-40f2-aa43-dae13b63bffb", "@id": "/publications/daea24a9-b600-40f2-aa43-dae13b63bffb/", "authors": ["Yan J", "Chen SA", "Local A", "Liu T", "Qiu Y", "Dorighi KM", "Preissl S", "Rivera CM", "Wang C", "Ye Z", "Ge K", "Hu M", "Wysocka J", "Ren B"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}], "number_of_experiments": 1, "@context": "/terms/", "aggregated-items": {"badges": [{"parent": "/biosamples/4DNBSMX7YM2H/", "embedded_path": "experiments_in_set.biosample.badges", "item": {"messages": ["Biosample missing doubling_number", "Biosample missing passage_number", "Biosample missing morphology_image", "Biosample is a stem cell line with unknown passage number missing karyotype"], "badge": {"commendation": null, "warning": "Biosample Metadata Incomplete", "uuid": "2b2cc7ff-b7a8-4138-9a6c-22884fc71690", "@id": "/badges/biosample-metadata-incomplete/", "badge_icon": "/static/img/badges/biosample-icon.svg", "description": "Biosample is missing metadata information required as part of the standards implemented by the 4DN Samples working group."}}}, {"parent": "/experiment-set-replicates/4DNESHGZUBL9/", "embedded_path": "badges", "item": {"messages": ["Replicate set contains only a single biological replicate"], "badge": {"commendation": null, "warning": "Replicate Numbers", "uuid": "24a64a84-3c33-4d76-aaf2-e5ef45eff347", "@id": "/badges/replicate-numbers/", "badge_icon": "/static/img/badges/replicates-orange-circle.svg", "description": "Issues with replicate numbers"}}}]}, "validation-errors": []}