Freeze substitution Hi-C, a convenient and cost-effective method for capturing the natural 3D chromatin conformation from frozen samples

Zheng, Wu; Yang, Zhaoen; Ge, Xianping; Feng, Yakai; Wang, Ye; Li, Chengwei; Luan, Yanan; Kun, Cai; Vakal, Serhii; You, Feng; Guo, Wei; Wang, Wei; Li, Fuguang

doi:10.1016/j.jgg.2020.11.002

“…Initially, 5-10 mg of snap-frozen tissue was placed into a tissueTUBE-TT05 (Covaris 520071) and cryopulverized using the Covaris CP02 cryoPREP Automated Dry Pulverizer, following the manufacturer’s procedure. The pulverized tissue was then subjected to freeze substitution 73 by submerging it in 1 ml of −80°C 0.01% formaldehyde (ThermoFisher 28906), 97% ethanol, and 2% water. Following this, samples were incubated on dry ice for 3 hours at a rotor spinning speed of approximately 100 rpm.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, 5-10 mg of snap-frozen tissue was loaded in tissueTUBE-TT05 (Covaris 520071) and cryopulverized using the Covaris CP02 cryoPREP Automated Dry Pulverizer following manufacturer's procedure. Pulverized tissues were treated with freeze substitution 45 by submerging in 1ml -80 o C 0.01% formaldehyde (ThermoFisher 28906), 97% ethanol, and 2% water. Samples were incubated in dry ice for 3 hours on a rotor with spinning speed around 100 rpm, and then placed in a CoolCell Container (Corning), transferred to a -20 o C freezer for overnight incubation.…”

Section: Multi-digested Hi-c (Mhi-c)mentioning

confidence: 99%

Global loss of promoter-enhancer connectivity and rebalancing of gene expression during early colorectal cancer carcinogenesis

Zhu

¹

,

Lee

²

,

Weimer

³

et al. 2022

Preprint

0

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Although 3D genome architecture is essential for long-range gene regulation, the significance of distal regulatory chromatin contacts is challenged by recent findings of low correlation between contact propensity and gene expression. To better understand the role of long-range interactions between distal regulatory elements during the early transformation from healthy colon to colorectal cancer, here we performed high resolution chromatin conformation capture for 33 samples including non-neoplastic mucosa, adenomatous polyps and adenocarcinomas, mostly from Familial Adenomatous Polyposis (FAP) patients. We identified hundreds of thousands of chromatin micro-structures, such as architectural stripes and loops, which originated from active cis-regulatory elements. Surprisingly, these structures progressively decayed throughout cancer progression, particularly at promoters. Meta-analyses revealed that this decay was independent of alterations in DNA methylation and chromatin accessibility. Interestingly, the degree of interaction loss was poorly correlated with gene expression changes. Instead, genes whose expression were disproportionately lower and higher than their relative promoter interaction in mucosa shifted their expression in polyps and adenocarcinomas to yield a more direct relationship between strength of interaction and gene expression. Our work provides the first high resolution 3D conformation maps during early cancer formation and progression, and provides novel insights into transcriptional readouts associated with fine-scale chromatin conformation alterations.

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“…Brie y, 5-10 mg of snap-frozen tissue was loaded in tissueTUBE-TT05 (Covaris 520071) and cryopulverized using the Covaris CP02 cryoPREP Automated Dry Pulverizer following manufacturer's procedure. Pulverized tissues were treated with freeze substitution 45 To stop restriction digestion, the suspension was incubated in a 62 o C heating block for 20 min followed by cool down. End repair was carried out by adding a 30 μl solution containing 0.5 mM biotin-14-dATP (Active Motif 14138), 0.5 mM biotin-14-dCTP (AAT bio 17019), 0.5 mM dTTP, 0.5 mM dGTP, and 4 μl Klenow DNA polymerase (NEB M0210L) to the mixture, and incubated for 1 hour at 37 o C with rotation.…”

Section: Multi-digested Hi-c (Mhi-c)mentioning

confidence: 99%

Global loss of fine-scale chromatin architecture and rebalancing of gene expression during early colorectal cancer development

Snyder

¹

,

Zhu

²

,

Lee

³

et al. 2022

Preprint

0

View full text Add to dashboard Cite

Although 3D genome architecture is essential for long-range gene regulation, the significance of distal regulatory chromatin contacts is challenged by recent findings of low correlation between contact propensity and gene expression. To better understand the role of long-range interactions between distal regulatory elements during the early transformation from healthy colon to colorectal cancer, here we performed high resolution chromatin conformation capture for 33 samples including non-neoplastic mucosa, adenomatous polyps and adenocarcinomas, mostly from Familial Adenomatous Polyposis (FAP) patients. We identified hundreds of thousands of chromatin micro-structures, such as architectural stripes and loops, which originated from active cis-regulatory elements. Surprisingly, these structures progressively decayed throughout cancer progression, particularly at promoters. Meta-analyses revealed that this decay was independent of alterations in DNA methylation and chromatin accessibility. Interestingly, the degree of interaction loss was poorly correlated with gene expression changes. Instead, genes whose expression were disproportionately lower and higher than their relative promoter interaction in mucosa shifted their expression in polyps and adenocarcinomas to yield a more direct relationship between strength of interaction and gene expression. Our work provides the first high resolution 3D conformation maps during early cancer formation and progression, and provides novel insights into transcriptional readouts associated with fine-scale chromatin conformation alterations.

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Interrogating Global Chromatin Interaction Network by High-Throughput Chromosome Conformation Capture (Hi-C) in Plants

Wang

¹

,

Niu

²

,

Hou

³

2022

Plant Gametogenesis

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Freeze substitution Hi-C, a convenient and cost-effective method for capturing the natural 3D chromatin conformation from frozen samples

Cited by 5 publications

References 34 publications

Global loss of promoter-enhancer connectivity and rebalancing of gene expression during early colorectal cancer carcinogenesis

Global loss of promoter-enhancer connectivity and rebalancing of gene expression during early colorectal cancer carcinogenesis

Global loss of fine-scale chromatin architecture and rebalancing of gene expression during early colorectal cancer development

Interrogating Global Chromatin Interaction Network by High-Throughput Chromosome Conformation Capture (Hi-C) in Plants

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