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Review
. 2020 Jan 1:170:75-81.
doi: 10.1016/j.ymeth.2019.06.002. Epub 2019 Jun 12.

Advancements in mapping 3D genome architecture

Daniel J McKay  1 , Alexis V Stutzman  2 , Jill M Dowen  3
Affiliations
Review

Advancements in mapping 3D genome architecture

Daniel J McKay et al. Methods. .
No abstract available

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Figures

Figure 1.
Figure 1.. Detection of compartmental domains and DNA loops mediated by Cohesin and CTCF.
A. A chromatin molecule with three distal DNA sequences (A, B, and C) brought into close physical proximity by a protein of interest (grey). B. A diagram showing interaction frequency data obtained from HiC that identifies A and B compartmental domains (red and blue) across a window of the genome. Compartmental domains are identified in the red/blue track and can be observed in the HiC heatmap as a plaid pattern that stretches far off the linear distance axis. Binding of the architectural proteins Cohesin and CTCF is based on ChIP-seq data. C. An example DNA loop formed by Cohesin and CTCF proteins. Two convergently oriented CTCF bound sites are brought into proximity by an extruding Cohesin ring. This loop appears as a punctate spot at the top of a triangle in the HiC heatmap. D. Schematic of proximity-based ligation assays. Fragmentation of crosslinked chromatin is achieved by enzymatic or mechanical shearing. Biotinylated nucleotides or oligos are ligated to the ends and DNA is circularized with a ligation step. DNA is purified, sheared, and biotinylated fragments are captured by affinity purification to enrich for ligation junctions (not shown). From these fragments a library is created and high-throughput sequencing is performed to identify regions of the genome that are in close proximity in 3D space.
Figure 2.
Figure 2.. Schematic of SPRITE protocol.
A. Diagram of the split/ligate/pool strategy for building a unique 36 bp barcode on the ends of chromatin fragments. Chromatin is divided across a 96-well plate and one of six 6 bp barcodes is ligated onto the ends. The chromatin is then pooled together in a single tube before being split across another 96-well plate. This cycle of split/ligate/pool is performed a total of six times to build a 36 bp barcode. B. Crosslinked chromatin is fragmented with mechanical and enzymatic shearing. The chromatin is split across a 96-well plate, 6 bp tags are ligated, and all samples are pooled together. Five additional cycles of splitting, ligating and pooling yield 36 bp barcodes that are identical for all covalently-linked DNA fragments (termed “clusters”). DNA is purified, a library is built, and high-throughput sequencing is performed. This approach allows for efficient capture and identification of multiple simultaneously occurring DNA interactions that are informative of 3D genome organization.
Figure 3.
Figure 3.. Schematic of Trac-looping protocol.
Crosslinked chromatin is mixed with assembled tetrameric transposome complexes to insert defined DNA sequences into genomic sites. Insertion into a DNA site in cis tends to occur in open chromatin and is used to measure accessibility while insertion in trans links two different DNA molecules together based on their proximity revealing information about 3D DNA organization. Chromatin is enzymatically fragmented, DNA is purified by affinity capture, and a ligation creates circularized molecules. Finally, a library is built and sequenced. This approach is a ligation-free method of capturing DNA interactions between regions of accessible chromatin.
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References

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