In the limited space of the nucleus, most of the genome is tightly folded, leaving accessible only the parts that need to be transcribed. There is “huge interest” in determining which elements of the genome are active in a given cell type, says Howard Chang of Stanford University. This was Chang’s motivation for developing a technique called ATAC-seq (assay for transposase-accessible chromatin)—in which DNA tags (acting as transposons) are enzymatically integrated into open regions of the genome and then used to identify those regions through sequencing.
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Chang described ATAC-seq in 2013, but says, “we were breaking the cell open to get this information, so we didn’t have any sense of the 3-D organization of the [accessible] elements.”
Fast-forward three years, and Chang has now developed ATAC-see. This technique uses the same enzymatic approach as ATAC-seq to integrate DNA tags, but fluorophores conjugated to the tags allow for their visualization in 3-D, fixed nuclei. And, once the cells have been observed, sequencing can identify the tagged regions.
Using ATAC-see, Chang’s group has discovered that in neutrophils, unlike in most cells, accessible chromatin tends to be located at the nuclear periphery. This arrangement seems to facilitate formation of neutrophil extracellular traps—strings of discharged genomic DNA that are used to catch and kill pathogens, but that also enable cancer metastasis.
The team has also used ATAC-see to examine how accessibility changes across the genome during the cell cycle.
“One of the big challenges in the field of chromatin study is to integrate data obtained by imaging with data obtained by genomic methods,” says Job Dekker of the University of Massachusetts Medical School who was not involved in the study. “So what I like about this assay is that it combines these two things.” (Nature Methods, doi:10.1038/nmeth.4031, 2016)
|ACCESSIBLE CHROMATIN ASSAY||HOW IT WORKS||TECHNICAL EASE||VISUALIZATION||NUMBER OF CELLS REQUIRED FOR SEQUENCING|
|DNase-seq||DNase I preferentially digests DNA that is free of nucleosomes (accessible). Nucleotide linkers are ligated to the digested strands and used for isolating and sequencing the digested regions.||Difficult, especially optimizing digestion conditions||No||Millions|
|ATAC-see||A transposase enzyme inserts fluorescently labeled DNA sequencing adaptors (tags) into accessible chromatin, and the tags are used to visualize, amplify, and sequence the DNA.||Relatively easy for those familiar with standard molecular techniques||Yes||50,000. ATAC-seq (the protocol lacking fluorescent visualization) has been performed on single cells, so this might be possible.|