New Technique Enables Observation of Accessible Chromatin

A test tube-based genome-labeling technique has been brought under the microscope.

By | January 1, 2017

ATAC DO: The next generation of a technique called ATAC-seq, which captures and sequences active regions of DNA (1), allows for the visualization of these regions as well. In ATAC-see, a so-called transposome (2) uses a transposase enzyme (yellow) to insert a pair of fluorescent DNA tags into open regions of chromatin (3). Upon insertion, the DNA is cleaved (4) and the tags are visible under a microscope (5) human cells’ open chromatin labeled red). These tagged sections of DNA are then sequenced.© GEORGE RETSECK; XINGQI CHEN, STANFORD UNIVERSITY

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.

See “Reveling in the Revealed

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.


 

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