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NEB-Led Team Introduces Open Chromatin Profiling Method Centered on Nicking Enzyme

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NEW YORK (GenomeWeb) – A New England Biolabs-led team has introduced a new sequencing-based method for profiling open chromatin in fixed or non-fixed cells. 

The investigators introduced the "nicking enzyme-assisted sequencing," or NicE-seq, protocol in a paper published in Genome Biology this week. Along with collaborators from the Cancer Science Institute of Singapore, they applied this approach to human colorectal cancer cells before and after chemotherapy for proof-of-principle experiments comparing NicE-Seq to existing chromatin profiling methods.

In general, the NicE-seq protocol relies on a sequence-specific nicking enzyme that targets so-called "CCD" sequences — two consecutive cytosines followed by an adenine, guanine, or thymine base. The samples are then treated with a DNA polymerase that is active in fixed or unfixed cells to incorporate biotin tags at the nick-prone, open chromatin regions. The sample is then sonicated, captured with streptavidin beads, and sequenced to generate reads mapping to open chromatin regions.

"Now you know every region that was accessible or open is now biotin tagged," said senior author Sriharsa Pradhan, a researcher at New England Biolabs. "Once you purify the DNA, you can selectively capture this DNA to rebuild open chromatin sites and transcription factor occupancy at single-nucleotide resolution."

The nicking enzyme used in the protocol is still tricky to purify in sufficient quantities and is not available commercially at the moment, but Pradhan noted the enzyme will likely be available in the NEB catalogue in the coming months, once purification procedures are hammered out and quality control tests are complete.

"I'm hoping that in three or four months this is going to be in the [NEB] catalogue," Pradhan said.

He expects to see a simplified protocol on the company's web page, rather than a full kit — at least for the foreseeable future — since a kit would require still more quality control and optimization steps. In the meantime, he is open to providing it to scientists who want to take a crack at the NicE-seq protocol.

Several sequencing-based methods have been developed for assessing open chromatin, the team noted. For example, DNase-seq is used to find sites that are hyper-sensitive to the DNase I enzyme and, consequently, believed to fall in parts of the genome where nucleosomes are relatively few and far between. Although the method is useful, Pradhan explained, it requires many cells and is not particularly amenable to cells that have not been fixed.

Likewise, he noted that some of the other sequencing-based methods to characterize open chromatin — from FAIRE-seq to ATAC-seq — tend to have limitations related to the sample size needed, sequencing depth needed, experimental efficiency, and/or requirements for cells that are either fixed or unfixed.

"We wanted to have a technology where we can actually work on fixed cells as well as native cells, or non-fixed cells," Pradhan said. "Also, we want to lower the cell number and get all of the information with less sequencing."

With that in mind, the team turned to a sequence-specific nicking enzyme known as Nt.CviPII that nicks, but does not cut, sequences in the CCD context. Such sequences fall across the genome, but become inaccessible in regions with closed chromatin.

"This [enzyme] has selective nicking, so it gives us a little bit more time to manipulate the cells' nuclear architecture," Pradhan explained, noting that the Nt.CviPII enzyme tends to nick nuclear DNA, but destroys mitochondrial DNA that might otherwise produce background reads that could interfere with the interpretation of open chromatin in the nucleus.

With sonication, the researchers usually chop the captured, nick-containing sequences into fragments that range from 200 to 300 bases apiece, which are amenable to producing Illumina paired-end short reads that are then stitched together computationally. They are also working on modifications to NicE-seq that may make it useful with long-read platforms from Pacific Biosciences and/or Oxford Nanopore.

"Those are the types of things we're trying to do to see if we can profile much larger chunks of DNA," Pradhan said, provided there are ways to address issues such as error rate or throughput when generating the long reads.

With the current NicE-seq protocol, the team is typically able to identify open chromatin regions genome-wide with 20 million to 30 million reads. Based on the experiments done so far, the group estimates that the same level of chromatin profiling would require some 200 million to 500 million reads if assessed with an ATAC-seq protocol.

The number of required reads does notch up as fewer cells are used, Pradhan cautioned. In general, he and his colleagues have managed to routinely look at 250 or so cells, and the signal remains relatively robust when between 250 and 250,000 cells are available. When attempting to stretch down to 25 cells, though, they found that more reads — roughly five times as many — were needed to get enough signal over background noise, though profiling was still possible.

Across the 250- to 250,000-cell sample range, Pradhan noted that it is typically possible to use relatively little of the nicking enzyme reagent, which is expected to help in keeping the cost of NicE-seq experiments down. The samples can also be multiplexed and run together on a 96-well plate, he said.

The team believes NicE-seq will have applications for everything from basic to applied research. Pradhan and his colleagues are particularly interested in profiling mouse embryonic development, chromatin patterns across the cell cycle, and the way epigenetic features in the cell relate to open or closed chromatin sites. They are also teaming up with investigators at Dana-Farber Cancer Institute to apply NicE-seq to compare the chromatin landscapes in tumor and matched normal samples.

For their Genome Biology study, the researchers applied NicE-seq to several human cell lines, including the HCT116 human colorectal cancer line.

After hammering out the NicE-seq protocol and optimizing nicking enzyme concentrations, they demonstrated that it highlighted regions of the genome that overlapped with transcription start sites, RNA polymerase II binding, active histone marks, and other features associated with open chromatin.

The NicE-seq peaks largely overlapped with those described for projects such as ENCODE and with chromatin sites found in a series of experiments on HCT116 and other cell lines using methods such as DNase-seq, chromatin immunoprecipitation sequencing, or ATAC-seq, the researchers reported.

Even so, each method also uncovered unique regions, prompting the authors to speculate that such differences "may be attributable to sequence and structural bias of DNaseI or Nt.CviPII or biased tagging of DNA by the transposases used in ATAC-seq."

"Therefore," they noted, "for limited number of cells, both NicE-seq and ATAC-seq may be performed to negate sequence bias."

In the HCT116 CRC line context specifically, the team compared NicE-seq profiles and 5-methylcytosine levels before and after treatment with the DNA demethylating chemotherapeutic drug decitabine, exploring the possibility that the demethylation associated with this treatment might lead to new open chromatin regions and enhanced transcriptional activity at genes that were once silenced.

As demethylation dipped — down by 75 percent in less than one week of treatment — the researchers saw a shift in the open chromatin sites from baseline, with only a fraction of the sites maintained across treatment.

Based on their findings, the authors argued that NicE-seq "is a straightforward method that can be performed on potentially any cell type from any species with a sequenced genome."

With a modified version of the assay, the researchers labeled open chromatin in cells from the HeLa cell line so that these regions could be visualized microscopically rather than sequenced.

The team is exploring options for applying the open chromatin profiling approach to formalin-fixed, paraffin-embedded clinical samples or tissue sections. It is also continuing to tinker with the NicE-seq protocol in the hopes of making it faster, easy to use, and applicable to smaller and smaller samples sizes, including individual cells.

"We want [the method] to be generally applicable," Pradhan said. "It should be so easy that you don't have to struggle."

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