Despite the current advent of a genomics revolution driven by the decreasing cost of sequencing, and massively parallel sequencing techniques, profiling of the epigenome has lagged significantly. This is due to limitations in methodologies used for mapping chromatin fragments to the specific sequences in the genome. Current techniques such as chromatin immunoprecipitation with sequencing (ChIP-seq) and its variations have low signal, high background and epitope masking due to cross-linking. They also require large numbers of cells. Enzyme-tethering methods for unfixed cells provide alternatives to ChIP, where a specific protein of interest is targeted in situ and profiled genome-wide.
Dr. Steve Henikoff’s laboratory in the Basic Sciences Division developed one of these methods, called CUT&RUN, which maps the position of a chromatin-associated protein by successive binding of a specific antibody, followed by tethering a Protein A/Micrococcal Nuclease (pA-MNase) fusion protein in permeabilized cells. The activated MNase cuts near the protein’s binding site and generates DNA fragments that are made into sequencing libraries to be sequenced. Although CUT&RUN is able to generate high quality data, the overall procedure is costly, time consuming, labor intensive, and not suited for single-cell platforms.
To overcome the limitations of ChIP-seq and CUT&RUN, Dr. Henikoff and members of his laboratory developed Cleavage Under Targets and Tagmentation (CUT&Tag), and published their work in a recent issue of Nature Communications. Led by Dr. Hatice Kaya-Okur, a postdoc in the Henikoff lab, the authors used a transposome consisting of a hyperactive Protein A-Tn5 transposase (pA-Tn5) fusion protein loaded with sequencing adapters, capable of tethering in situ within live cells, to generate amplified, sequence-ready libraries within a day. This method can also be used in a high-throughput format, and on single cells.
To develop CUT&Tag, the authors utilized an antibody to lysine-27-trimethylation of the histone H3 tail (H3K27me3), an abundant histone modification that marks silenced chromatin regions, in intact permeabilized human K562 cells. The entire protocol takes place within a single tube; approximately 8 million reads were mapped to the human genome showing distinct large chromatin domains marked by the selected target, H3K27me3. The signal-to-noise of CUT&Tag relative to CUT&RUN and ChIP-seq were compared. CUT&Tag gave higher signals compared to CUT&RUN despite having similarly low background noise. CUT&Tag can also map active sites in chromatin and transcription factor binding. Importantly, CUT&Tag is able to profile low cell numbers and single cells, thereby allowing chromatin profiling to discriminate single cell types. All this is made possible by the unfixed in situ approach employed in CUT&Tag, along with more efficient use of reagents and improved signal-to-noise ratio.
This technological feat did not come without challenges. Dr. Kaya-Okur describes some obstacles she had to overcome: “In our preliminary experiments, we noticed that in addition to the genome-wide binding sites of our target protein, we were also getting the accessible chromatin. This is an inherent feature of the Tn5 transposase enzyme. We were able to minimize this artifact by performing stringent washes.”
CUT&Tag can be easily implemented in a standardized, cost-effective manner, making it possible for high-throughput applications. The ability to reveal regulatory information in genomes by chromatin profiling provides valuable insight to gene expression. Dr. Kaya-Okur explains the significance of this work: “The high efficiency and the exceptionally low backgrounds of CUT&Tag compared to ChIP-seq make it worth considering for almost all chromatin profiling applications. We expect that with its simplicity and suitability for automation and high-throughput single-cell assays, CUT&Tag will soon make epigenome profiling a routine research procedure even for clinical applications.”
So, what comes next? Dr. Kaya-Okur reveals more to come: “We are currently improving single-cell CUT&Tag and developing different versions of CUT&Tag. We have several interesting biological questions that we want to address and CUT&Tag (and its versions) will serve as great tools.”
Kaya-Okur HS, Wu SJ, Codomo CA, Pledger ES, Henikoff JG, Ahmad K, Henikoff S. 2019. CUT&TAG for efficient epigenomic profiling of small samples and single cells. Nat Commun, Apr 29; 10(1):1930.
This work was supported by the Howard Hughes Medical Institute, the National Institutes of Health, and the Fred Hutch Summer Undergraduate Research Program.