Chromatin Analysis Methods

Chromatin, comprised of histones wrapped with DNA to form nucleosomes, plays an important role in the accessibility of DNA sequences to transcription factors or other regulatory DNA-binding proteins. Completely inaccessible DNA is effectively silenced, while accessible DNA may be in use by the cell. The organization and efficient packing of nucleosomes creates a complex chromatin architecture that protects and regulates DNA. Thus, chromatin structure and function can reveal information about disease mechanisms, epigenetic modifications, developmental processes, cell cycle regulation, and many other areas. This article will look at the methods scientists use to study the structure and function of chromatin, including ATAC-seq, ChIP, 3C-based, and single-cell techniques.

ATAC-seq

A common method of determining accessible regions of chromatin, assay for transposase-accessible chromatin with sequencing (ATAC-seq) works by inserting sequencing adapters into accessible regions of DNA using the transposase Tn5. Following amplification by qPCR, the products are sequenced with next-generation sequencing (NGS).

ATAC-seq provides a useful overview of all the DNA accessible in one snapshot in time. Marwan Alsarraj, the Biopharma Segment Manager at Bio-Rad Laboratories, Digital Biology Group, notes ATAC-seq’s ease-of-use, with its robust enzymatic digestion, lack of harsh cell treatments, and no need for antibodies. “ATAC-seq provides a genome-wide view of open chromatin regions, indicative of potentially active gene switches and TF-binding sites, whereas CUT&RUN, CUT&Tag, and ChIP-seq use antibodies specific to chromatin-binding proteins,” he says. “This results in different biological information and interpretation.” Bio-Rad offers a droplet-based single-cell ATAC-seq (scATAC-seq) product for researchers studying epigenetics at the single-cell level.

A disadvantage is that it doesn’t give you information about what is actually happening at open chromatin—just that something could potentially occur. “It doesn’t tell you if that transcription factor is really bound to the chromatin, is that gene really active, or is it poised to be activated?” says Chris Fry, Ph.D., Senior Director of Epigenetics, Assays and Workflow Products at Cell Signaling Technology. “And just as importantly, what cofactors, what co-activators or co-repressors are active?” Other disadvantages include requiring access to NGS and its associated costs, and cell number restrictions.

DNase-seq & MNase-seq

Like ATAC-seq, DNase-seq and MNase-seq are methods that determine the accessibility of DNA at a particular moment, instead of relying on nuclease activity. DNase-seq uses DNase to digest accessible sequences (the euchromatin, which is unprotected) into fragments for subsequent sequencing by NGS. MNase-seq uses micrococcal nuclease to digest away regions of euchromatin, leaving the protected heterochromatin (wound into nucleosomes) remaining for NGS analysis.

These are well-established protocols that can be adapted to use in conjunction with other chromatin analysis methods such as ChIP-seq. A disadvantage is that these methods can require a lot of sample, which can be challenging when using rare cell types.

ChIP-seq and other variations

Chromosome immunoprecipitation (ChIP) is another common technique that can be followed by other techniques to create ChIP variations (i.e., ChIP-seq, ChIP-PCR, ChIP-PET, ChIP-loop).

ChIP begins by crosslinking chromatin (and any other proteins in proximity to it), capturing information about 3D conformational associations that would be missed by simply looking at sequencing information. Fragments can then be precipitated using specific antibodies to DNA-associated proteins, and subsequently sequenced by NGS. Unlike ATAC-seq, ChIP can provide more mechanistic details by analyzing specific histone modifications. “Just by sampling 4 to 5 histone mods, you get a feel for what’s active, what’s not active, what’s poised to be active,” says Fry. “You can also look for transcription factors, cofactors, and other proteins.”

Though ChIP is a workhorse technique, its usefulness depends on the context of the experiment—in some cases, an alternative method of chromatin analysis might be better. “The main limitation of ChIP-seq is that it requires a large amount of input material, cells, or tissue to produce a strong enough signal over background noise, as well as the use of cross-linking during an initial fixation step,” says Michael Spelios, a scientist at EpigenTek. He notes that their customers mainly use ChIP methods such as ChIP-PCR and ChIP-seq, as well as the enzyme-based CUT&RUN and CUT&Tag assays to study DNA-protein interactions.

Another potential limitation of ChIP-based methods is the need for high-quality antibodies. “ChIP-based methods are powerful but their performance is limited by the reliability of the antibody,” says Ibrahim Jivanjee, Director of Product Management and Marketing at Arima Genomics. “Arima has validated specific antibodies to help scientists more readily use HiChip in their experiments.”

3C-based methods and variations

Chromatin conformation capture (3-C) is a proximity ligation method used to study chromatin architecture (genes that are widely separated by bases can be in close physical proximity in 3-dimensional space). Chromatin is digested following crosslinking, then the associated genes analyzed by qPCR and then NGS. Higher throughput variations include circularized 3-C (known as 4-C); carbon copy 3-C (known as 5-C), and genome-wide 3C (known as Hi-C).

Arima Genomics’ Hi-C technology generates a high-resolution, genome-wide map of interacting loci that includes 3D genome organization. “Unlike the two-dimensional chromatin accessibility information provided by ATAC-seq, Arima Hi-C provides chromosome interaction information, allowing scientists to better understand how chromosome interactions affect gene regulation,” says Jivanjee. “DNase- and 3C-based conformation methods can also provide interaction data; however, users often suffer sample-to-sample variability (specifically for nuclease-based methods) and they do not provide the comprehensive long-range interaction information of an Arima-optimized Hi-C based approach.”

CUT&RUN, CUT&Tag

Newer enzyme-based methods are becoming increasingly useful. The CUT&RUN method begins by permeabilizing cells and labeling them with a specific antibody. Then you add an enzymatic fusion protein (made from protein A/G and MNase) that will locate your specific antibody and cut the chromatin at that site. The resulting snipped out chromatin diffuses out of the cells for purification and sequencing.

One advantage of CUT&RUN is its lower sample requirement, making it suitable for primary cells and other cell types limited in number. “While ChIP requires typically 1 to 4 million cells per IP, for CUT&RUN, you can get away with as low as 5,000 to 10,000 cells for histones, and 10,000 to 20,000 cells for transcription factors and cofactors,” says Fry. Another advantage compared to ChIP is CUT&RUN’s lower background noise, because you purify only the chromatin you cut out selectively, rather than all of it. Fry also notes that sequencing for CUT&RUN is cheaper and faster than for ChIP: “We typically do 3 to 5 million in-depth reads for sequencing CUT&RUN, while with ChIP it’s typically 10 to 30 million reads.”

Fry notes that the number of validated antibodies for CUT&RUN is still limited, but believes it will grow. “CUT&RUN opens up the door to working with primary cell lines, so I think it’s only a matter of time that this will become more adopted over ChIP,” he says.

The CUT&Tag method is similar to CUT&RUN, but it uses a Tn5 transposase instead of MNase in the fusion protein. This allows it to cut the DNA, and ligate in barcode-containing oligos for cell-specific labeling. The ability to cut and label DNA in situ can simplify NGS library preparation, which is advantageous for single-cell and multiplexing applications. But CUT&Tag is limited to certain target types. It works well with histones, but “only with transcription factors and cofactors that either bind chromatin tightly, or bind broadly across large regions of chromatin,” says Fry. In comparison, CUT&RUN works with all three target types at low cell numbers, whereas ChIP works with all three target types but with higher cell numbers.

A disadvantage of both these methods is the potential for A/T sequence bias by the fusion protein, notes Spelios. “CUT&Tag displays the same digestion bias [as CUT&RUN] and is less specific due to off-target accessibility of chromatin caused by the Tn5 transposase enzyme,” he says. “EpigenTek’s brand of CUT&RUN and CUT&Tag assay kits circumvents these issues via a unique cleavage enzyme mix that has low sequence bias and minimizes immunocapture/sequencing background, allowing for much lower input requirements.”

Single-cell chromatin analysis

Any experiment using 1000s of cells gives an average result, so single-cell studies can be uniquely informative—especially when chromatin changes too quickly to be captured in one snapshot. “The benefits of such techniques are apparent in terms of analyzing rare cell populations, precious samples, and tissue specimens that are composed of multiple cell types with different functions,” says Spelios.

Increasingly, researchers are taking advantage of the possibility of single-cell multi-omics using assays with multimodal readouts. For example, researchers can assay the epigenome and the proteome simultaneously in the same cell using ATAC with select antigen profiling by sequencing (ASAP-seq). “All these multimodal techniques, especially at a single-cell level, assist in gaining insight into more complex biological interactions,” says Alsarraj.

Researchers are already using Arima chemistry in single-cell studies. “There is a lot of potential for discovery by understanding 3D genomics at the single-cell level,” says Jivanjee. “Of course, one of the primary challenges with any single-cell approach is integrating the information gained with other multi-omic technologies to develop new understanding of disease mechanisms.”