Image Courtesy of Sophia Zhao.
Within each cell of the thirty trillion that comprise your body, bundles of thread-like chromatin float in a nebulous shape defined by the nucleus. This collection of chromatin contains the human genome—the catalog of genetic material that encodes every cell, from the neurons in your brain to the keratinocytes lining your skin. But if each nucleus contains the same catalog of genetic information, what differentiates one cell from another? The answer lies in the process of gene regulation—which genes are activated and which are repressed. This concept is fundamental to the expanding field of epigenetics: the study of how cellular mechanisms can change the reading of genetic code without altering the sequence of nucleotides itself.
Previous technologies have allowed scientists to study these epigenetic changes on a single-cell level by analyzing gene or protein expression. However, these methods required scientists to dissociate the tissue section into individual cells and to break those cells down further for analysis. In doing so, researchers lost spatial information that indicated where the epigenetic regulations were occurring within the tissue—details that were key to understanding cellular function.
Researchers in the Fan Lab at Yale and the Gonçalo Castelo-Branco Group at the Karolinska Institute in Sweden have developed a novel technique called spatial-CUT&Tag. The method allows them to map out epigenetic gene regulation in the original tissue section using grids composed of 20-micrometer pixels, an area equivalent to a single neuron in the brain. This technique represents a huge leap forward in the field of spatial omics and was recently published in Science.
“What has been missing in terms of [past] technology is that you don’t really see single-cell information in a kind of genome-scale, unbiased way, [while it is] still in the original tissue environment,” said Rong Fan, a Yale professor of biomedical engineering and the principal investigator at the Fan Lab. “Over the past couple of years, people realized how important that tissue spatial information is in the development of technology for spatial transcriptomics. Now, we can see [gene regulation] pixel by pixel, just like your TV.”
The Importance of spatial-CUT&Tag in visualizing histone modifications
Fan and his colleagues focused on using spatial-CUT&Tag to identify a specific mechanism for epigenetic regulation, called histone modification.
To understand the process of histone modification, we first have to visualize how DNA is packaged within the nucleus. The average nucleus of a human cell is only six micrometers in diameter, less than half of the width of a human hair, but it must contain approximately six feet of DNA. To optimize space within the cell, the ribbon of DNA is first coiled around clusters of positively-charged proteins called histones, like a thread strung with beads. These histones are then grouped together, forming long ropes called chromatin fibers, which are condensed into chromosomes.
In histone modification, however, these histones are altered with chemical groups that cause sections of the DNA to unwind, leaving certain genes exposed and easily accessible to transcription complexes. Depending on whether the genes are unwound or wound, gene transcription may be activated, causing the production of proteins associated with the gene, or inhibited, resulting in the ‘silencing’ of the gene. Using spatial-CUT&Tag, the researchers achieved enough precision to see the histone modifications themselves in individual cells.
“This is completely mind-blowing. You can see the mechanism that controls gene expression, rather than just the expression of the individual genes, pixel by pixel in a tissue matrix. So, once you have that, the greater impact is [that] you’ll know what type of cells there are and what gene expression determines types of tissue [formation],” Fan said.
Previously, one of the most commonly used methods to detect specific histone modifications was ChIP-Seq, which “pulls down” specific histones from the cellular mixture using antibodies and analyzes the DNA associated with those histones. However, Fan said that ChIP-Seq is a tedious, time-consuming process that requires a large sample of cells. Spatial-CUT&Tag expedites this process and provides an unimaginably large amount of data in comparison.
“[With spatial-CUT&Tag], the data now is equivalent to doing thousands of ChIP-Seq, but precisely from every tiny pixel of your tissue and doing thousands of those covering the entire tissue section. That was just science fiction a number of years ago,” Fan said.
Achieving this level of precision took three years of work, and the final version of the novel spatial-CUT&Tag technology required a combination of three existing techniques: microfluidic deterministic barcoding, CUT&Tag chemistry, and next-generation sequencing (NGS).
How spatial-CUT&Tag works
To carry out spatial-CUT&Tag, the researchers first performed standard CUT&Tag on mouse embryos and brain tissues. CUT&Tag is a common procedure used to analyze interactions between histones and DNA and determine which proteins are associated with which DNA binding sites. This process required tagging specific histone targets with antibodies, Y-shaped proteins with conformations that perfectly bind to histones of interest, marking them for further analysis. The next step of CUT&Tag involved cleaving the DNA strands coiled around the histone, isolating these protein-associated genes. Finally, synthesized DNA strands, called adapters, were added to the ends of the sectioned genes. These adapters proved to be important later on, as they served as “landing docks” for the attachment of lab-made DNA “barcodes.”
The next step, called microfluidic deterministic barcoding, allowed scientists to track epigenetic modifications back to their locations within the original tissue sample. This novel technology, developed by the Fan Lab three years ago, provided researchers with crucial spatial epigenetic data that were lost when performing regular CUT&Tag. Microfluidic deterministic barcoding involves labeling cells containing specific histone modifications with “barcodes”, which are unique combinations of DNA strands that the scientists can track to reconstruct a visual map of epigenetic modifications. To carry out microfluidic barcoding, the researchers developed two microfluidic devices.
The microfluidic devices contained fifty microfluidic channels each and were positioned atop each other at perpendicular angles to create a grid containing 2,500 intersection points, with the tissue sample placed underneath. The researchers then flowed fifty unique DNA strands through the microfluidic channels in each device, creating 2,500 distinctive combinations of DNA strands, or “barcodes.” As Fan explained, each microfluidic channel forms a long, straight road with a particular street name, and every cell settled at an intersection sits like a unique house along this road. The “barcode” functions as a cell’s address: a sign-post declaring its location.
“If we know the address code of every single pixel there, we know where that house, [or the cell], is located. Basically, we give every single tiny piece of tissue in a whole tissue section a unique address code,” Fan said.
In the procedure, the “barcodes” adhered to the adapters attached to the selected genes during the first step of CUT&Tag. The researchers then photographed the model to ensure that they could align the arrangements of different cell types in the tissue with the spatial data they gathered from spatial-CUT&Tag. Afterward, they performed next-generation sequencing, which allowed them to determine the DNA sequences of the selected genes. With this data, they conducted a robust computational analysis that required much trial and error to perfect.
“Computationally, there are no existing pipelines to analyze the spatial epigenomics data. It took a lot of effort to borrow the modules developed by others and generate the whole pipeline we needed to analyze the data,” said Yanxiang Deng, a post-doc in the Fan Lab and the first author of the Science paper.
After months of trouble-shooting and optimization, they compiled their results. The researchers found that the epigenomic map developed by spatial-CUT&Tag accurately distinguished between the distinct cell types present in embryonic and postnatal mouse tissues. In fact, in one experiment, the epigenomic data formed distinct stripes that correlated with the cortical layers in a mouse brain, demonstrating that spatial-CUT&Tag could differentiate cell types based on histone modifications. They validated the results by running ChIP-Seq tests on the same tissue samples.
Implications in cancer research and next steps
Looking ahead, Fan and Deng want to expand their study of histone modifications to other epigenetic mechanisms, including DNA methylation. The researchers are also thinking about developing 3D epigenomic maps, updating the 2D grids generated with spatial-CUT&Tag data.
“We’re working on combining with other modalities because these technologies are working [on] one molecular layer each time. We are working on co-profiling different layers of molecules […] to understand gene regulation networks,” Deng said.
Deng acknowledged that the development of spatial-CUT&Tag opens up a wide range of biological applications—most notably, identifying epigenetic mechanisms underlying cancer.
Cancerous cells may arise due to epigenetic alterations, and the difference between a healthy and malignant cell can be traced back to histone modifications. “Potentially, if you’re looking at a disease, [you can use spatial-CUT&Tag to determine] what actually drives the disease,” Fan said. “[You can see what is happening at] the mechanistic level and can target specific histone modifications in specific loci to develop new drugs that really target the root of the disease initiation.”
Yet there is still much to be explored; even with the development of spatial-CUT&Tag, questions about the future of cancer epigenetic research linger.
“Now the door is open,” Fan said. “[With spatial-CUT&Tag], you can begin to gather data and think about how to target the cancer with a completely new approach and [find] a potentially much more efficacious and much more personalized [treatment] one day.”
About the Author: Hannah Han is a first-year prospective HSHM and MCDB double-major in Grace Hopper College. Beyond writing and editing for YSM, Hannah conducts breast cancer research at the Yale School of Medicine, volunteers for Splash and HAPPY, and contributes to various literary publications on campus.
Acknowledgments: The author would like to thank Professor Fan and Dr. Deng for their time and enthusiasm about their work.
Extra Reading:
Deng, Y., Bartosovic, M., Kukanja, P., Zhang, D., Liu, Y., Su, G., Enninful, A., Bai, Z., Castelo-Branco, G., & Fan, R. (2020). “Spatial-CUT&Tag: Spatially resolved chromatin modification profiling at the cellular level.” Science, 375(6581): 681–686, https://doi.org/10.1126/science.abg7216.
Liu, Y., Yang, M., Deng, Y., Su, G., Enninful, A., Guo, C. C., Tebaldi, T., Zhang, D., Kim, D., Bai, Z., Norris, E., Pan, A., Li, J., Xiao, Y., Halene, S., & Fan, R. (2020). “High-Spatial-Resolution Multi-Omics Sequencing via Deterministic Barcoding in Tissue.” Cell, 183(6): 1665–1681, https://doi.org/10.1016/j.cell.2020.10.026.