New Technique scCUT&Tag Helps Evaluate Histone Modifications at the Single Cell Level
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Scientists at the Karolinska Institute in Sweden have developed a technique that is a further elaboration of the "cleavage under targets and tagmentation" (CUT&Tag) method, which is used to examine protein-DNA interactions. They combined CUT&Tag with single-cell library preparation to form “single-cell CUT&Tag” (scCUT&Tag) – a process that can analyze individual epigenetic modifications in tens of thousands of single cells at the same time.
Single-cell research allows scientists to identify the cell-to-cell variation present in an organism. There are a few different single-cell technologies in the research field already, including ATAC-seq and single-cell DNA Methyl-Seq. Newly developed methods like CUT&RUN and CUT&Tag allow the investigation of chromatin modifications by in situ chromatin cleavage or tagmentation, but nothing specifically for measuring modifications of histones.
One of the most widely used methods for studying protein-DNA interactions has been ChIP paired with ChIP-Seq. But this approach comes with many limitations like false-positive signals, poor resolution of interaction sites, and a large number of input samples.
The biggest advantage scCUT&Tag offers is that it is unbiased when examining tissues, allowing it to determine the origin of specific cell populations and peaks of histone modifications at enhancers and promoters. This capability is significant because knowing which histone modifications affect DNA at particular points could open the door for understanding the relationship between gene expression and disease.
"This technique will be an important tool for examining what makes cells different from each other at the epigenetic level" says Dr. Marek Bartosovic, lead author of the study. Until this recent finding, it has been near impossible to examine individual histone modifications in a singular cell. scCUT&Tag offers the opportunity to study transcription factor binding and chromatin modifications at the single-cell level in a high-throughput fashion.
Dr. Bartosovic and his team coupled bulk CUT&Tag with an existing single-cell ATAC-seq protocol in order to examine the epigenetic modifications in oligodendrocytes of a young mouse model. Oligodendrocytes (OLG) are myelinating cells that are responsible for insulating the axons of nerve cells. OLG cell differentiation is an extremely complex process, and as a result, these cells are left highly vulnerable to modifications.
The team was interested in the histone modification characteristics of active gene bodies, promoters, and enhancers, as well as inactive regions. They focused on histone marks H3K4me3 and H3K27ac, and H3K36me3, and H3K27me3 for the inactive regions.
They found an increase in the breadth of H3K4me3, which is consistent with gene expression, cell identity, and transcriptional consistency across multiple cell types. They also noticed an increase in H3K4me3, particularly at population-specific marker gene promoters compared to other marker gene promoters. Further investigation revealed an increase in H3K4me3 at promoters when oligodendrocyte precursor cells (OPCs) differentiated into mature oligodendrocytes (MOLs).
Integration of scCUT&Tag data with single-cell gene expression.A., Co-embedding of H3K4me3 scCUT&Tag data data1. B. Co-embedding of the H3K4me3 of OPC and MOL clusters. C. Metagene activity scores of OLG lineage subclusters. D. Ridgeline plots depicting histograms of number of unique reads per population of four histone modifications. E. Co-embedding of scCUT&Tag data of three active histone modifications (H3K4me3, H3K27ac and H3K36me3) in a single 2D UMAP space.
Determining TF binding using ChIP-seq with low input samples is extremely difficult. The team applied scCUT&Tag to investigate the transcription factors (TFs) OLIG2 and RAD21, which are specific for glial cell populations and chromatin structure, respectively.
They weren't able to gather the same amount of data for TFs that they did for histones, but scCUT&Tag allowed them to characterize the cell identity for all cells except for OPCs and vascular cells. OLIG2 was found to bind to OLG-specific promoters and enhancers, and therefore wouldn’t paint an accurate picture for different cell types. Contrastingly, RAD21 was found to bind to cell-type-specific promoters and enhancers, which helped to provide insight regarding enhancer–promoter connectivity into establishing cell identities.
scCUT&Tag analysis of transcription factor binding.A,B., 2D UMAP embedding of OLIG2 (a) and RAD21 (b) scCUT&Tag data with coloring by the number of unique reads per cell. C,D., UMAP embedding of the OLIG2 (c) and RAD21 (d) scCUT&Tag with coloring by cell type. E.Pseudobulk profiles of OLIG2 and RAD21 scCUT&Tag data aggregated by cell type around the marker gene regions
In all, they determined that the profiles formed using scCUT&Tag were successful in determining cell identity and promoter–enhancer connectivity. scCUT&Tag provides seemingly endless possibilities in the field of epigenetic research, including the ability to predict gene regulation networks—a feat that has previously been impossible in the epigenomic field.
Dr. Gonçalo Castelo-Branco, a co-author on the study, says the next step is to employ the new technique in humans: "Next, we would like to apply single-cell CUT&Tag in the human brain, both in development and in various diseases. For instance, we would like to investigate which epigenetic processes contribute to neurodegeneration during multiple sclerosis and whether we would be able to manipulate these processes in order to alleviate the disease."
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