A significant aspect of gene regulation is the structure of chromatin, which is a combination of DNA and proteins that helps package our genetic material inside cells. Modifications to chromatin—known as epigenetic marks—play a vital role in controlling how genes are expressed. However, studying these modifications, particularly in repetitive regions of the genome, has been challenging for researchers.
What Are Chromatin Modifications?
Chromatin modifications can be thought of as chemical flags attached to the DNA and histone proteins that influence how genes are turned on or off. These modifications are essential for various cellular processes, such as development, differentiation, and response to environmental changes. Unfortunately, mapping these modifications in complex and repetitive regions of the genome has been difficult due to their intricate nature.
Introducing scNanoSeq-CUT&Tag
To tackle this challenge, scientists at Peking University have developed a new method called scNanoSeq-CUT&Tag. This innovative approach adapts an existing technique known as CUT&Tag, which stands for “Cleavage Under Targets and Tagmentation.” Essentially, CUT&Tag allows researchers to identify where specific proteins (like transcription factors and histones) bind to the DNA. By integrating this method with nanopore sequencing technology, scNanoSeq-CUT&Tag can profile chromatin modifications across the entire genome, all at the level of individual cells.
Overview of the scNanoSeq-CUT&Tag method
a, Illustration of key steps for the scNanoSeq-CUT&Tag method. Ab, antibody. b, Comparison of unique reads per cell detected between the SMS-based scNanoSeq-CUT&Tag method (with median unique reads per cell: H3K4me3, 12,967; H3K27ac, 9,351; H3K36me3, 13,373; H3K27me3, 9,482; H3K9me3, 9,193; CTCF, 10,527; RAD21, 10,482, across six human cell lines) and several published NGS platform-based methods without downsampling the data. The number of single cells sequenced for each dataset is indicated: scNanoSeq-CUT&Tag (H3K4me3, n = 3,546; H3K27ac, n = 2,092; H3K36me3, n = 1,950; H3K27me3, n = 1,902; H3K9me3, n = 2,157; CTCF, n = 2,979; RAD21, n = 2,585), CoBATCH (n = 3,465), Paired-Tag (n = 50,754), ACT–seq (n = 1,246), scCUT&Tag (n = 46,810). c, Comparison of the fraction of reads in peaks per cell detected for each chromatin mark between the scNanoSeq-CUT&Tag method and the NGS platform-based scCUT&Tag method without downsampling the data. In the box plots in b and c, the middle line denotes the median, box lines represent the lower and upper quartiles (Q1 and Q3, respectively), and whiskers represent the minimum and maximum, with range limited to 1.5× interquartile range (IQR). d, Integrative Genomics Viewer (IGV) track view showing the chromatin signals of seven chromatin marks at specific target regions from pseudobulk scNanoSeq-CUT&Tag K562 cell profiles and ChIP–seq K562 data from ENCODE profiles. For scNanoSeq-CUT&Tag, each chromatin mark has two biological replicates.
How Does It Work?
The beauty of scNanoSeq-CUT&Tag lies in its ability to analyze chromatin modifications with remarkable precision. Here’s how it works:
Single-Cell Resolution: The method allows scientists to examine chromatin modifications in individual cells. This is important because cells can behave differently even within the same tissue.
Profiling Histone Marks and Transcription Factors: scNanoSeq-CUT&Tag can accurately identify patterns of histone modifications (the chemical changes to histone proteins) and where transcription factors (proteins that regulate gene expression) are located in the genome.
Distinguishing Cell Types: By examining chromatin modifications, researchers can differentiate between various types of cells. This is vital for understanding the unique roles different cells play in the body.
Allele-Specific Mapping: The method can map chromatin modifications to specific copies of genes (alleles), providing insights into how gene expression may differ between alleles within the same cell.
Analysis of Repetitive Elements: scNanoSeq-CUT&Tag can also analyze chromatin modifications for repetitive DNA sequences, which are often overlooked in genetic studies but are important for understanding genomic structure and function.
Significance of scNanoSeq-CUT&Tag
The introduction of scNanoSeq-CUT&Tag is a game-changer for researchers studying chromatin modifications. It opens the door to investigating previously underexplored areas of the genome, especially complex and repetitive regions that have been difficult to profile accurately. By providing a detailed view of how genes are regulated at the single-cell level, this method can enhance our understanding of fundamental biological processes and diseases.
scNanoSeq-CUT&Tag represents a powerful tool for scientists aiming to unlock the secrets of chromatin modifications. By enabling the detailed analysis of these crucial epigenetic marks in individual cells, it paves the way for new discoveries in genetics, development, and disease treatment, contributing to our overall understanding of cellular function and regulation.
Availability – All the codes used in this project are available on GitHub with the link https://github.com/Guo-Yuqing/scNanoSeq-CUT-Tag