Scientists at the Massachusetts Institute of Technology (MIT) have invented a new single-cell nascent RNA sequencing technique that lets them observe the timing of gene and enhancer activation in cells to gain insights into how gene transcription is controlled and coordinated. Details of the technique are provided in a new Nature paper titled, “Single-cell nascent RNA sequencing unveils coordinated global transcription.”
According to the paper, the technique is called scGRO-seq and it uses click chemistry “to assess genome-wide nascent transcription in individual cells in a quantitative manner” and “unveil coordinated transcription throughout the genome.” The technique was developed in the laboratory of Philip Sharp, PhD, an MIT Institute Professor Emeritus, a member of MIT’s Koch Institute for Integrative Cancer Research, and senior author on the study.
“When people start using genetic technology to identify regions of chromosomes that have disease information, most of those sites don’t correspond to genes,” Sharp said. “We suspect they correspond to these enhancers, which can be quite distant from a promoter, so it’s very important to be able to identify these enhancers.”
Previous studies have documented how enhancers are transcribed into enhancer RNA or eRNA. Scientists suspected that this transcription occurs when the enhancers interact with their target genes. Measuring eRNA transcription levels could be a way to determine when an enhancer is active as well as which genes it targets.
“That information is extraordinarily important in understanding how development occurs, and in understanding how cancers change their regulatory programs and activate processes that lead to de-differentiation and metastatic growth,” said D.B Jay Mahat, PhD, a postdoctoral research fellow in the Sharp lab and the lead author on the Nature paper. “You want to be able to determine, in every cell, the activation of transcription from regulatory elements and from their corresponding gene. And this has to be done in a single cell because that’s where you can detect synchrony or asynchrony between regulatory elements and genes.”
Measuring eRNA is ephemeral, produced in small quantities, and lacks a poly-A tail making it difficult to capture and sequence. One way to capture eRNA is to add a nucleotide containing a biotin tag to stop transcription when it is incorporated into RNA. The tag is used to pull the RNA out of the cell. However, this technique only gives information about pools of cells.
Sharp and his team came up with a way to make the technique work with single cells. They turned to click chemistry, a technique that uses two molecules tagged with “click handles” that can react with each other. The scientists designed nucleotides tagged with one click handle and allowed them to be incorporated into eRNA strands. They used the complementary click handle to pull them out for amplification and sequencing. Using this method, the scientists estimate that they are able to pull out about 10% of eRNA from a given cell. It’s enough to get a snapshot of the enhancers and genes that are being actively transcribed.
To show that the technique provides information about transcription, the researchers tested it in over 2,600 individual mouse embryonic stem cells. They reported being able to estimate when a particular region will be transcribed based on the length of the RNA strand and the polymerase speed as well as which genes and enhancers were being transcribed. They also confirmed several sets of known gene-enhancer pairs and generated a list of 50,000 possible pairs.
The team is now testing their approach using other types of cells. Specifically, they are working with researchers at Boston Children’s Hospital to explore immune cell mutations that have been linked to lupus. “It’s not clear which genes are affected by these mutations, so we are beginning to tease apart the genes these putative enhancers might be regulating, and in what cell types these enhancers are active,” Mahat said. “This is a tool for creating gene-to-enhancer maps, which are fundamental in understanding the biology, and also a foundation for understanding disease.”
They may also attempt to confirm a theory, developed by Sharp and other colleagues, which posits that gene transcription is controlled by condensates—clusters of enzymes and RNA. “We picture that the communication between an enhancer and a promoter is a condensate-type, transient structure, and RNA is part of that. This is an important piece of work in building the understanding of how RNAs from enhancers could be active,” Sharp said.
