Being able to see precisely when various genes are turned on and off, as well as spatially visualizing the whole transcriptome at once, has been challenging for single-cell analysis. However, now, Caltech investigators have just released findings on a major advance that allows scientists to image 10,421 genes at once within individual cells using a new technique, dubbed intron seqFISH (sequential fluorescence in situ hybridization). Results from the new study were published recently in Cell, in an article entitled “Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH.”
Previously, researchers could only image four to five genes at a time in cells with microscopy. This work builds off previous advances from the research team, including an earlier version of seqFISH from 2014 and research from 2017 that profiled over 10,000 genes under a microscope. Scaling seqFISH up to a genomic level now enables the imaging of over 10,000 genes—about half of the total number of genes in mammals—within single cells.
“...we demonstrate a multiplexed single-molecule in situ method, intron seqFISH, that allows imaging of 10,421 genes at their nascent transcription active sites in single cells, followed by mRNA and lncRNA [long noncoding RNA] seqFISH and immunofluorescence,” the authors wrote. “This nascent transcriptome-profiling method can identify different cell types and states with mouse embryonic stem cells and fibroblasts.”
For genetic instructions to be turned into an actual functioning protein, transcription must first occur. This process often occurs in pulses, or "bursts." First, a gene will be read and copied into a precursor messenger RNA, or pre-mRNA, like jotting a quick, rough draft. This molecule then matures into a mRNA, akin to editing the rough draft. During the "editing" process, certain regions called introns are cut out of the pre-mRNA.
The Caltech team chose to focus on labeling introns because they are produced so early in the transcription process, giving a picture of what a cell is doing at the precise moment of gene expression. Using the newly developed intron seqFISH technique, each intron is labeled with a unique fluorescent barcode, enabling it to be seen with a microscope. Seeing introns reveals which genes are currently turned on in individual cells, how strongly they are expressed, and where they are located.
Previous work, which developed the barcoding technique, focused on labeling mRNA itself, providing a measurement of how gene expression changed over several hours as the mRNA developed. Looking at introns enabled the researchers to examine, for the first time, so-called nascent transcriptomes. This led them to discover that the transcription of genes oscillates globally across many genes on a "surprisingly short" timescale compared to the time it takes for a cell to divide and replicate itself, which takes from 12 to 24 hours. This means that over the course of a two-hour period, many genes within a cell will burst on and off.
“The nascent sites of RNA synthesis tend to be localized on the surfaces of chromosome territories, and their organization in individual cells is highly variable,” the authors stated. “Surprisingly, the global nascent transcription oscillated asynchronously in individual cells with a period of 2 hours in mouse embryonic stem cells, as well as in fibroblasts. Together, spatial genomics of the nascent transcriptome by intron seqFISH reveals nuclear organizational principles and fast dynamics in single cells that are otherwise obscured.”
There are several reasons why the oscillation phenomenon had not been observed previously. First, because these two-hour oscillations are not synchronized amongst different cells, the fluctuations are averaged out by methods that require many cells. Second, the high accuracy of the seqFISH method allows the researchers to be certain that what they observe represents real biological fluctuations, rather than technical noise. Last, these two-hour oscillations are obscured when mRNAs, rather than introns, are measured, because mRNA molecules have a longer lifetime, three to four hours, in mammalian cells.
Additionally, because introns stay where the gene is physically located, fluorescently imaging introns allows researchers to visualize where genes are located within the chromosome, the large structure that DNA folds into within the cell's nucleus. In this work, the team was surprised to discover that most active protein-encoding genes are located on the surface of the chromosome, not buried inside of it.
"This technique can be applied to any tissue," remarked senior study investigator Long Cai, Ph.D., a research professor in biology at Caltech and a collaborator on the Human Cell Atlas, a project that aims to define all cell types in the human body. "Intron seqFISH can help identify cell types and also what the cells are going to do, in addition to giving us a look at the chromosome structure in the same cells."