Scientists have developed a method to simultaneously observe the active genes and enhancers in a cell, which could be useful in identifying which enhancers are responsible for controlling specific genes. This discovery may also lead to the identification of new targets for treating genetic disorders. The human genome contains approximately 23,000 genes, but only a small portion of them are active within a cell at any given time. The regulatory network that governs gene expression involves enhancers, which are regions of the genome located far from the genes they regulate.MIT researchers have created a new method to observe the timing of gene and enhancer activation in cells, addressing the challenge of understanding complex gene-enhancer interactions. By identifying when a gene is activated alongside a specific enhancer, the researchers can infer that the enhancer is influencing the gene’s activity.
Understanding the relationships between enhancers and genes in various cell types could aid in pinpointing potential drug targets for genetic disorders. Genomic research has revealed mutations in non-protein-coding regions that are associated with a range of diseases.Could these be unidentified boosters? “When individuals begin using genetic technology to pinpoint areas of chromosomes that contain disease information, most of those locations do not correspond to genes. We suspect they correspond to these boosters, which can be quite far from a promoter, so it’s crucial to be able to pinpoint these boosters,” says Phillip Sharp, an MIT Institute Professor Emeritus and member of MIT’s Koch Institute for Integrative Cancer Research. Sharp is the senior author of the new study, which will be published in Nature. MIT Research Assistant D.B. Jay Mahat is the lead author of the paper.Searching for eRNA
Less than 2% of the human genome is made up of protein-coding genes. The remaining part of the genome consists of various elements that regulate the expression of those genes. Enhancers, which are believed to activate genes by physically interacting with gene promoter regions through the formation of a temporary complex, were first identified approximately 45 years ago.
In 2010, scientists found that these enhancers are transcribed into RNA molecules, which are referred to as enhancer RNA or eRNA. Researchers believe that this transcription occurs when the enhancers are actively involved.interacting with their target genes. This raised the possibility that measuring eRNA transcription levels could help researchers determine when an enhancer is active, as well as which genes it’s targeting.
“That information is extremely 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,” Mahat says.
However, this kind of mapping has proven difficult to perform because eRNA is produced in very small quantities and does not last long in the cell. Additionally, eRNA lacks a modificati, known as a poly-A tail, which is the “hook” that most methods utilize to extract RNA from a cell.
One method for capturing eRNA involves adding a nucleotide to cells that stops transcription when it becomes part of RNA. These nucleotides also have a biotin tag that can be used to isolate the RNA from a cell. However, this current method only works on large groups of cells and does not provide information about individual cells.
While brainstorming new ideas for capturing eRNA, Mahat and Sharp considered using click chemistry, a technique that can be used to link two molecules together if they are both labeled with “click handles”.
When nucleotides labeled with a click handle are incorporated into growing eRNA strands, researchers can easily capture and purify the strands. By using a tag containing the complementary handle, the eRNA can be fished out and then amplified and sequenced. Although some RNA is lost at each step, approximately 10 percent of the eRNA from a given cell can be successfully extracted, according to Mahat’s estimates.
This technique allowed the researchers to obtain a snapshot of the enhancers and genes that are actively transcribed at a specific time.
me in a cell.
“The goal is to identify the activation of transcription from regulatory elements and from their related gene in each cell. This needs to be performed in a single cell because it allows us to detect synchrony or asynchrony between regulatory elements and genes,” explained Mahat.
Timing of gene expression
By testing their method in mouse embryonic stem cells, the scientists determined that they could estimate the start of transcription for a specific region based on the length of the RNA strand and the speed of the polymerase (the enzyme responsible for transcription).The researchers utilized a new method to measure the transcriptional velocity of RNA polymerase — that is, how quickly the polymerase transcribes per second. This enabled them to identify which genes and enhancers were transcribed at the same time.
Using this method, the researchers were able to determine the timing of the expression of cell cycle genes in more precise detail than previously possible. They also confirmed several known gene-enhancer pairs and compiled a list of approximately 50,000 potential enhancer-gene pairs that they can now work to validate.
Understanding which enhancers regulate which genes would be beneficial in developing new treatments for genetic diseases. Last year, the UThe US Food and Drug Administration has given the green light to the first gene therapy treatment for sickle cell anemia. This treatment works by interfering with an enhancer that activates a fetal globin gene, reducing the production of sickled blood cells.
The team at MIT is now using the same method for other types of cells, particularly focusing on autoimmune diseases. They are collaborating with researchers at Boston Children’s Hospital to study mutations in immune cells that have been associated with lupus, many of which are found in non-coding regions of the genome.
<p”Since it is not known which genes are affected by these mutations, we are beginning to unravel the mystery.”Mahat explains that the enhancers identified in the study may be involved in regulating specific genes and may be active in certain types of cells. This tool for creating gene-to-enhancer maps is important for understanding the biology of gene regulation and provides a basis for understanding diseases,” Mahat states.
The study’s findings also support a theory developed by Sharp, along with MIT professors Richard Young and Arup Chakraborty, which suggests that gene transcription is controlled by condensates, or membraneless droplets. These condensates are composed of large clusters of enzymes and RNA, which may include eRNA produced at enhancer sites, according to Sharp.
The researcher explains that they believe the interaction between an enhancer and a promoter forms a temporary condensate-like structure, and RNA plays a role in this process. This study is a significant contribution to our understanding of the potential activity of RNAs from enhancers,” he says.
