In his quest to understand how each cell of an organism interprets the same genome in a different way, researcher Sascha Duttke wondered whether there might be any undiscovered rules of biology.
The human genome contains information about our development, functioning, growth, and reproduction, and all of it takes up only about 2 MB of space.
“That led us to wonder: maybe some of the magic is in the CD player, too?” Duttke, an assistant professor at the College of Veterinary Medicine, Washington State University, wrote in an email. “In this analogy, the CD is our genome and the CD player is the regulatory machinery,” and the transcription factors are important components in the player.
Inspired by a toddler
Transcription factors are proteins that bind to specific portions of the DNA and control the rate at which the cell transcribes genetic information from DNA to RNA. The cell then makes proteins by ‘reading’ the RNA.
Groundbreaking new work by Duttke and his colleagues has shown that the fate of a gene being transcribed depends on the location of the transcription factor binding site relative to the location where transcription begins.
The results, published in the journal Nature, provide insights into how different spatial arrangements of the same transcription factors can have different effects.
The findings can “help filter and refine genomic tools and algorithms that predict gene expression”, which can inform new diagnostic and therapeutic strategies for diseases like cancers caused by mutations in regulatory elements, Meenakshi Ghosh, a structural biologist-turned clinical scientist, said.
“Watching my toddler destroy a puzzle by forcing in the right colour but the wrong shaped piece made me think: maybe we’ve been focusing too much on transcription factor binding sites and protein interactions, and not enough on how everything fits together spatially and in an even bigger picture,” Duttke said.
Before or after?
The team investigated whether the arrangement of transcription factors relative to the transcription start site could influence gene expression.
When presented with the DNA, the activator transcription factor binds to it at specific points, the binding sites. These points are different from the transcription start site.
Team members developed tools to help them analyse patterns in the building blocks of the DNA that are typically found at the start sites. They subjected cells specially cultured in the lab to a form of RNA sequencing that could detect these sites in RNA. Then they identified the preferred locations at which transcription factors bound relative to an active start site.
The researchers found the binding sites for activator transcription factor NRF1 were located before the start sites and for factor YY1 it was located after the start site. Curiously, NRF1 is an activator whereas YY1 is both an activator and a repressor, a factor that stops transcription.
Next they checked how the relative position of the start site affected how the transcription factor behaved. When they knocked down the gene that cells used to make NRF1, the cells transcribed less DNA only when NRF1’s binding site was located before the transcription start site. If its binding site was located after, the absence of NRF1 increased the transcription rate.
Natural genetic variations
These results were “surprising,” Duttke said. “If you look in textbooks or even Wikipedia, transcription factors are usually grouped into either activators or repressors. The fact that some factors can do both was considered unusual.”
Organisms often carry natural genetic variations at the binding sites. The researchers assessed how these variations influenced the start of transcription. They analysed more than 4 million variations and 80,000 start sites in mice cells and found opposing transcription outcomes depending on whether the variations affected the factors before or after the start site. For instance, only mutations affecting NRF1 binding before the start site reduced the transcription rate.
The researchers also synthetically inserted binding sites for six factors at different distances from the start sites in some DNA sequences. They observed similar position-dependent outcomes. For example, adding an NRF1 binding site ahead of the start increased transcription, consistent with its activator function. Inserting it after the start site reduced transcription.
‘Spatial grammar’
Last, the researchers studied the relevance of these effects in human diseases. They identified start sites from genomic sequences from 67 people and combined this information with databases that describe disease risk linked to specific genetic variants. Consistent with previous results, they observed position-dependent effects of disease-associated variants based on the location of the start sites and the binding sites.
“Uncovering this spatial ‘grammar’ was a true eureka moment for many scientists like us who are working to understand how DNA encodes the instructions for turning genes on and off,” Duttke said, adding it would be “exciting” to explore how interactions between different factors affect this spatial grammar.
These results have “vast potential applications”, including helping researchers identify and predict disease-associated mutations, called polymorphisms, that occur outside genes and provide a basis for therapeutic interventions.
“How many of those polymorphisms contribute to disease is currently largely unknown,” he said. “The discovery of the spatial grammar may help to change that.”
The light of evolution
This study is “pretty cool,” Ghosh said. “It adds crucial new insights about how positioning and spacing relative to [start sites] can impact the ability of [factors] to either activate or repress gene expression.”
She added that the results can also improve our understanding of evolution and how organisms regulate gene expression to adapt to environmental changes.
Duttke said he would like to understand more about how this grammar evolved and how it helped create complex organisms like humans. He quoted the title of geneticist Theodosius Dobzhansky’s famous 1973 essay to make his point: “Nothing in biology makes sense except in the light of evolution”.
Sneha Khedkar is a biologist turned freelance science journalist.
Published – October 09, 2024 05:30 am IST
