In the mammalian genome, the number of large non-coding RNAs (lncRNAs) produced rivals the number of proteins. But while lncRNAs are now believed to have a major impact on cell identity, very little is known about their role in gene regulation.
New research from the California Institute of Technology suggests that lncRNAs can alter expression of a gene by uniquely exploiting that gene’s 3-D surroundings through binding to nearby target sites.
“This provides one of the first mechanisms for thinking about how lncRNAs can localize to chromatin, and in a sense, what makes lncRNAs unique,” said Mitchell Guttman, Assistant Professor in the Division of Biology and Biological Engineering at the California Institute of Technology, who led the study. “I think in that respect, it’s going to have a major impact on how we think about lncRNAs and how they work.”
Guttman and colleagues found that lncRNAs can modify the target sites they bind to, change chromosome architecture, and draw in new regions of the genome so that lncRNA molecules can spread to distant sites. Their findings were published in ScienceExpress on July 12.
The researchers focused on a specific lncRNA called Xist (X-inactive specific transcript). Xist, one of the first lncRNAs to be identified, binds to and effectively turns off portions of the X-chromosome. This critical process, known as X-chromosome inactivation, prevents a potentially lethal double dose of X-linked gene products in females.
“We’ve known for about 20 years that Xist is the gene responsible for this, so understanding how an RNA gene can orchestrate this process has really become not just essential for understanding X-chromosome inactivation, but has become this paradigm for understanding lncRNAs,” said Guttman.
To study the effect of Xist as it coats the X-chromosome, the team developed a technique called RNA Antisense Purification (RAP), which maps the interactions between lncRNAs and chromatin at high resolution. Guttman said that, even with RAP, the main challenge was finding a way to tightly control Xist RNA activation in stem cells.
“If you use a normal stem cell population and induce it to differentiate, cells will start differentiating at different times, so all the Xist localization would be a heterogeneous mix,” explained Guttman.
To solve this problem, expression of Xist was placed under control of an inducible doxycycline promoter, allowing the researchers to turn on expression at exactly the same time in every cell. With Xist under control, Guttman’s team could now observe its spread across the chromosome, providing the first clear picture of early binding sites.
Guttman’s team found that, within the 3-D architecture of the nucleus, Xist first localizes to those areas on the chromosome nearest the DNA sequence coding for the lcnRNA. To verify their observations, they relocated the Xist coding sequence to the other side of the chromosome to examine whether Xist’s early targeting patterns would change.
“If it was the three-dimentional structure that was dictating [Xist’s localization patterns], then it should recapitulate the three-dimensional position of that new integration site…and sure enough when you move it, the entire localization pattern of Xist remodeled,” said Guttman. “That really established the fact that Xist uses its 3-D spatial structure to find its early targets.”
Guttman says that while Xist utilizes 3-D positioning to find targets, this lncRNA subsequently moves beyond those initial sites by recruiting chromatin-modifying proteins, allowing it to spread across the whole X-chromosome. Xist then forms compartments containing X-chromosome genes that can then be co-regulated.
Now Guttman wants to determine whether other lncRNAs work through similar mechanisms.
“Xist is special because it coats a whole chromosome. Obviously other lncRNAs don’t do that, but understanding how other lncRNAs may use 3-D spatial position to find more specialized targets and how other lncRNAs may bring addition targets into compartments is a question I’m now fascinated by.”