Pioneering technique reveals new layer of human gene regulation

A technique can determine for the first time how often and where exactly a molecular event called “rollback” occurs in the genetic material (genome) of any species, a new study shows.

Published online February 9 in Molecular cell, The study results support the theory that rollback represents a widespread form of gene regulation, which influences thousands of human genes, many of which are involved in fundamental life processes like cell division and development in the uterus.

Led by researchers at the NYU Grossman School of Medicine, the work revolves around genes, segments of DNA molecular “letters” arranged in a certain order (sequence) to encode the blueprints of most organisms. In humans as in bacteria, the first step in gene expression, transcription, occurs when a protein “machine” called RNA polymerase II travels down the DNA chain, reading genetic instructions in one direction. .

In 1997, Evgeny Nudler, Ph.D. and colleagues published a paper showing that RNA polymerase can sometimes move backward along the string it is reading, a phenomenon they called “backtracking.” Since then, studies have shown that rollback sometimes occurs in living cells shortly after RNA polymerase begins synthesizing RNA or when it encounters damaged DNA to make way for the incoming repair enzymes.

Later work suggested that the retrogradation and repair mechanisms had to work quickly and dissipate, otherwise they could collide with DNA polymerase and cause breaks in the DNA chains causing cell death.

Now, a new study led by Nudler’s team at NYU Langone Health reveals that their new technique, long-term sequencing (LORAX-seq), can directly detect where rollback events begin and end. By complementing past indirect or limited approaches, the new method reveals that many such events go back further than previously thought and, in doing so, last longer.

The results also suggest that persistent backtracking occurs frequently across genomes, occurs more often near certain types of genes, and has functions well beyond DNA repair.

“The surprising stability of backtracking over longer distances suggests that it represents a ubiquitous form of genetic regulation in species from bacteria to humans,” says Nudler, lead author of the study and Julie Wilson Anderson Professor at the Department of Biochemistry and Molecular Biology. Pharmacology at NYU Langone.

“If further work extends our findings to different developmental programs and disease conditions, the rollback could be akin to epigenetics, the discovery of which revealed a surprising new layer of gene regulation without changing the DNA code .”

At the center of life?

RNA polymerase II translates the DNA code into a related material called RNA, which then directs the construction of proteins. To do this, the complex moves down DNA chains in one direction, but reverses in certain scenarios. Previous studies have shown that when RNA polymerase II reverses, it expels (expels) from its interior channel the tip of the RNA chain that it has built based on the DNA code.

Because prolonged backtracking is likely to cause detrimental collisions, transcription is thought to be rapidly restored by the transcription factor IIS (TFIIS), which promotes the severing (cleavage) of extruded and “backed-off” RNA. This clears the way for RNA polymerase II to resume its direct reading of the code.

However, other previous studies have shown that when the polymerase moves back beyond a certain distance (e.g., 20 nucleobases DNA building blocks), the pulled back RNA can attach to the channel through which it is extruded, thus holding it in place longer. Locked and recoiled complexes are less likely to be rescued by TFIIS-induced cleavage and more likely to delay transcription of the involved gene.

This has led to the theory that backtracking, in addition to playing a key role in DNA repair pathways, can increase or decrease gene action as a major regulatory mechanism.

According to the researchers, TFIIS likely occurs at low concentrations in living cells and competes with hundreds of other proteins to reach and remove retrograded RNA so that transcription can continue.

In the current study, the team instead used a high concentration of purified TFIIS (without competing proteins) to precisely remove any piece of retrograded RNA wherever it appears in a cell’s genetic code. This made the clipped snippets accessible to technologies that read the code sequences and provide clues about their locations and functions.

The research team also discovered that genes that control histones – “spools” of proteins that DNA strings surround in the chromatin that organizes gene expression – are highly prone to persistent backtracking.

The authors hypothesize that the degree to which this occurs, along with associated changes in the transcription of certain genes, could control the timing of the large-scale histone accumulation needed during cell division to rebuild the chromatin. They also suggest that persistent rollback could influence the timely transcription of genes essential for tissue development.

“In addition to its potentially useful functions, persistent backtracking could also lead to DNA damage and other genetic dysfunctions contributing to disease,” says Kevin Yang, author of the first study and a graduate student in the lab of the Dr. Nudler.

“We believe that measuring rollback in the context of aging or cancer, for example, could help us understand why dysfunctions occur in the cellular stress response and cell replication, and suggest new therapeutic approaches .”

In addition to Yang and Nudler, study authors from NYU Langone Health’s Department of Biochemistry and Molecular Pharmacology were Aviram Rasouly, Vitaly Epshtein, Criseyda Martinez, Thao Nguyen and Ilya Shamovsky. Nudler is also an investigator at the Howard Hughes Medical Institute.