February 26, 2024

Pioneering technique reveals new layer of human genetic regulation

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DNA, which has a double helix structure, can have many mutations and genetic variations. Credit: NIH

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DNA, which has a double helix structure, can have many mutations and genetic variations. Credit: NIH

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

Published online February 9 at molecular cell, The study results support the theory that backtracking represents a widespread form of gene regulation, which influences thousands of human genes, including many involved in basic life processes such as cell division and development in the womb.

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

In 1997, Evgeny Nudler, Ph.D., and colleagues published a paper that showed that RNA polymerase can sometimes slide backward along the strand it is reading, a phenomenon they called “backtracking.” Studies since then have shown that backtracking occasionally occurs in living cells shortly after RNA polymerase begins RNA synthesis or when it encounters damaged DNA to make room for repair enzymes to enter.

Subsequent work suggested that the rewinding and repair machinery had to function quickly and dissipate, or it could collide with DNA polymerase to cause breaks in the DNA strands that induced cell death.

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

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

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

“If further work expands our findings to different developmental programs and pathological conditions, backtracking could be similar to epigenetics, the discovery of which revealed a surprising new layer of gene regulation without altering the DNA code.”

Central to 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 the DNA strands in one direction, but moves backwards in certain scenarios. Previous studies have shown that as RNA polymerase II recoils, it forces out (expels) from its inner channel the end of the RNA chain it has been building based on the DNA code.

Because prolonged backtracking is prone to causing damaging collisions, transcription is thought to be rapidly restored by the transcription factor IIS (TFIIS), which promotes the cutting (cleavage) of extruded and “backtracked” RNA. This paves the way for RNA polymerase II to resume its direct code reading.

Other previous studies, however, have shown that when the polymerase backtracks beyond a certain distance (e.g., 20 nucleobase DNA building blocks), the backtracked RNA can attach itself to the channel through which it is extruded, keeping it in place longer. Blocked and backward complexes are less likely to be rescued by TFIIS-driven cleavage and are more likely to delay transcription of the gene involved.

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

According to the researchers, TFIIS likely occurs in low concentrations in living cells and competes with hundreds of other proteins to obtain and cut back RNA so that transcription can continue.

In the current study, the team used a high concentration of purified TFIIS (without competing proteins) to precisely cut any piece of backtracked RNA anywhere it occurred in a cell’s genetic code. This made the clipped snippets available to technologies that read 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 strands wrap around within the chromatin that organizes gene expression – are highly prone to persistent backtracking.

The authors theorize that the degree to which this happens, with related changes in the transcription of certain genes, may control the timing of the large-scale histone accumulation required during cell division to rebuild chromatin. They also suggest that persistent backtracking may influence the timely transcription of genes vital for tissue development.

“Along with its potentially useful functions, persistent backtracking can also result in DNA damage and other genetic dysfunctions that contribute to disease,” says study first author Kevin Yang, a graduate student in Dr.

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

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

More information:
Persistence of backtracking by human RNA polymerase II, Molecular Cell (2024). DOI: 10.1016/j.molcel.2024.01.019. www.cell.com/molecular-cell/fu… 1097-2765(24)00055-8

Diary information:
Molecular Cell

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