A recent study led by Charles Danko, an associate professor at Cornell University, has unveiled the significance of a small but crucial molecular pause in gene activity that may have played a pivotal role in the evolution from simple cells to complex multicellular organisms. This phenomenon, known as promoter-proximal pausing, occurs shortly after RNA polymerase II, the cell’s molecular “copy machine,” begins its work. The polymerase temporarily halts, typically after synthesizing 20 to 60 nucleotides, awaiting further signals before proceeding.
The research, which is detailed in a study co-authored by John Lis, the Barbara McClintock Professor in the Department of Molecular Biology and Genetics at Cornell, aims to explore how this regulatory step evolved across various species. Promoter-proximal pausing was first identified in the 1980s, primarily through studies focused on yeast, which did not exhibit this process, leading many to underestimate its importance.
“Many researchers believed pausing was not a critical mechanism due to the lack of evidence in yeast,” Danko noted. However, subsequent investigations revealed that this pause is present not only in Drosophila but also in human cells, prompting questions about its evolutionary origins and significance.
Using a technique called PRO-seq, developed in Lis’ lab, the researchers mapped the pausing process across a diverse range of organisms, from bacteria to animals. They discovered that a rudimentary version of this pause existed in single-celled organisms. Over time, as evolution progressed, the pause became longer and more precisely regulated in multicellular animals, largely due to the emergence of new protein complexes, particularly the Negative Elongation Factor (NELF).
Danko explained that the NELF complex consists of four subunits, with two core units found in many eukaryotes and two others that appeared later. This evolutionary adaptation allows RNA polymerase to pause for more extended periods, granting cells enhanced control over gene expression. “This finding provides valuable context regarding the evolution of pausing systems,” he stated.
To assess the role of NELF in gene regulation, Danko and his team collaborated with researchers at the Memorial Sloan Kettering Cancer Center. They depleted two NELF subunits in mouse cells, which resulted in RNA polymerase progressing too rapidly along genes. Consequently, many genes did not respond adequately to heat stress, which is intended to trigger the transcription of essential heat shock genes.
“We observed that without NELF, a significant number of genes failed to be up-regulated as they should be under stress conditions,” Danko explained. He likened NELF’s function to adjusting the volume on a stereo, allowing cells to fine-tune the intensity of gene expression.
This research underscores the importance of promoter-proximal pausing in maintaining cellular function. Disruptions in this regulatory checkpoint can lead to diseases, including cancer. “Understanding the mechanisms driving transcription is crucial for uncovering their connections to various diseases,” Danko emphasized. “Without this knowledge, we risk merely cataloging gene changes associated with diseases without grasping their true significance.”
The paper also included contributions from several Cornell faculty members, namely Anna-Katerina Hadjantonakis, a professor at Weill Cornell Medicine; Ilana L. Brito, an associate professor in the Meinig School of Biomedical Engineering; and John Lis. The research received funding from the National Institutes of Health.
These findings not only illuminate the evolutionary pathways of gene regulation but also enhance our understanding of cellular responses to environmental challenges, paving the way for future research in genetics and disease prevention.
