Nav: Home

Scientists illuminate a hidden regulator in gene transcription

May 26, 2016

Gene transcription is the process by which DNA is copied and synthesized as messenger RNA (mRNA) -- which delivers its genetic blueprints to the cell's protein-making machinery.

Now researchers at MIT and the Howard Hughes Medical Institute (HHMI) have identified a hidden, ephemeral phenomenon in cells that may play a major role in jump-starting mRNA production and regulating gene transcription.

In a paper published in the online journal eLife, the researchers report using a new super-resolution imaging technique they've developed, to see individual mRNA molecules coming out of a gene in a live cell. Using this same technique, they observed that, just before mRNA's appearance, the enzyme RNA polymerase II (Pol II) gathers in clusters on the same gene for just a few brief seconds before scattering apart.

When the researchers manipulated the enzyme clusters in such a way that they stayed together for longer periods of time, they found that the gene produced correspondingly more molecules of mRNA. Clusters of Pol II therefore may play a central role in triggering mRNA production and controlling gene transcription.

Ibrahim Cisse, assistant professor of physics at MIT, explains that because of their transient nature, enzyme clusters have largely been regarded as a mystery, and scientists have questioned whether such clustering is purposeful or merely coincidental. These new results, he says, suggest that, although short-lived, enzyme clustering can have a significant impact on major biological processes.

"We think these weak and transient clusters are a fundamental way for the cell to control gene expression," says Cisse, who is senior author on the paper. "If a small mutation changes the cluster's lifetime ever so slightly, that can also change the gene expression in a major way. It seems to be a very sensitive knob that the cell can tune."

What's more, Cisse says scientists can now explore Pol II clusters as targets to "stall or induce a burst of transcription" and control the expression of certain genes, to explore cancer drugs and other gene therapies.

The paper's co-authors include Won-Ki Cho, lead author and postdoc in the Department of Physics; Namrata Jayanth and Jan-Henrik Spille, also postdocs in physics; Takuma Inoue and J. Owen Andrews, graduate students in physics; and William Conway, an undergraduate in physics and biology; as well as researchers from HHMI's Janelia Research Campus: Brian English, Jonathan Grimm, Luke Lavis, and Timothee Lionnet who is also co-senior author with Cisse.

Imaging at super-resolution

Pol II enzymes only cluster together for very short periods of time, on the order of several seconds. These clusters are also extremely small, on the scale of 100 nanometers in width. Because they are so tiny and fleeting, Pol II clusters and other weak and transient interactions have largely been hidden from view, essentially invisible to conventional imaging techniques.

To see these interactions, Cisse and his colleagues developed a super-resolution imaging technique to visualize cellular processes at the single-molecule level. The team's technique builds on two existing super-resolution methods -- photo-activation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). Both techniques involve tagging molecules of interest and lighting them up one by one to determine where each molecule is in space. Scientists can then merge every molecule's position to create one super-resolution image of the cellular region.

While incredibly precise, these imaging techniques rely on the assumption that every molecule remains stationary. Molecules that come and go, and quickly cluster and scatter, are difficult to track. To catch Pol II clusters in action, Cissé and his team tweaked existing super-resolution imaging techniques, looking not just at a single enzyme's position, but also at how frequently molecules were detected. The higher the frequency of detection, the higher the chance that a cluster has formed.

The team applied their technique to image cells, using a camera that recorded one frame every 50 milliseconds, running continuously for up to 10,000 frames.

A transient lifetime

They then created a cell line that included a bright fluorescent tag for mRNA, as well as a fluorescent tag of a different color for Pol II enzymes. The team applied its super-resolution technique to image a particular gene inside the cell, called beta-actin, which has been characterized extensively. In experiments with live cells, the researchers observed that, while previously transcribed mRNA molecules lit up on the gene, new Pol II clusters appeared on the same gene, for about 8 seconds, before disassembling.

From these experiments, the group was uncertain as to whether the clusters had any impact on mRNA production, since the time it takes from the start of transcription to the complete production of mRNA takes significantly longer -- about 2.5 minutes. Could a cluster, appearing for just a fraction of that time, have any effect on mRNA?

To answer this question, the team stimulated the cells with a chemical cocktail which they knew would affect gene transcription and mRNA output. In these cells, they found that, just before the mRNA peak appeared, clusters formed on the gene and actually remained stable for as long as 24 seconds -- a fourfold increase in a cluster's typical lifetime. What's more, the number of mRNAs increased by a similar amount.

After repeating the experiment in 207 living cells, the team found that the lifetime of Pol II clusters was directly related to the number of mRNA produced from the same gene.

Cisse speculates that perhaps Pol II clusters acts as an efficient driver of gene transcription, speeding up an otherwise inefficient process.

"It makes sense that you wouldn't want an efficient initiation process, because you don't want to randomly turn on any gene just because there was a random collision," Cisse says. "But you also want to have a way to change the initiation from an inefficient to an efficient process, for example when you want to express a gene in response to some environmental stimuli. We think that these transient clusters are probably the way that the cell can render transcription initiation efficient."

Next, Cisse plans to follow up his studies on Pol II clusters to determine what are the forces holding them together, as well as how they're formed, and whether other molecular factors cluster with similar effects.

"I suspect there are new biophysical phenomena that come from weak and transient interactions," Cisse says. "This is an underexplored area in biology, and because the interactions are so elusive we understand very little about how the regulatory processes happen inside a living cell."

This research was funded, in part, by the National Institutes of Health Director's New Innovator Award to Cisse, and additional support from the National Cancer Institute, the MIT physics department start-up funds, and the Howard Hughes Medical Institute.
-end-
Additional background

ARCHIVE: Automating single-molecule image studies

ARCHIVE: Extending super-resolution techniques

ARCHIVE: Faculty highlight: Ibrahim Cisse

Massachusetts Institute of Technology

Related Transcription Articles:

Excess transcription factor Heat Shock Factor 1 can delay embryonic neural migration
Transcription factor Heat Shock Factor 1, which the developing brain releases to shield the vital organ from the ravages of environmental stress, actually can contribute to impairing the embryonic brain when too much Hsf1 is produced, research led by Children's National Health System scientists indicates.
Transcription factor expression tied to medial amygdala neuronal ID, sex-specific response
Neurons derived from two different types of precursor cells that later develop into neurons in the medial amygdala -- one of the interconnected structures in the brain involved in emotion, motivation and memory -- help to program innate reproductive and aggressive behaviors into the brain, research led by Children's National Health System indicates.
FASEB Science Research Conference: Mechanism and Regulation of Prokaryotic Transcription
This SRC will focus on the process of transcription in prokaryotes, from structure/function investigations to transcription networks and system level regulation.
Focus on quiescent cells brings to light essential role of RNAi in transcription control
A CSHL team demonstrates for the first time that most cells cannot survive in a quiescent state unless an epigenetic mechanism called RNA interference (RNAi) is up and running.
New molecular mechanism revealed for genetic mutations in aggressive cancer cells
Scientists at the University of Birmingham have described a previously unknown molecular mechanism that could lead to the genetic mutations seen in certain types of aggressive cancer cells, involving a cell's own transcription machinery.
DNA structure influences the function of transcription factors
Spatial arrangement of the binding site and neighboring segments modulates gene activity.
Disturbances in blood cell gene transcription may lead to leukemia
Researchers have succeeded in shedding light on the pathogenesis of DNA breakpoints that are associated with leukemia.
Collisions during DNA replication and transcription contribute to mutagenesis
Replication-transcription head-on collisions contribute to mutagenesis.
Scientists illuminate a hidden regulator in gene transcription
Gene transcription is the process by which DNA is copied and synthesized as messenger RNA (mRNA) -- which delivers its genetic blueprints to the cell's protein-making machinery.
Researchers unveil new, detailed images of DNA transcription
An unprecedented molecular view of the critical early events in gene expression, a process essential for all life, has been provided by researchers at Georgia State University, the University of California at Berkeley and Northwestern University.

Related Transcription Reading:

Best Science Podcasts 2019

We have hand picked the best science podcasts for 2019. Sit back and enjoy new science podcasts updated daily from your favorite science news services and scientists.
Now Playing: TED Radio Hour

Setbacks
Failure can feel lonely and final. But can we learn from failure, even reframe it, to feel more like a temporary setback? This hour, TED speakers on changing a crushing defeat into a stepping stone. Guests include entrepreneur Leticia Gasca, psychology professor Alison Ledgerwood, astronomer Phil Plait, former professional athlete Charly Haversat, and UPS training manager Jon Bowers.
Now Playing: Science for the People

#524 The Human Network
What does a network of humans look like and how does it work? How does information spread? How do decisions and opinions spread? What gets distorted as it moves through the network and why? This week we dig into the ins and outs of human networks with Matthew Jackson, Professor of Economics at Stanford University and author of the book "The Human Network: How Your Social Position Determines Your Power, Beliefs, and Behaviours".