To further the quantitative understanding of cellular decision making, Dr. Gregory Reeves and his team in the chemical engineering department have worked to interpret how a transcription factor dictates the alteration of gene expression in cells.
The team's work, recently published in Science Advances , focuses on a protein called Dorsal, which is a version of nuclear factor-κB (NF-κB) — a crucial transcription factor controlling cellular processes and decision making while regulating cell immunity and development.
"NF-κB is involved in several medically relevant cellular behaviors, such as inflammation, innate immunity and wound healing," Reeves said. "This level of understanding could lead to the ability to control these cellular processes ourselves, because mistakes in NF-κB activity can lead to disease states, such as cancer."
NF-κB has various states inside the cell's nucleus. It has the potential to bind with DNA, clump together, and be active or inactive. Reeves' team has shown that gene regulation is happening at this level.
"We can distinguish between the molecules that are moving slowly versus those that are moving quickly, as well as those not moving at all," Reeves said. "We can do this using a fluctuation spectroscopy method that shows us how much Dorsal is moving around."
The goal is to create a map that relates how much Dorsal is in the nucleus to how much of it is bound to DNA. Understanding this map of how Dorsal binds to DNA leads to a predictive level of understanding and can reveal how to manipulate this pathway for therapeutic purposes.
In using special imaging techniques to identify the different states of Dorsal in the cell, the team was able to obtain mathematical models that reflect an accurate picture of how much Dorsal binds to the DNA, as well as the amount of Dorsal clumped together.
In previous studies, Reeves' team was only taking snapshots with their imaging technique. They decided to view cells for a longer duration.
The work encompasses multiple length and time scales, allowing them to obtain a nucleus-wide view of the mechanism connecting Dorsal to DNA.
"When we looked at how much Dorsal is freely moving around, it seemed to be independent of how much was in the nucleus," Reeves said. "This map would reveal the free amount of Dorsal in the nucleus. Once we create it, others would be free to use it so they can advance their understanding of gene regulation."
The relationship between how much NF-κB is in the nucleus versus how much of it does its job on DNA becomes straightforward when applying these methods. This allowed Reeves and the team to evaluate different parts of the embryos.
The team found that the amount of NF-κB that is free to move around is constant in different parts of the embryo. The amount bound to DNA, however, is not constant. This means the relationship between the two is not linear. "With this knowledge of how Dorsal is interacting with the DNA, we have a better understanding of how much we would need to activate the NF-κB pathway, if we needed to intervene for therapeutic purposes," Reeves said.
