In a recent study published in Development on May 24, 2023, Zainab Afzal, Ph.D., a former University of Kansas Medical Center graduate student in the Krumlauf Lab, along with collaborators from the Stowers Technology Centers, uncovered the dynamic properties that three shared retinoic acid responsive enhancers have on regulating the transcription—the creation of gene messages to be translated into proteins—of the Hoxb gene cluster, which is responsible for specifying tissue identity along the head-to-tail axis of the mouse body plan.

The team sought to answer two questions. First, within single cells, how do shared, overlapping enhancers situated in the middle of a long stretch of DNA containing nine Hoxb genes coordinately regulate these genes over short and long distances? Second, do they turn on Hoxb genes simultaneously or one by one in one cell?

The answers were surprising.

“It’s amazing what we don’t know.” – Robb Krumlauf

To answer if the enhancers impact Hoxb genes selectively or in coordination with each other, patterns of transcription for several of these genes spanning the entire cluster were analyzed. This was accomplished by taking snapshots of transcription activity at a single point in developmental time on several sections of mouse embryos.

Together, these three enhancers are required for controlling the correct level and activity of gene expression at precise points in time and space in the developing embryo. However, each of these regulatory elements have distinct tendencies, with two of the three having a more global role in regulating the Hoxb cluster while one has a more proximal preference for nearby genes.

By analyzing whole tissue sections at the single cell level, nearly all measurements indicated that only one gene was active at a time in a cell. Thus, Hoxb genes are not turned on simultaneously in a single cell, but rapidly and dynamically activated independently.

“These results suggest that the genes might be in a transcriptional hub with a 3D structure that positions them in close proximity to the enhancers despite their linear position on the chromosome,” said Krumlauf. “In hubs, the enhancers may be positioned to dart in and out to engage different target genes and rapidly coordinate gene activity for genes that are far apart on DNA.”

Ground Control to Major Tom: It’s time for a technology boost

The technologies and techniques developed and used in this study were integral for addressing these fundamental questions about transcription. The Krumlauf Lab in conjunction with several scientists from the Technology Centers, developed several fluorescent probes. Each probe was designed to emit a specific-colored spot when bound to actively transcribing RNA for three Hoxb genes spanning the entire cluster (Hoxb1, 4, and 9).

Zooming in on the nucleus, the physical location of gene transcription in a cell, in different sections of a mouse embryo, the probe images were divided among several scientists for a manual fluorescent spot count. Using the results from each person’s tally to train a computer to “learn” this counting technique allowed the team to develop a deep learning algorithm that was just as accurate, if not better, than the manual method—and much faster.

Additionally, while the three enhancers were known to be required for Hoxb gene transcription, how each one contributes was addressed by making function-disrupting mutations for each, and then examining mouse lines harboring one, two, or all three mutations.

A result that the team did not expect was that single enhancer mutations actually increased gene activity across the Hoxb cluster. Even more astonishing was that transcription activity returned to expected levels when two of the three enhancers were mutated. Hoxb expression was null when all three were disrupted.

“The results from the single and double mutations were really surprising to us,” said Krumlauf. “Somehow the cooperativity between the enhancers was altered and it may be better to have neither than to have one without the other balancing it.”

Space: the final frontier

Collective findings from the study indicate that the basic mechanisms of transcription that were thought to be understood are regulated in a much more complex way. Combining new technologies and rethinking previously presumed principles will provide a far greater understanding of how cell and tissue identity is determined in the right way, at the right time to build an organism.

NASA astrophysicist Jane Rigby told the audience at the 2023 American Association for the Advancement of Science annual meeting, “There are two ways to succeed in astronomy: either be really smart or be there at the right time.”

Krumlauf takes a strikingly similar stance on the study of transcription; a combination of intelligence and observation at the right time both factor in. “We are really trying to understand the dynamics of how transcription happens in real time, and the Technology Center experts were just as fascinated by these questions as we were,” said Krumlauf. “For me, whenever you have smart people interested in a problem, throwing out ideas and struggling through setbacks, you come up with a solution.”

Additional authors include Jeffery Lange, Ph.D., Christof Nolte, Ph.D., Sean McKinney, Ph.D., Christopher Wood, Ph.D., Ariel Paulson, Ph.D., Bony De Kumar, Ph.D., Jay Unruh, Ph.D., and Brian Slaughter, Ph.D.

This work was funded by institutional support from the Stowers Institute for Medical Research.

Read more here on Development's Research Highlight and an interview with Krumlauf and Afzal:
RARE peak into Hoxb transcriptional regulation
The people behind the papers - Robb Krumlauf and Zainab Afzal