Skip to main content


Pieces of the genomic puzzle

Stowers scientists are discovering new dynamics for genetic elements during development

21 September 2023

A spiral clock represents the time progression of fruit fly embryogenesis. Chromatin shown on the clock’s rim signifies the importance of chromatin changes for the developmental progression. Stable Diffusion AI software was used together with Adobe Illustrator. Artwork courtesy of Vivekanadan Ramalingam, Zeitlinger Lab, Stowers Institute for Medical Research.

We still do not understand how a relatively simple blueprint like a genome sequence can construct and maintain as complex an organism as a human being. Not only is it a complex puzzle, but also one for which we are missing many pieces. Simpler organisms provide us with an opportunity to not only identify but also test the roles of those missing pieces. Thus, if we want to understand the human genome beyond just knowing its sequence, it is essential for us to carry out foundational biology research.

Investigating the common fruit fly reveals much about embryogenesis, the developmental program dictating how a single fertilized cell becomes a functional organism. It is a delicate, deliberate, and extraordinarily exact orchestration. Literally. For example, a single rogue gene can cause a fruit fly to have legs growing out of its head.

New research from the Stowers Institute for Medical Research is uncovering novel regulatory dynamics during development that cast fresh roles for sequence elements called promoters. The dynamic changes were unanticipated since these elements are considered to always be open for transcription, which produces an RNA molecule from a DNA sequence. The new findings suggest that assembling the puzzle of development is far from finished.

“Many people study early embryogenesis and how transcription first begins,” said Stowers Investigator Julia Zeitlinger, Ph.D. “We discovered a second act, where a protein called Lola-I opens previously inaccessible promoters, thereby dictating when new genes become available for transcription in the middle of embryogenesis.”

Single molecule fluorescence in situ hybridization (smFISH) microscopy image shows expression of lola-I gene transcripts (magenta) and a fluorescent stain (DAPI) for specific DNA regions (turquoise) throughout the late-stage Drosophila embryo.

The study, published on September 21, 2023, in Nature Communications, expands our knowledge of how the DNA controlling developmental programs across species is accessed and precisely executed. The unexpected finding that promoters can be dynamically regulated and prepared for activation may inform our understanding of human promoters during development and disease.

Behind the scenes: Meet the developmental players 

Starting with the same genome, cells proliferate, or multiply, and become diversified during development. Different genes are expressed, or turned on, to produce cell-specific products that shape their structure and function to form the tissues and organs of a maturing animal.

Located adjacent to genes, promoters are DNA elements that serve as the gathering places for the cellular machinery that initiates transcription, the first step in gene expression that produces the RNA instructions for protein production. Like a wide receiver, promoters tend to be generally accessible and receptive to activation signals from enhancers that are located nearby or far away.

Enhancers, in contrast, only become physically accessible around the time in which they are needed. Since their function is very specific and a gene has many of them to regulate the gene’s expression throughout the course of development, it makes sense that the accessibility of enhancers is regulated.

Making enhancers accessible is the role of proteins called pioneer factors. In this first step of enhancer regulation, chromatin—the DNA-protein complex that packages DNA to fit inside a cell—is modified to expose DNA. Once pioneer factors have undressed the DNA, specific proteins and enzymes can bind to the enhancer and direct the next step of regulation, in which an activation signal is sent to the promoter.

However, new findings from the Zeitlinger Lab suggest that certain promoters are also regulated like enhancers. A dedicated pioneer factor makes them accessible only during a certain stage of development—mid to late embryogenesis—in the fruit fly, Drosophila melanogaster. The researchers were then able to study the mechanisms by which promoters respond to this type of regulation.

“A common belief is that promoters are always open, but we show that a subset of promoters only start to become receptive to signals during mid-embryogenesis.” Zeitlinger explains. “This type of regulation has not been described before and blurs the boundary between enhancers and promoters.”

Graphical illustration showing that the pioneering transcription factor Lola-I binds DNA later during embryogenesis, indicating a second stage of gene activation later during development. Image courtesy of Mark Miller, Stowers Institute for Medical Research.

Vivekanadan Ramalingam, Ph.D., a former predoctoral researcher in the Zeitlinger Lab, provided proof that Lola-I functions as a promoter pioneer factor during the later stages of embryogenesis. He showed that Lola-I opens chromatin at promoters in a similar way as at enhancers, but the consequences for promoters are different. When newly accessible, these promoters now recruit the enzyme required for transcription, and this prepares them for activation.

Promoter pioneer factors are required but not sufficient for gene activation

The discovery began with an unusual observation.

Since promoters are presumed to always be open, the team was surprised to find that a significant subset of promoters did not possess the enzyme required for transcription from the start. Rather, they saw that these promoters gradually acquired it beginning at the midpoint of development.

“Early on, we recognized that Lola-I must be responsible for this change,” said Zeitlinger. “But we didn’t know how or why.”

Using a multidisciplinary approach consisting of genomics tools, imaging techniques, and fruit flies with lola gene mutations, the researchers discovered that Lola-I was functioning as a pioneer factor at promoters. By binding to the edge of chromatin, it was opening up promoters and allowing the enzyme for transcription to come and initiate transcription.

But while Lola-I causes transcription to be initiated from these promoters throughout the embryo, the enzyme typically does not produce a full RNA that instructs protein production. Rather, the enzyme “pauses” shortly after it starts and only proceeds to produce a burst of transcription in some tissues.

Single molecule FISH microscopy image shows decreased activation of Lola-I’s target gene Gip (yellow) with co-localized expression of non-target genes PPO1/2 in Drosophila embryos with mutations in lola-I (right) compared to normal embryos (left). Top insets show the nascent Gip signal in the nuclei; bottom insets show the single Gip RNAs in the cytoplasm.

This means that Lola-I does not activate transcription on its own but functions as a preparatory step toward activation. By allowing transcription to begin but not to fully proceed, Lola-I makes the promoters now receptive to the tissue-specific instructions from enhancers. Without Lola-I, the researchers found, the promoter’s ability to produce tissue-specific bursts of transcription was drastically reduced.

“It felt like the story or central piece was always there,” said Ramalingam. “Exploring it from many different angles allowed us to really nail the story, to develop a clear picture of how Lola-I functions to make a new set of genes available for activation.”

Next steps: From fruit flies to humans 

The study’s findings may lead to learning more about gene expression regulation during human development and disease.

“With the latest genomics tools and AI techniques, we’re facing both a challenge and an opportunity to really understand the human genome,” said Ramalingam. “This finding contributes a piece to that fundamental framework through which we can solve this enormous puzzle that could explain a lot of human biology.”

Zeitlinger added, “This has always been the goal for what we do in the lab: to decode the human genome, to understand disease and disease susceptibility. This particular piece is relevant—the additional availability of previously hidden promoters and the increasingly ambiguous boundary distinguishing promoters from enhancers are likely principles that generalize to the human genome. But there is more work to be done to put everything together.”

Ramalingam dedicates this research to the memory of his mother.

Additional authors include Xinyang Yu, Ph.D., Brian Slaughter, Ph.D., Jay Unruh, Ph.D., Kaelan Brennan, Anastasiia Onyshchenko, Jeffery Lange, Ph.D., Malini Natarajan, Ph.D., and Michael Buck, Ph.D.

This work was funded by the National Institutes of Health (NIH) (award: DP2OD004561) and institutional support from the Stowers Institute for Medical Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Newsletter & Alerts