From one gene switch, many possible outcomes

Within all complex, multicellular living systems such as plants and humans, there exists a set of genetic elements that can be likened to the blueprints, tools, and specialized personnel at a construction site for an expanding development. Plant biologists like Aman Husbandsat the University of Pennsylvaniastudy a family of skilled subcontractors, known as the HD-ZIPIII transcription factors (TFs). These subcontractors are tasked with deciding which blueprints, or genes, to follow as they guide the development of a plant's form and features, such as its plumbing-analogous to the vasculature-and structural components like roots and leaf shapes.

However, despite sharing overlapping blueprints and access to the same tools, each HD-ZIPIII family member, like CORONA (CNA) and PHABULOSA (PHB), has a unique way of interpreting these blueprints and using their tools. These differences lead to distinct and measurable outcomes in the structures they help create.

"Now, the million-dollar question," Husbands says, "is, 'How do you get these functionally divergent outcomes?'"

In a paper published in Nature Communications,Husbands and the team focused on two near-identical paralogs, PHB and CNA, to uncover the mechanism behind this divergence.

"We found that while these two transcription factors bind to the same regions of DNA, they regulate different genes, resulting in unique developmental outcomes," Husbands says. "This surprising discovery points to a small but crucial feature of the transcription factors: their START domain," a lipid-binding region within the TFs, which the researchers liken to a foreman's decision-making tool, dictating how the blueprints are executed at each site.

By swapping the START domains of PHB and CNA, the researchers demonstrated that this single change could alter their function, effectively rewriting the developmental instructions.

"The implications are pretty significant, not just for other plant biologists and researchers in this space," says first author Ashton Holub, a former postdoctoral researcher in the Husbands Lab. "In synthetic biology or gene therapy, transcription factors with off-target effects can cause some concerning unintended consequences, so, by understanding and being able to manipulate mechanisms like the START domain, we can someday fine-tune genetic tools to minimize risks and achieve precise outcomes."

Where CNA and PHB work and what they do

The researchers initially explored the functional divergence between the CNA and PHB by performing qPCR, a quantitative technique used to measure the abundance of RNA molecules and, by extension, gene-expression levels. Initially, they focused on examining two genomic targets they expected CNA and PHB to regulate based on the team's previous researchand assumptions about TFs based on other literature.

However, Holub says, the qPCR results revealed an unexpected finding. Although one type of location-based test (ChIP-qPCR) found that CNA and PHB were binding to the same target sites, another that examines the effects of the binding activity (RT-qPCR) showed they didn't always produce a regulatory effect. "We saw binding at these spots but no changes in gene expression," he says, "which really forced us to think more broadly and explore the full genome rather than just a couple of sites."

To address this paradox, they turned to ChIP-seq to systematically map all the binding sites of CNA and PHB across the genome, thus allowing the researchers to see the broader landscape of where TFs were binding. To complement this, they used RNA-seq (transcriptome profiling) to measure changes in gene expression at a genome-wide scale. This combination of techniques enabled the team to determine not just where CNA and PHB were binding but also which genes were being activated or repressed as a result.

Holub says, "qPCR showed us the anomaly, and ChIP-seq and RNA-seq gave us the complete story."

The START Domain: A critical decision-maker

The results pointed the researchers toward a key feature of CNA and PHB: their START domain, a lipid-binding part of the proteins that imbues them with certain transcriptional abilities.

"One of the interesting things about these TFs is that they have this START domain, which you also see in other proteins across the tree of life," Husbands says. "These domains are important for development, stress responses, and even disease. When we saw them in these TFs, we hypothesized that they were the reason CORONA and PHB might function differently."

To test this hypothesis, the researchers generated chimeric CNA proteins by swapping its START domain with those from PHB, or even from species separated by hundreds of millions of years of evolution. "Our experiments confirmed that the START domain was the critical determinant," says Sarah Choudury, a postdoctoral researcher in the Husbands Lab. "It wasn't where these TFs bound that changed; it was how they regulated the genes they bound to."

By deleting, mutating, and swapping START domains, the researchers demonstrated that this small region acted as a decision-making tool, dictating whether a gene was activated or suppressed. Even small changes in the START domain had significant impacts, illustrating how this mechanism contributes to the diversity of gene regulation.

In noting how the START domain enables a single set of binding sites to generate a wide range of developmental instructions, Husbands flipped the familiar Latin phrase "e pluribus unum" (out of many, one) on its head, remarking, "Out of one, many. Out of one bound network, you can get a diversity of regulatory programs."

Husbands and the team are now exploring how this mechanism operates in other transcription factor families, as well as in species beyond the model used for this research, Arabidopsis thaliana.

"We're testing whether this kind of differential regulation is a generalizable feature across evolution," Holub says. "If it's happening in plants, there's every reason to believe it could be happening in animals, too."

The team wants to understand the finer details of how START domains interact with other cellular components to influence gene regulation. "There's so much we still don't know," Choudury says. "What about transcription factors that don't have START domains? Are there parallel mechanisms at play? And how do these domains sense and respond to the environment?"

Aman Husbands is the Mitchell J. Blutt and Margo Krody Blutt Presidential Assistant Professor of Biology in the Department of Biology in the School of Arts & Sciences.

Ashton Holub is a former postdoctoral researcher in the Husbands Lab at Penn Arts & Sciences and now a fellow at Nationwide Children's Hospital.

Sarah Choudury is a postdoctoral researcher in the Husbands Lab at Penn Arts & Sciences.

Other authors are Ricardo Urquidi Camacho and Courtney E. Dresden of Penn Arts & Sciences and Ekaterina P. Andrianova and Igor B. Zhulin of Ohio State University.

This work was supported by the National Science Foundation (grants 2039489 and 2310356).