Scientists have made intriguing discoveries about how transcription factors, which serve as genetic switches for various genes, control the growth and development of plants. Their research highlights how minor modifications in a lipid-binding section known as the START domain can significantly impact gene regulation, opening new avenues for crop improvement, synthetic biology, and targeted gene treatments.
Researchers from the University of Pennsylvania, including Aman Husbands from the School of Arts & Sciences, have highlighted novel ways transcription factors—setting genetic switches—guide plant development. Their discoveries indicate that small variations within a lipid-binding section called the START domain can significantly influence gene regulation, paving the way for advancements in agriculture, synthetic biology, and precise gene therapy approaches.
In complex living organisms like plants and humans, there are essential genetic components that can be compared to construction blueprints, tools, and skilled workers at a development site. Plant biologists, like Aman Husbands at the University of Pennsylvania, study a group of specialized players known as HD-ZIPIII transcription factors (TFs). These TFs are responsible for determining which genetic blueprints to follow to shape a plant’s structure and characteristics, including its vascular system and features such as roots and leaf form.
Interestingly, although all members of the HD-ZIPIII family share similar blueprints and tools, each—like CORONA (CNA) and PHABULOSA (PHB)—interprets these blueprints differently, yielding unique and observable results in the structures they help forge.
“The pressing question,” Husbands states, “is, ‘How can we explain these different functional outcomes?'”
In a study published in Nature Communications, Husbands and colleagues investigated two nearly identical paralogs, PHB and CNA, to identify the mechanism behind this divergence.
“We discovered that, despite these two transcription factors binding to the same DNA regions, they regulate different genes, leading to distinct developmental results,” Husbands explains. “This unexpected finding draws attention to a seemingly minor yet vital aspect of the transcription factors—the START domain,” which serves as a basic tool for decision-making regarding how the plans are executed at various sites.
By switching the START domains of PHB and CNA, the researchers showed that this single modification could dramatically change their functions, essentially rewriting the developmental instructions.
“The potential implications are significant, not only for plant biologists but also for researchers in related fields,” says Ashton Holub, first author and former postdoctoral researcher in the Husbands Lab. “In synthetic biology or gene therapy, transcription factors may lead to unwanted off-target effects. Understanding and manipulating mechanisms such as the START domain can enable us to fine-tune genetic tools, minimizing risks and achieving accurate outcomes in the future.”
Understanding the roles of CNA and PHB
The researchers began by probing the functional differences between CNA and PHB using qPCR, a quantitative method to assess RNA molecule quantities, thus reflecting gene expression levels. Initially, they examined two genomic targets that CNA and PHB were expected to regulate based on previous research and established assumptions regarding TFs.
However, Holub mentions that the qPCR results revealed something unexpected. Although a location-based test (ChIP-qPCR) found both CNA and PHB binding to the same target locations, a different test evaluating the impact of this binding (RT-qPCR) suggested they were not always effective in producing regulatory effects. “Although we detected binding at these locations, there were no shifts in gene expression,” he says. “This discrepancy prompted us to broaden our perspective and explore the entire genome rather than a few sites.”
In response to this paradox, the team applied ChIP-seq to comprehensively map all the binding sites of CNA and PHB throughout the genome, giving them an overview of the transcription factors’ binding landscape. Additionally, they used RNA-seq (transcriptome profiling) to gauge gene expression changes on a genome-wide scale. This combination of methods allowed them to ascertain not only where CNA and PHB were binding but also which genes were being turned on or off as a result.
Holub notes, “qPCR highlighted the anomaly, while ChIP-seq and RNA-seq painted the complete picture.”
The START Domain: A pivotal decision-maker
The research led the team to identify a crucial characteristic of CNA and PHB: their START domain, a lipid-binding aspect of the proteins that endows them with specific transcriptional capabilities.
“An intriguing aspect of these transcription factors is that they possess a START domain, also identified in other proteins across various life forms,” Husbands explains. “These domains play a significant role in development, responses to stress, and even disease. When we observed them in these transcription factors, we proposed that they might explain the differing functions of CORONA and PHB.”
To verify this hypothesis, the researchers created chimeric CNA proteins by swapping the START domain with those from PHB or even from species that diverged hundreds of millions of years ago. “Our experiments confirmed that the START domain was the critical factor influencing their functions,” states Sarah Choudury, a postdoctoral researcher in the Husbands Lab. “What changed was not the sites these transcription factors attached to but how they directed the genes they bound.”
Through deletion, mutation, and swapping of START domains, the researchers demonstrated that this small segment acts as a decision-making component, determining whether a gene is activated or suppressed. Minor alterations to the START domain led to significant effects, highlighting how this mechanism contributes to the variety of gene regulation.
Reflecting on how the START domain allows a single set of binding sites to generate a broad spectrum of developmental instructions, Husbands cleverly turned the common Latin phrase “e pluribus unum” (out of many, one) on its head, asserting, “Out of one, many. From one binding network, diverse regulatory programs can emerge.”
Husbands and his colleagues are now investigating how this mechanism functions in other families of transcription factors and in species beyond the primary model used in this study, Arabidopsis thaliana.
“We are probing whether this type of differential regulation is a common feature across evolution,” Holub remarks. “If it occurs in plants, there is every reason to suspect it might be present in animals as well.”
The research team seeks to grasp the intricacies of how START domains interact with other cellular elements to affect gene regulation. “There’s a vast amount we still need to uncover,” Choudury observes. “What about transcription factors lacking START domains? Are there parallel mechanisms involved? How do these domains detect and respond to environmental changes?”
Aman Husbands serves as 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, previously a postdoctoral researcher in the Husbands Lab at Penn Arts & Sciences, is currently a fellow at Nationwide Children’s Hospital.
Sarah Choudury is a postdoctoral researcher at the Husbands Lab at Penn Arts & Sciences.
Other contributors include Ricardo Urquidi Camacho and Courtney E. Dresden from Penn Arts & Sciences, alongside Ekaterina P. Andrianova and Igor B. Zhulin from Ohio State University.
This research received funding from the National Science Foundation (grants 2039489 and 2310356).