Understanding how genes are regulated in living organisms is crucial to understanding how cells develop and function. At the heart of this process are transcription factors (TFs), proteins that help turn specific genes on or off by binding to particular regions of DNA known as enhancers. Visualizing these interactions within the cell nucleus has been a major challenge, requiring sophisticated techniques to see where and how these proteins interact. Now, a new technique makes it possible to observe these protein interactions directly in the salivary gland nuclei of Drosophila larvae, shedding light on the intricate dance of molecules that drives gene regulation.
A new experimental method, led by Dr. Samir Merabet and Dr. Solène Vanderperre of the Lyon Institute of Functional Genomics at the University of Lyon, has been developed by researchers to study protein interactions within the nuclei of the salivary glands of Drosophila larvae. This novel method, called BiFOR, combines bimolecular fluorescence complementation (BiFC) with the bacterial DNA labelling system ANCHOR. Their findings have been published in the journal Cells.
Dr. Merabet explained that this new technique allows for the precise quantification of dimeric protein complexes in specific enhancers of the nuclei of Drosophila salivary glands. The aim of the study was to decipher the molecular signals underlying TF specificity in vivo, a crucial aspect of genetic regulation.
The researchers employed BiFC, a technique that has been widely used to reveal protein-protein interactions (PPIs) in several model systems, including living Drosophila embryos. However, visualizing PPIs at the level of specific enhancers or genomic regions required the emergence of new DNA labeling methods. The introduction of the ANCHOR system allows for the precise localization and quantification of these interactions without disrupting transcriptional regulation.
Significant results were obtained using the well-characterized enhancer of the salivary gland selector gene. fork head (fkh250) as a model. This enhancer is regulated by the Hox protein Sex combs reduction (Scr) in association with the cofactor Extradenticle (Exd). The researchers demonstrated that Scr/Exd complexes are specifically enriched with the fkh250 enhancer in salivary gland nuclei, confirming previous in vitro and in vivo findings.
The study revealed that BiFC signals were significantly enriched with ParB1–mCherry, a component of the ANCHOR system, demonstrating a preferential localization in the fkh250 enhancer. Quantification of these signals showed that the enrichment was specific to Scr/Exd complexes, as no significant binding was observed with another Hox/Exd complex or with Scr alone (the Exd cofactor is necessary to help Scr recognize its target). fkh250 enhancer).
The sensitivity and specificity of the BiFOR technique were further confirmed by analyzing two additional variants of the fkh250 Enhancer: A mutant version (fkh250Mutual) and a consensus version (fkh250CONS). He fkh250Mutual The enhancer, with mutations that eliminate Hox/Exd binding, did not show significant enrichment of BiFC signals, while the fkh250CONS The enhancer, which allows recognition by different Hox/Exd complexes, showed significant enrichment.
This study lays the experimental groundwork for future applications of the BiFOR strategy, which could be applied to other tissues during Drosophila development and potentially to other model organisms. The findings highlight the potential of BiFOR as a powerful tool to visualize and quantify the dynamics of protein complexes at specific DNA regions, providing deeper insights into the molecular mechanisms of gene regulation.
Dr Merabet said: “Our work demonstrates that BiFOR can reproduce the targeted recognition of specific enhancers via a dimeric protein complex in salivary gland nuclei.” The versatility and sensitivity of the technique make it a promising approach for future studies aimed at unraveling the complexities of transcription factor interactions and gene regulation.
Dr. Merabet and Dr. Vanderperre’s research marks a significant advance in the field of molecular biology, as it provides a new methodology to explore the dynamic interactions of proteins within the nucleus. This innovative approach has the potential to uncover new aspects of gene regulation and transcription factor specificity, paving the way for future discoveries in developmental biology and genetics.
Journal reference
Vanderperre, Solène and Samir Merabet. “Visualization of the association of dimeric protein complexes at specific enhancers in the nuclei of Drosophila larval salivary glands.” Cells, 2024. DOI: https://doi.org/10.3390/cells13070613
About the Author
Dr. Samir Merabet Samir Merabet is a research director at the CNRS (Centre National de Recherche Scientifique). He completed his PhD in Marseille at the Institut de Biologie de Développement de Marseille (IBDM, France) and his postdoctoral work at the Biozentrum (Basel, Switzerland). He established his group “Ontogenesis and molecular interactions” at the IGFL (Institut de Génomique Fonctionnelle de Lyon, France) in 2012. Samir Merabet has always been fascinated by a conserved family of developmental regulators, the Hox proteins. His PhD and postdoctoral work were dedicated to understanding their intrinsic molecular properties in development and evolution. Since settling at the IGFL, Samir Merabet’s group is developing innovative tools to capture and study protein-protein interactions of Hox proteins and other important developmental regulators in different model systems, including living beings. Drosophila embryos or larvae and human cells.
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