Understanding how genes are regulated within living organisms is crucial for comprehending 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 significant challenge, requiring sophisticated techniques to see where and how these proteins interact. A new technique now makes it possible to observe these protein interactions directly in the nuclei of Drosophila larval salivary glands, shedding light on the intricate dance of molecules that drives gene regulation.
A new experimental approach has been developed by researchers from the Institut de Génomique Fonctionnelle de Lyon at the University of Lyon, led by Dr. Samir Merabet along with Dr. Solène Vanderperre, to study protein interactions within the nuclei of Drosophila larval salivary glands. This novel method, termed BiFOR, combines Bimolecular Fluorescence Complementation (BiFC) with the bacterial ANCHOR DNA-labelling system. 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 on specific enhancers in Drosophila salivary gland nuclei. The study’s objective was to decrypt the molecular cues underlying TF specificity in vivo, a crucial aspect of gene regulation.
The researchers employed BiFC, a technique that has been extensively used to reveal protein-protein interactions (PPIs) in various model systems, including live Drosophila embryos. However, visualizing PPIs at the level of specific target enhancers or genomic regions required the advent of new DNA-labelling methods. The introduction of the ANCHOR system enables 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 forkhead (fkh250) as a model. This enhancer is regulated by the Hox protein Sex combs reduced (Scr) in association with the Extradenticle (Exd) cofactor. The researchers demonstrated that Scr/Exd complexes are specifically enriched on the fkh250 enhancer in salivary gland nuclei, confirming previous in vitro and in vivo findings.
The study revealed that the BiFC signals were significantly enriched with ParB1–mCherry, a component of the ANCHOR system, demonstrating preferential localization on 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 Scr alone (The Exd cofactor is required to help Scr recognizing its target fkh250 enhancer).
The BiFOR technique’s sensitivity and specificity were further confirmed by analyzing two additional variants of the fkh250 enhancer: a mutant version (fkh250MUT) and a consensus version (fkh250CONS). The fkh250MUT enhancer, with mutations abolishing Hox/Exd binding, showed no significant enrichment of BiFC signals, while the fkh250CONS enhancer, which allows recognition by different Hox/Exd complexes, displayed significant enrichment.
This study sets the experimental basis 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 for visualizing and quantifying protein complex dynamics on specific DNA regions, providing deeper insights into the molecular mechanisms of gene regulation.
Dr. Merabet stated, “Our work demonstrates that BiFOR can recapitulate the specific recognition of target enhancers through a dimeric protein complex in salivary gland nuclei.” The technique’s versatility and sensitivity make it a promising approach for future studies aiming to unravel the complexities of transcription factor interactions and gene regulation.
The research by Dr. Merabet and Dr. Vanderperre marks a significant advancement in the field of molecular biology, providing a new methodology to explore the dynamic interactions of proteins within the nucleus. This innovative approach holds 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 on Specific Enhancers in the Salivary Gland Nuclei of Drosophila Larva.” Cells, 2024. DOI: https://doi.org/10.3390/cells13070613
About the Author
Dr. Samir Merabet is a Research Director of the CNRS (Centre National de Recherche Scientifique). He did his PhD in Marseille at the Institut de Biologie de Développement de Marseille (IBDM, France) and post-doctoral 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 post-doctoral work were dedicated to the understanding of their intrinsic molecular properties in development and evolution. Since his installation at the IGFL, the group of Samir Merabet is developing innovative tools for capturing and studying protein-protein interactions of Hox proteins and other major regulators of development in different model systems, including living Drosophila embryos or larvae and human cells.
