Search

The gene regulatory network governing anterior–posterior axis formation in Drosophila is a well-established paradigm to study transcription in developmental biology. The rapid temporal dynamics of gene expression during early stages of development, however, are difficult to track with standard techniques. We optimized the bright and fast-maturing fluorescent protein mNeonGreen as a real-time, quantitative reporter of enhancer expression. We derive enhancer activity from the reporter fluorescence dynamics with high spatial and temporal resolution, using a robust reconstruction algorithm. By comparing our results with data obtained with the established MS2-MCP system, we demonstrate the higher detection sensitivity of our reporter. We used the reporter to quantify the activity of variants of a simple synthetic enhancer, and observe increased activity upon reduction of enhancer–promoter distance or addition of binding sites for the pioneer transcription factor Zelda. Our reporter system constitutes a powerful tool to study spatio-temporal gene expression dynamics in live embryos., Ceolin et al. optimise the fluorescent protein mNeonGreen to generate a reporter to measure enhancer expression in living Drosophila embryos. They also find that reduced enhancer–promoter distance or addition of binding sites for the pioneer transcription factor Zelda increases enhancer activity.

Hunchback (Hb) is considered a context-dependent transcription factor, able to activate or repress different enhancers during Drosophila embryo segmentation. The mechanism driving the contextdependent activity of Hb is however not well understood. Here we measure the activity of a large set of 20 synthetic enhancers that we design to elucidate the effect of Hb binding sites in Drosophila segmentation. We obtain quantitative data on the spatiotemporal dynamics of activity of all synthetic enhancers in-vivo, by using a quantitative and sensitive reporter system we recently developed. Our data reveal the dual role of Hb binding sites in segmentation enhancers: on the one hand, Hb act as a typical short range repressor by binding to its cognate sequences; on the other hand, we report a novel effect of a sequence containing multiple Hb binding sites, which is able to increase enhancer activity independently from Hb binding. This sequence, which contains multiple Poly-dA stretches, increases the activity of enhancers driven by different activators, possibly by disfavoring nucleosome occupancy.AUTHOR SUMMARYThe control of gene expression is a fundamental process that allows cells to respond to external stimuli and take on various identities in complex organisms. Enhancers are DNA sequences that play a key role in this process. In the simplest model of an enhancer, small parts of its sequence can be specifically bound by proteins called transcription factors and the occupancy pattern of these proteins on the enhancer determines the expression level of a specific gene. In this research work we have studied enhancers in the context of the development of a fruit fly embryo. We have built synthetic enhancer sequences containing binding sites for a few specific factors and measured their activity in living embryos using fluorescence microscopy. Our results revealed that binding sites for a particular protein, Hunchback, are able to influence the activity of the enhancer even independently from Hunchback binding to them. This discovery might help to explain the complex effects that have been observed when studying Hunchback binding sites in natural enhancers.

Summary The DNA of eukaryotes is wrapped around histone octamers to form nucleosomes. Although it is well established that the DNA sequence significantly influences nucleosome formation, its precise contribution has remained controversial, partially owing to the lack of quantitative affinity data. Here, we present a method to measure DNA-histone binding free energies at medium throughput and with high sensitivity. Competitive nucleosome formation is achieved through automation, and a modified epifluorescence microscope is used to rapidly and accurately measure the fractions of bound/unbound DNA based on fluorescence anisotropy. The procedure allows us to obtain full titration curves with high reproducibility. We applied this technique to measure the histone-DNA affinities for 47 DNA sequences and analyzed how the affinities correlate with relevant DNA sequence features. We found that the GC content has a significant impact on nucleosome-forming preferences, but 10 bp dinucleotide periodicities and the presence of poly(dA:dT) stretches do not., Graphical Abstract, Highlights • Robotics permits full titration series to measure histone-DNA binding affinities • Fluorescence anisotropy used as a fast, sensitive readout of bound/unbound DNA • Free energies span three orders of magnitude, less for naturally occurring sequences • GC content is a major determinant of measured binding free energies, Molecular Biology; Molecular Interaction; Biological Sciences Research Methodologies

// Bianca Cali 1,2 , Stefano Ceolin 3 , Federico Ceriani 1,3 , Mario Bortolozzi 1,3 , Andrielly H.R. Agnellini 1,2 , Veronica Zorzi 1,3 , Andrea Predonzani 4 , Vincenzo Bronte 1,5 , Barbara Molon 4,* and Fabio Mammano 1,3,6,* 1 Foundation for Advanced Biomedical Research, Venetian Institute of Molecular Medicine, Padua, Italy 2 University of Padua, Department of Surgery Oncology and Gastroenterology, Oncology and Immunology Section, Padua, Italy 3 University of Padua, Department of Physics and Astronomy, Padua, Italy 4 IRCCS, Istituto Oncologico Veneto, Padua, Italy 5 Verona University Hospital, Department of Pathology and Diagnostics, Immunology Section, Verona, Italy 6 Present address: CNR, Institute of Cell Biology and Neurobiology, Monterotondo (RM), Italy * These authors contributed equally to this work Correspondence to: Vincenzo Bronte, email: // Barbara Molon, email: // Fabio Mammano, email: // Keywords : cancer, photodynamic therapy, nitric oxide, calcium signaling, connexins Received : January 24, 2015 Accepted : February 14, 2015 Published : March 12, 2015 Abstract Ionizing and nonionizing radiation affect not only directly targeted cells but also surrounding “bystander” cells. The underlying mechanisms and therapeutic role of bystander responses remain incompletely defined. Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca 2+ wave initiated in the irradiated cell. The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca 2+ -dependent enzymatic production. We detected similar signals in tumors grown in dorsal skinfold chambers applied to live mice. Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca 2+ and NO in the bystander effects triggered by photodynamic therapy.