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Final Activity Report Summary - REGUCROW (Functional genomics of transcriptional regulation in crowded media)

The project was conceived as an extension of the work already accomplished through the previous EIF fellowship (MEIF-CT-2004-007320) in the CBS (CNRS, Montpellier, France), in the context of a multidisciplinary project entailing collaboration between structural biologists, biophysicists and microbiologists from France and Spain. In the course of the EIF research, we investigated the structure, dynamics and function of key transcriptional regulators operating in Bacilli, which had been recently identified as result of another European project (BIO-CT95-0278), in which our collaborators at INRA Paris-Grignon had participated. During this period, we gained some understanding of the molecular mechanisms of the function of crucial regulators involved in bacterial proliferation or virulence (CcpN, CggR and PlcR), using biophysical methods, and particularly state-of-the-art single molecule fluorescence micro-spectroscopy approaches.

Upon my return in Spain, we have set a new project in the laboratory (IQFR, CSIC), to continue with the biophysical characterisation of these bacterial regulators, and of key human transcriptional regulatory proteins, by using a combination of methods including fluorescence micro-spectroscopy approaches. These approaches allow characterising protein interactions inside live cells with single molecule sensitivity, although their application to the investigation of biological problems is still under development. Taking advantage of the experience acquired through the postdoctoral EIF training in France, we have incorporated these new methods in our Institute in Spain (IQFR, CSIC). By using single molecule approaches in combination with analytical ultracentrifugation methods, we have made some progress in the understanding of the physical basis of the regulatory function of CcpN (control catabolite protein of gluconeogenic genes), a transcriptional repressor crucial for bacterial survival in response to the availability of nutrients.

Moreover, we have developed a powerful method for the determination of protein-DNA binding stoichiometries, usually challenging, based on a sophisticated single molecule fluorescence approach. In parallel, the methodology applied to the investigation of the bacterial regulators is being extended to human transcriptional regulators, the PPARs (peroxisome proliferators-activated receptors), which are nuclear receptors targets for the treatment of serious diseases such as cancer and diabetes. After years of intense biomedical research in the field, the functional mechanisms of the regulation by the PPARs have not been fully elucidated. We are currently analysing the energetics and dynamics of the interactions of these regulators with their ligands and with another nuclear receptor, RXR, in dilute solution, and in model macromolecular crowding conditions. Besides, we are also going to look at those interactions in their natural environment, the cell.

After the EIF period, we set a permanent collaboration with the groups of Drs. Royer and Declerck in France, which has been very fruitful during this year in terms of transfer of knowledge between the two countries. Moreover, in the context of this project we are also working in collaboration with Spanish groups (CIB, CSIC) with experience in cell biology (Dr. Pérez-Sala) and in other biophysical methods (Dr. Rivas).

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