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Quantifying transcription factor search mechanism by three-dimensional single molecule tracking

Final Report Summary - SMT OF TFS (Quantifying transcription factor search mechanism by three-dimensional single molecule tracking)

Transcription factors (TFs) regulate when and how specific genes needs to be expressed by a cell in response to specific external stimuli, by binding to specific sequences of DNA in proximity of the genes that need to be activated or repressed. One example is provided by the tumor suppressor p53, an important TF that determines the cellular response to stress signals that challenge the integrity of genetic information such as DNA damage. In particular, different sources of DNA damage activate specific pathways that converge to this transcription factor, and p53 is capable of recognizing the offending stimuli and activate different transcriptional programs, that range from the arrest of the cell cycle to the induction of cellular death (apoptosis). Understanding how p53 can translate different stress signals into different transcriptional responses is crucial as this TFs (mutated in almost 70% of known tumors) play a fundamental role in chemotherapeutic and radiotherapeutic approaches based on the induction of DNA damage to kill cancer cells.

Despite being one of the most widely studied TFs, many questions remain unanswered about the microscopical behavior of p53. In our project, we have focused on studying the search mechanism of this TF or in other words on the mechanism used by p53 to recognize the sequences that need to be regulated among the many binding sites available in the genome. We have hypothesized that p53 could modulate its search mechanism and its binding affinity to regulatory regions depending on the source of genotoxic stress and that such differential modulation could represent a mechanism by which p53 could regulate different transcriptional responses. This hypothesis known as “latency model” for p53 has been supported by test-tube experiments, but the data available from biochemistry experiments in cells is at the moment controversial.

To the scope of studying the p53 dynamic behavior with unprecedented detail we developed a fluorescence microscope capable of identifying and tracking individual TFs in three-dimensional environments: When starting the project single molecule imaging was mostly applied to in-vitro and in-membrane environments due to limitations associated to obtaining information over an extended amount of time from molecules moving in 3D. The first limitation is given by the out-of focus blur that affects single molecule imaging in three-dimensional samples: we overcame this problem by using an improved oblique illumination approach, allowing a significant reduction of the background signal. The second limitation is represented by the limited amount of time spent by each individual molecules in the focal plane of the high numerical aperture objectives typically used in single molecule imaging. We have devised an approach for extending the focal depth of high NA objectives and we have demonstrated that the approach can be used for tracking individual objects (particles) in 3D.

We have then developed analytical and numerical methods to reliably quantify the movement of single TFs and the interactions in the nucleus of living cells, and applied these methods characterize the dynamic behavior of the tumor suppressor p53 in basal conditions and upon the induction of different kinds of stress.
The interdisciplinary efforts, related to the development of ad-hoc technology and biological materials and to the production of reliable methods to quantify TFs dynamics, have resulted in the following specific results and deliverables.

- We have developed a microscope with single molecule sensitivity and we demonstrated that it can be used for various applications including TIRF (Total Internal Reflection Fluorescence Microscopy), 2D single molecule tracking, 3D single particle tracking and super-resolution localization microscopy imaging. The instrument has been used for the development of the project and is now a technology available to the institute, which will be used for multiple intramural and extramural collaborations.

- We have shown that our microscope together with the developed analysis tools can be used to reliably quantify TFs dynamics, in terms of their diffusion and their binding to DNA. The method has been cross-validated with more conventional ensemble average techniques, such as FRAP (Fluorescence Recovery After Photobleaching) and FCS (Fluorescence Correlation Spectroscopy).

- We have characterized the basal microscopical dynamics of p53. The TF binds only transiently to DNA (on a timescale of seconds) with less than 20% of the protein being bound at any time.

- We have dissected the mechanism used by p53 to identify the specific sites on DNA. Mutation analyisis supports the hypothesis that p53 finds its targets by first contacting the DNA non-specifically viia its C-terminal tail, in agreement with a sliding model for p53 search.

- We have demonstrated that p53 modulates its affinity to DNA and its search mechanism following DNA damage and that different sources of genotoxic stress can induce differential modulations of p53 affinity. This result represent a first demonstration of the “latency” hypothesis for p53 in living cells: our final aim will be to show that the modulation of the binding affinity of the TF can determine the specific regulation of target genes, which can contribute explaining how the dynamic behavior of p53 can control the cellular fate.

Single molecule approaches are predicted to become more and more widespread in the following years: for the moment, however, these approaches are restricted to a limited group of specialized scientists with technical background: only the development of interdisciplinary projects will allow single molecule methods to become widespread. We believe that the capability of integrating quantitative approaches such as those described here in the research lines of important biomedical centers like the San Raffaele University will boost European competitiveness in research: in this project, our effort has been to develop a technology that would be useful for the whole institute, while inducing the bidirectional transfer of knowledge, by participating to the institution seminars, by training students and teaching in Ph.D. classes and schools. The collaborative environment of San Raffaele has allowed to receive constant feedback on our research and to establish intramural collaborations that will be the basis of my future work here at San Raffaele. While developing important knowledge and tools for my future research and for the institute, the project has produced results that shed light on the microscopical dynamic behavior of p53. We believe that this information will be valuable in the future to better understand how to control the cellular responses to anti-cancer therapies.