Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus

Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time

Une approche quantitative de la microscopie en molécule unique dans le noyau des cellules

The cell nucleus is a chemical reactor. Nuclear components interact with each other to express genes, duplicate the chromosomes for cell division, and protect DNA from alteration. These reactions are regulated along the cell cycle and in response to stress. One of the fundamental nuclear processes, transcription, enables the production of a messenger RNA from a template DNA sequence. While mandatory for the cell, transcription nevertheless may involve a very small number of molecules. Indeed, a single gene would have only few copies in the genome. During my PhD, I studied nuclear processes in human cells nuclei at the single molecule level with novel imaging techniques. I developed new statistical tools to quantify nuclear components movement that revealed a dynamic nuclear architecture. Since the 90s, simple methods have been developed for the observation of single molecules in the cell. These experiments can be conducted in an ordinary inverted microscope. We used these methods to monitor nuclear molecules called transcription factors (TF) that regulate transcription. From TF dynamics, we concluded that nuclear exploration by transcription factors is regulated by their chemical interactions with partners. The organization of the components of the nucleus guide transcription factors in their search of a gene. As an example of this organization, we then studied chromatin, the de-condensed form of nuclear DNA, proving that it displays the characteristics of a self-organized fractal structure. This structure changes in response to cellular fate and stress. In yeast, we showed that the interminglement of chromatin constrained DNA locus movement in a reptation regime. All these results show the interdependence of the structure of the nucleus and of its chemical reactions. With combination of realistic modeling and high resolution microscopy, we have enlightened the specificity of the nucleus as a chemical reactor. This thesis has also enabled the development of accurate methods for the statistical analysis of single molecule data.Le noyau cellulaire est le siège de réactions chimiques dont le but est l’expression de gènes, la duplication du génome et du maintien et l’intégrité de l’information génétique. Ces réactions sont régulées au cours du cycle cellulaire ou en réponse à un stress. Parmi elles, la transcription permet qu’une séquence d’ADN soit reproduite sous forme d’ARN messager. La transcription est un exemple frappant de processus fondamental pour la cellule impliquant parfois un nombre très faible de molécules. En effet, il n’y a souvent dans un même génome que quelques copies d’un même gène. Le but de cette thèse est d’imager les processus nucléaires dans des cellules humaines à l’échelle de la molécule unique et d’en extraire les grandeurs caractéristiques. Depuis les années 90, des inventeurs de génie ont développé des méthodes simples à partir de microscopes inversés ordinaires pour observer des molécules individuelles jusque dans le noyau des cellules. Nous avons utilisé ces méthodes pour suivre des facteurs de transcription qui régulent la transcription d’un gène. Nos mesures montrent que, bien que hiératique, l’exploration du noyau par les facteurs de transcriptions est régulée par leurs propriétés chimiques. L’agencement des composants du noyau guide les facteurs de transcription dans la recherche d’un gène. Comme exemple de cet agencement, nous nous sommes ensuite intéressés à l’organisation de l’ADN dans le noyau pour montrer qu’elle présentait les caractéristiques d’une structure auto-organisée, une structure fractale. Cette structure change en réponse aux aléas de la vie de la cellule. Dans une dernière étude, nous avons suivi un locus dans le noyau d’une levure. La structure du noyau, qui est révélée par notre méthode, contraint la diffusion du locus à un régime de reptation. Tous ces résultats montrent combien la structure du noyau et les réactions chimiques qui y ont lieu sont interdépendantes. Cette thèse a également permis le développement de méthodes de quantification précises des réactions cellulaires à l’échelle de la molécule unique

Single cell correlation fractal dimension of chromatin: a framework to interpret 3D single molecule super-resolution.

Chromatin is a major nuclear component, and it is an active matter of debate to understand its different levels of spatial organization, as well as its implication in gene regulation. Measurements of nuclear chromatin compaction were recently used to understand how DNA is folded inside the nucleus and to detect cellular dysfunctions such as cancer. Super-resolution imaging opens new possibilities to measure chromatin organization in situ. Here, we performed a direct measure of chromatin compaction at the single cell level. We used histone H2B, one of the 4 core histone proteins forming the nucleosome, as a chromatin density marker. Using photoactivation localization microscopy (PALM) and adaptive optics, we measured the three-dimensional distribution of H2B with nanometric resolution. We computed the distribution of distances between every two points of the chromatin structure, namely the Ripley K(r) distribution. We found that the K(r) distribution of H2B followed a power law, leading to a precise measurement of the correlation fractal dimension of chromatin of 2.7. Moreover, using photoactivable GFP fused to H2B, we observed dynamic evolution of chromatin sub-regions compaction. As a result, the correlation fractal dimension of chromatin reported here can be interpreted as a dynamically maintained non-equilibrium state

Dynamics of Nuclear Protein Exploration Revealed by Intracellular Single Particle Tracking PALM

Cellular regulation of eukaryotic cells involves molecular interactions of factors diffusing within the cellular volume. Understanding the gene expression regulation requires thus elucidating the spatio-temporal dynamics of intranuclear proteins at the single molecule level. However, live cell imaging of single molecules in eukaryotic cells has remained mostly restricted to processes occurring in the plasma membrane, readily accessible by TIRF microscopy as opposed to intra-nuclear processes. We report an intracellular single particle tracking method using photoactivated localization microscopy that enables the study of protein dynamics inside live eukaryotic cells. So far single particle tracking PALM (sptPALM) (Manley et al, 2008) has been restricted to cellular systems for which imaging can be performed using total internal reflection microscopy (TIRF), and believed to be limited to slow diffusing systems (∼ 0.1 um2/s). Here we demonstrate an approach that reduces the background of out-of-focus fluorophores by a tight control of the photoactivation, thus allowing the detection and characterization of single protein dynamics directly in the nucleus of living cells. Applying this method to several nuclear proteins, we captured a wide range of diffusive behaviors from very rapid diffusion (> 10 μm2/s) to bound chromatin associated states (< 0.1 μm2/s). We measured the single molecule dynamics for a diverse set of proteins, from free fluorophores (Dendra2) with no known interactions in the nucleoplasm, to DNA binding (c-Myc), RNA binding (Fibrillarin), and protein-protein interacting complexes (p-TEFb). We observe that, overall, nuclear exploration is not governed by a unique nucleoplasmic geometry but rather a protein-specific variable. Our approach provides a versatile tool for single molecule in vivo studies in eukaryotes