Aurélien Bancaud

Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin

The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin‐interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target‐search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.

High-throughput chromatin motion tracking in living yeast reveals the flexibility of the fiber throughout the genome

Chromosome dynamics are recognized to be intimately linked to genomic transactions, yet the physical principles governing spatial fluctuations of chromatin are still a matter of debate. Using high-throughput single-particle tracking, we recorded the movements of nine fluorescently labeled chromosome loci located on chromosomes III, IV, XII, and XIV of Saccharomyces cerevisiae over an extended temporal range spanning more than four orders of magnitude (10(-2)-10(3) sec). Spatial fluctuations appear to be characterized by an anomalous diffusive behavior, which is homogeneous in the time domain, for all sites analyzed. We show that this response is consistent with the Rouse polymer model, and we confirm the relevance of the model with Brownian dynamics simulations and the analysis of the statistical properties of the trajectories. Moreover, the analysis of the amplitude of fluctuations by the Rouse model shows that yeast chromatin is highly flexible, its persistence length being qualitatively estimated to <30 nm. Finally, we show that the Rouse model is also relevant to analyze chromosome motion in mutant cells depleted of proteins that bind to or assemble chromatin, and suggest that it provides a consistent framework to study chromatin dynamics. We discuss the implications of our findings for yeast genome architecture and for target search mechanisms in the nucleus.

A fractal model for nuclear organization: current evidence and biological implications

Chromatin is a multiscale structure on which transcription, replication, recombination and repair of the genome occur. To fully understand any of these processes at the molecular level under physiological conditions, a clear picture of the polymorphic and dynamic organization of chromatin in the eukaryotic nucleus is required. Recent studies indicate that a fractal model of chromatin architecture is consistent with both the reaction-diffusion properties of chromatin interacting proteins and with structural data on chromatin interminglement. In this study, we provide a critical overview of the experimental evidence that support a fractal organization of chromatin. On this basis, we discuss the functional implications of a fractal chromatin model for biological processes and propose future experiments to probe chromatin organization further that should allow to strongly support or invalidate the fractal hypothesis.

Fluorescence perturbation techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, photoactivation, photoconversion, and FLIP.

The technique of fluorescence recovery after photobleaching (FRAP) was introduced in the mid-1970s to study the diffusion of biomolecules in living cells. For several years, it was used mainly by a small number of biophysicists who had developed their own photobleaching systems. Since the mid-1990s, FRAP has gained increasing popularity because of the conjunction of two factors: First, photobleaching techniques are easily implemented on confocal laser-scanning microscopes (CLSMs), and so FRAP has become available to anyone who has access to such equipment. Second, the advent of green fluorescent protein (GFP) has allowed easy fluorescent tagging of proteins and their observation in living cells. Thanks both to the versatility of modern CLSMs, which allow control of laser intensity at any point of the image, and to the development of new fluorescent probes, additional photoperturbation techniques have emerged during the last few years. After the photoperturbation event, one observes and then analyzes how the fluorescence distribution relaxes toward the steady state. Because the photochemical perturbation of suitable fluorophores is essentially irreversible, changes of fluorescence intensity in the perturbed and unperturbed regions are due to the exchange of tagged molecules between those regions. This article first discusses the materials required for performing FRAP experiments on a CLSM and the software for data analysis. It then describes general considerations on how to perform FRAP experiments as well as the necessary controls. Finally, different possible ways to analyze the data are presented.

A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells.

In mammals, silencing of one of the two X chromosomes in female cells provides dosage compensation between the sexes. The noncoding Xist RNA localizes over the inactive X chromosome and initiates gene silencing. Visualization of Xist in living cells is used to study the mechanism of localization and measure the dynamics of chromosome-bound Xist.

Right-Handed Nucleosome: Myth or Reality?

In their recent paper in Cell, Furuyama and Henikoff (2009) report that nucleosomes in centromeres may be right-handed, that is, they wrap DNA in a right-handed manner and induce positive supercoils. This raises intriguing new questions, such as how centromeric histone variants may be assembled into right-handed particles, and why chromatin would retain negative supercoiling in chromosome arms but positive supercoiling in centromeres. We wish to comment on these new findings in the context of topological insights that we have gained from recent in vitro experiments with centromeric nucleosomes and single chromatin fibers submitted to torsional constraints and from 3D modeling of chromatin dynamics.

Relevance and limitations of crowding, fractal, and polymer models to describe nuclear architecture.

Chromosome architecture plays an essential role for all nuclear functions, and its physical description has attracted considerable interest over the last few years among the biophysics community. These researches at the frontiers of physics and biology have been stimulated by the demand for quantitative analysis of molecular biology experiments, which provide comprehensive data on chromosome folding, or of live cell imaging experiments that enable researchers to visualize selected chromosome loci in living or fixed cells. In this review our goal is to survey several nonmutually exclusive models that have emerged to describe the folding of DNA in the nucleus, the dynamics of proteins in the nucleoplasm, or the movements of chromosome loci. We focus on three classes of models, namely molecular crowding, fractal, and polymer models, draw comparisons, and discuss their merits and limitations in the context of chromosome structure and dynamics, or nuclear protein navigation in the nucleoplasm. Finally, we identify future challenges in the roadmap to a unified model of the nuclear environment.

Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement

Genome dynamics are intimately linked to the regulation of gene expression, the most fundamental mechanism in biology, yet we still do not know whether the very process of transcription drives spatial organization at specific gene loci. Here, we have optimized the ANCHOR/ParB DNA-labeling system for real-time imaging of a single-copy, estrogen-inducible transgene in human cells. Motion of an ANCHOR3-tagged DNA locus was recorded in the same cell before and during the appearance of nascent MS2-labeled mRNA. We found that transcription initiation by RNA polymerase 2 resulted in confinement of the mRNA-producing gene domain within minutes. Transcription-induced confinement occurred in each single cell independently of initial, highly heterogeneous mobility. Constrained mobility was maintained even when inhibiting polymerase elongation. Chromatin motion at constant step size within a largely confined area hence leads to increased collisions that are compatible with the formation of gene-specific chromatin domains, and reflect the assembly of functional protein hubs and DNA processing during the rate-limiting steps of transcription.

Analysis of DNA Replication by Optical Mapping in Nanochannels.

DNA replication is essential to maintain genome integrity in S phase of the cell division cycle. Accumulation of stalled replication forks is a major source of genetic instability, and likely constitutes a key driver of tumorigenesis. The mechanisms of regulation of replication fork progression have therefore been extensively investigated, in particular with DNA combing, an optical mapping technique that allows the stretching of single molecules and the mapping of active region for DNA synthesis by fluorescence microscopy. DNA linearization in nanochannels has been successfully used to probe genomic information patterns along single chromosomes, and has been proposed to be a competitive alternative to DNA combing. Yet this conjecture remains to be confirmed experimentally. Here, two complementary techniques are established to detect the genomic distribution of tracks of newly synthesized DNA in human cells by optical mapping in nanochannels. Their respective advantages and limitations are compared, and applied them to detect deregulations of the replication program induced by the antitumor drug hydroxyurea. The developments here thus broaden the field of applications accessible to nanofluidic technologies, and can be used in the future as part for molecular diagnostics in the context of high throughput cancer drug screening.

BIABooster: Online DNA Concentration and Size Profiling with a Limit of Detection of 10 fg/μL and Application to High-Sensitivity Characterization of Circulating Cell-Free DNA

We describe a technology to perform sizing and concentration analysis of double stranded DNA with a sensitivity of 10 fg/μL in an operating time of 20 min. The technology is operated automatically on a commercial capillary electrophoresis instrument using electro-hydrodynamic actuation. It relies on a new capillary device that achieves online concentration of DNA at the junction between two capillaries of different diameters, thanks to viscoelastic lift forces. Using a set of DNA ladders in the range of 100-1500 bp, we report a sizing accuracy and precision better than 3% and a concentration quantification precision of ∼20%. When the technology is applied to the analysis of clinical samples of circulating cell-free DNA (cfDNA), the measured cfDNA concentrations are in good correlation with those measured by digital PCR. Furthermore, the cfDNA size profiles indicate that the fraction of low molecular weight cfDNA in the range of 75-240 bp is a candidate biomarker to discriminate between healthy subjects and cancer patients. We conclude that our technology is efficient in analyzing highly diluted DNA samples and suggest that it will be helpful in translational and clinical research involving cfDNA.