Biology subjects

Bystricky, K.

Publications and source records attributed to Bystricky, K..

6 recordsLinked to original sources

bioRxiv2018

Nanoscale mapping of DNA dynamics reveals activity-driven genome organization in living human cells

Bulk chromatin motion has not been analysed at high resolution. We present Hi-D, a method to quantitatively map dynamics of chromatin and abundant nuclear proteins for every pixel simultaneously over the entire nucleus from fluorescence image series. Hi-D combines reconstruction of chromatin motion, and classification of local diffusion processes by Bayesian inference. We show that DNA dynamics in the nuclear interior are spatially partitioned into 0.3 - 3 m domains in a mosaic-like pattern, uncoupled from chromatin compaction. This pattern was remodelled in response to transcriptional activity. Hi-D can be applied to any dense and bulk structures opening new perspectives towards understanding motion of nuclear molecules.

biophysics↗

bioRxiv2018

Real-time visualization and quantification of human Cytomegalovirus replication in living cells using the ANCHOR DNA labeling technology.

Human cytomegalovirus (HCMV) induces latent life-long infections in all human populations. Depending on geographic area and socio-economic conditions between 30 to nearly 100% of individuals are affected. The biology of this virus is difficult to explore due to its extreme sophistication and the lack of pertinent animal model. Here we present the first application of the ANCHOR DNA labeling system to a herpes virus, allowing real time imaging and direct monitoring of HCMV infection and replication in human living cells. The ANCHOR system is composed of a protein (OR) which specifically binds to a short, non-repetitive DNA target sequence (ANCH) and spreads onto neighboring sequences due to protein oligomerization. If OR protein is fused to GFP, this accumulation results in a site specific fluorescent focus. We have created a recombinant ANCHOR-HCMV harboring an ANCH target sequence and the gene encoding the cognate OR-GFP fusion protein. Infection of permissive cells with ANCHOR-HCMV enables visualization of the nearly complete viral cycle until cell fragmentation and death. Quantitative analysis of infection kinetics and of viral DNA replication revealed cell-type specific behavior of HCMV and sensitivity to inhibitors. Our results show that the ANCHOR technology is a very efficient tool for the study of complex DNA viruses and new highly promising biotechnology applications.\n\nIMPORTANCEThe ANCHOR technology is to date the most powerful tool to follow and quantify the replication of HCMV in living cells and to gain new insights into its biology. This technology is applicable to virtually any DNA virus or virus presenting a dsDNA phase, paving the way to infection imaging in various cell lines or even in animal models and opening fascinating fundamental and applied prospects. Associated to high content automated microscopy, this technology permitted rapid, robust and precise determination of Ganciclovir IC50 and IC90 on HCMV replication, with minimal hands-on investment. To search for new antiviral activities, the experiment is easy to up-grade towards efficient and cost-effective screening of large chemical libraries. The simple infection of permissive cells with ANCHOR-viruses in the presence of a compound of interest may even provide a first estimation about the stage of the viral cycle this molecule is acting upon.

microbiology↗

bioRxiv2017

Formation of correlated chromatin domains at nanoscale dynamic resolution during transcription

Intrinsic dynamics of chromatin contribute to gene regulation. How chromatin mobility responds to genomic processes and whether this response relies on coordinated movement is still unclear. Here, we introduce an approach called Dense Flow reConstruction and Correlation (DFCC) to quantify correlation of chromatin motion with sub-pixel sensitivity at the level of the whole nucleus. DFCC is based on reconstructing dense global flow fields of fluorescent images acquired in real-time. By simulating variations in microscopic and dynamic parameters, we demonstrate that our approach is robust and more accurate than other methods to estimate flow fields and spatial correlations of dense structures such as chromatin. We applied our approach to analyze stochastic movements of DNA and histones based on direction and magnitude at different time lags in human cells. We observe long-range correlations extending over several m between coherently moving regions over the entire nucleus. Spatial correlation of global chromatin dynamics was reduced by inhibiting elongation by RNA polymerase II and abolished in quiescent cells. Furthermore, quantification of spatial smoothness over time intervals up to 30 seconds points to clear-cut boundaries between distinct regions, while smooth transitions in small (<1 m) neighborhoods dominate for short time intervals. Clear transitions between regions of coherent motion indicate directed squeezing or stretching of chromatin boundaries suggestive of changes in local concentrations of actors regulating gene expression. The DFCC approach hence allows characterizing stochastically forming domains of specific nuclear activity.\n\nSignificance StatementControl of gene expression relies on modifications of chromatin structure and activity of the transcription machinery. However, how chromatin responds dynamically to this genomic process and whether this response is coordinated in space is still unclear. We introduce a novel approach called Dense Flow reConstruction and Correlation (DFCC) to characterize spatially correlated dynamics of chromatin in living cells at nanoscale resolution. DFCC allows us to detect chromatin domains in living cells with long range correlations over the entire nucleus. Furthermore, transitions between domains can be quantified by the newly introduced smoothness parameter of local chromatin motion. The DFCC approach permits characterizing stochastically forming domains of other DNA dependent activity in any cell type in real time imaging.

biophysics↗

bioRxiv2017

In vivo, chromatin is a fluctuating polymer chain at equilibrium constrained by internal friction

Chromosome mechanical properties determine DNA folding and dynamics, and underlie all major nuclear functions. Here we combine modeling and real-time motion tracking experiments to infer the physical parameters describing chromatin fibers. In vitro, motion of nucleosome arrays can be accurately modeled by assuming a Kuhn length of 35-55 nm. In vivo, the amplitude of chromosome fluctuations is drastically reduced, and depends on transcription. Transcription activation increases chromatin dynamics only if it involves gene relocalization, while global transcriptional inhibition augments the fluctuations, yet without relocalization. Chromatin fiber motion is accounted for by a model of equilibrium fluctuations of a polymer chain, in which random contacts along the chromosome contour induce an excess of internal friction. Simulations that reproduce chromosome conformation capture and imaging data corroborate this hypothesis. This model unravels the transient nature of chromosome contacts, characterized by a life time of [~]2 seconds and a free energy of formation of [~]1 kBT.

genetics↗

bioRxiv2017

Heterogeneous chromatin mobility derived from chromatin states is a determinant of genome organisation in S. cerevisiae

Spatial organisation of the genome is essential for regulating gene activity, yet the mechanisms that shape this three-dimensional organisation in eukaryotes are far from understood. Here, we combine bioinformatic determination of chromatin states during normal growth and heat shock, and computational polymer modelling of genome structure, with quantitative microscopy and Hi-C to demonstrate that differential mobility of yeast chromosome segments leads to spatial self-organisation of the genome. We observe that more than forty percent of chromatin-associated proteins display a poised and heterogeneous distribution along the chromosome, creating a heteropolymer. This distribution changes upon heat shock in a concerted, state-specific manner. Simulating yeast chromosomes as heteropolymers, in which the mobility of each segment depends on its cumulative protein occupancy, results in functionally relevant structures, which match our experimental data. This thermodynamically driven self-organisation achieves spatial clustering of poised genes and mechanistically contributes to the directed relocalisation of active genes to the nuclear periphery upon heat shock.\n\nOne Sentence SummaryUnequal protein occupancy and chromosome segment mobility drive 3D organisation of the genome.

systems biology↗

bioRxiv2017

Real-time chromatin dynamics at the single gene levelduring transcription activation

Genome dynamics relate to regulation of gene expression, the most fundamental process in biology. Yet we still do not know whether the very process of transcription drives spatial organization and chromatin conformation at specific gene loci. To address this issue, we have optimized the ANCHOR/ParB DNA labeling system for real-time imaging and quantitative analysis of the dynamics of a single-copy transgene in human cells. Transcription of the transgene under the control of the endogenous Cyclin D1 promoter was induced by addition of 17{beta}-estradiol. Motion of the ANCHOR3-tagged DNA locus was recorded in the same cell prior to and during appearance of nascent mRNA visualized using the MS2 system. We found that transcription initiation resulted in rapid confinement of the mRNA-producing gene. The confinement was maintained even upon inhibition of pol2 elongation. It did not occur when recruitment of pol2 or transcription initiation was blocked by anti-estrogens or Triptolide. These results suggest that preinitiation complex formation and concomitant reorganization of the chromatin domain constrains freedom of movement of an induced genes promoter within minutes. Confined diffusion reflects assembly of functional protein hubs and DNA processing during the rate-limiting steps of transcription.

cell biology↗