Interphase chromatin biophysics and mechanics: new perspectives and open questions.

The physical organization and properties of chromatin within the interphase nucleus are intimately linked to a wide range of functional DNA-based processes. In this context, interphase chromatin mechanics - that is, how chromatin, physically, responds to forces - is gaining increasing attention. Recent methodological advances for probing the force-response of chromatin in cellulo open new avenues for research, as well as new questions.

Sulfobetaine-Phosphonate Block Copolymer Coated Iron Oxide Nanoparticles for Genomic Locus Targeting and Magnetic Micromanipulation in the Nucleus of Living Cells.

Exerting forces on biomolecules inside living cells would allow us to probe their dynamic interactions in their native environment. Magnetic iron oxide nanoparticles represent a unique tool capable of pulling on biomolecules with the application of an external magnetic field gradient; however, their use has been restricted to biomolecules accessible from the extracellular medium. Targeting intracellular biomolecules represents an additional challenge due to potential nonspecific interactions with cytoplasmic or nuclear components.

Live-cell micromanipulation of a genomic locus reveals interphase chromatin mechanics.

Our understanding of the physical principles organizing the genome in the nucleus is limited by the lack of tools to directly exert and measure forces on interphase chromosomes in vivo and probe their material nature. Here, we introduce an approach to actively manipulate a genomic locus using controlled magnetic forces inside the nucleus of a living human cell. We observed viscoelastic displacements over micrometers within minutes in response to near-piconewton forces, which are consistent with a Rouse polymer model.

Oncogenic chimeric transcription factors drive tumor-specific transcription, processing, and translation of silent genomic regions.

Many cancers are characterized by gene fusions encoding oncogenic chimeric transcription factors (TFs) such as EWS::FLI1 in Ewing sarcoma (EwS). Here, we find that EWS::FLI1 induces the robust expression of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome. These neogenes (NGs) are virtually undetectable in large collections of normal tissues or non-EwS tumors and can be silenced by CRISPR interference at regulatory EWS::FLI1-bound microsatellites.

Molecular basis of CTCF binding polarity in genome folding.

Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin.

Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription.

Nucleosomes represent a challenge in regard to transcription. Histone eviction enables RNA polymerase II (RNAPII) progression through DNA, but compromises chromatin integrity. Here, we used the SNAP-tag system to distinguish new and old histones and monitor chromatin reassembly coupled to transcription in human cells.

High-throughput single-molecule screen for small-molecule perturbation of splicing and transcription kinetics.

In eukaryotes, mRNA synthesis is catalyzed by RNA polymerase II and involves several distinct steps, including transcript initiation, elongation, cleavage, and transcript release. Splicing of RNA can occur during (co-transcriptional) or after (post-transcriptional) RNA synthesis. Thus, RNA synthesis and processing occurs through the concerted activity of dozens of enzymes, each of which is potentially susceptible to perturbation by small molecules.

Dynamics of chromatin accessibility and long-range interactions in response to glucocorticoid pulsing.

Although physiological steroid levels are often pulsatile (ultradian), the genomic effects of this pulsatility are poorly understood. By utilizing glucocorticoid receptor (GR) signaling as a model system, we uncovered striking spatiotemporal relationships between receptor loading, lifetimes of the DNase I hypersensitivity sites (DHSs), long-range interactions, and gene regulation. We found that hormone-induced DHSs were enriched within ± 50 kb of GR-responsive genes and displayed a broad spectrum of lifetimes upon hormone withdrawal.

Kinetic competition during the transcription cycle results in stochastic RNA processing.

Synthesis of mRNA in eukaryotes involves the coordinated action of many enzymatic processes, including initiation, elongation, splicing, and cleavage. Kinetic competition between these processes has been proposed to determine RNA fate, yet such coupling has never been observed in vivo on single transcripts. In this study, we use dual-color single-molecule RNA imaging in living human cells to construct a complete kinetic profile of transcription and splicing of the β-globin gene.

Eukaryotic transcriptional dynamics: from single molecules to cell populations.

Transcriptional regulation is achieved through combinatorial interactions between regulatory elements in the human genome and a vast range of factors that modulate the recruitment and activity of RNA polymerase. Experimental approaches for studying transcription in vivo now extend from single-molecule techniques to genome-wide measurements. Parallel to these developments is the need for testable quantitative and predictive models for understanding gene regulation.

Quantifying the contribution of chromatin dynamics to stochastic gene expression reveals long, locus-dependent periods between transcriptional bursts.

Background: A number of studies have established that stochasticity in gene expression may play an important role in many biological phenomena. This therefore calls for further investigations to identify the molecular mechanisms at stake, in order to understand and manipulate cell-to-cell variability. In this work, we explored the role played by chromatin dynamics in the regulation of stochastic gene expression in higher eukaryotic cells.

Towards experimental manipulation of stochasticity in gene expression.

For decades, most of molecular biology was driven by the "central dogma" in which the phenotype is defined by the genotype following a fully deterministic point of view. However, during the last 10 years, a wealth of studies has demonstrated that a given genotype can generate multiple phenotypes in identical environmental conditions, mainly because of the inherently probabilistic nature of the transcription process. It has also been shown that cells can tune this variability at the molecular level.

Membrane microdomains emergence through non-homogeneous diffusion.

Background: In the classical view, cell membrane proteins undergo isotropic random motion, that is a 2D Brownian diffusion that should result in an homogeneous distribution of concentration. It is, however, far from the reality: Membrane proteins can assemble into so-called microdomains (sometimes called lipid rafts) which also display a specific lipid composition. We propose a simple mechanism that is able to explain the colocalization of protein and lipid rafts.

Enhanced stimulus encoding capabilities with spectral selectivity in inhibitory circuits by STDP.

The ability to encode and transmit a signal is an essential property that must demonstrate many neuronal circuits in sensory areas in addition to any processing they may provide. It is known that an appropriate level of lateral inhibition, as observed in these areas, can significantly improve the encoding ability of a population of neurons. We show here a homeostatic mechanism by which a spike-timing-dependent plasticity (STDP) rule with a symmetric timing window (swSTDP) spontaneously drives the inhibitory coupling to a level that ensures accurate encoding in response to input signals within a certain frequency range.

On the spontaneous stochastic dynamics of a single gene: complexity of the molecular interplay at the promoter.

Background: Gene promoters can be in various epigenetic states and undergo interactions with many molecules in a highly transient, probabilistic and combinatorial way, resulting in a complex global dynamics as observed experimentally. However, models of stochastic gene expression commonly consider promoter activity as a two-state on/off system. We consider here a model of single-gene stochastic expression that can represent arbitrary prokaryotic or eukaryotic promoters, based on the combinatorial interplay between molecules and epigenetic factors, including energy-dependent remodeling and enzymatic activities.

Large multiprotein structures modeling and simulation: the need for mesoscopic models.

Recent observational techniques based upon confocal microscopy make it possible to observe cells at a scale that has never been probed before: the mesoscopic scale. In the eukaryotic cell nucleus, many objects demonstrating phenomena occurring at this scale, such as nuclear bodies, are current subjects of investigations. But from a modeling perspective, this scale has not been widely explored, and hence there is a lack of suitable models for such studies.

A long-term evolutionary pressure on the amount of noncoding DNA.

A significant part of eukaryotic noncoding DNA is viewed as the passive result of mutational processes, such as the proliferation of mobile elements. However, sequences lacking an immediate utility can nonetheless play a major role in the long-term evolvability of a lineage, for instance by promoting genomic rearrangements. They could thus be subject to an indirect selection.