No abstract

Little is known about how chromatin folds in its native state. Using optimized in situ hybridization and live imaging techniques have determined compaction ratios and fiber flexibility for interphase chromatin in budding yeast. Unlike previous studies, ours examines nonrepetitive chromatin at intervals short enough to be meaningful for yeast chromosomes and functional domains in higher eukaryotes. We reconcile high-resolution fluorescence in situ hybridization data from intervals of 14 -100 kb along single chromatids with measurements of whole chromosome arms (122-623 kb in length), monitored in intact cells through the targeted binding of bacterial repressors fused to GFP derivatives. The results are interpreted with a flexible polymer model and suggest that interphase chromatin exists in a compact higher-order conformation with a persistence length of 170 -220 nm and a mass density of Ϸ110 -150 bp͞nm. These values are equivalent to 7-10 nucleosomes per 11-nm turn within a 30-nm-like fiber structure. Comparison of long and short chromatid arm measurements demonstrates that chromatin fiber extension is also influenced by nuclear geometry. The observation of this surprisingly compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests that the passage of RNA polymerase II requires a very transient unfolding of higher-order chromatin structure.higher-order structure ͉ 30-nm fiber ͉ nucleosomes G enetic studies indicate that the spatial positioning of the genome in interphase contributes to the regulation of nuclear functions, yet the principles that govern the organization of interphase chromosomes (Chrs) are largely unknown. At the simplest level, DNA is folded through interaction with histones forming the nucleosome core particle (NCP), which yields a 6:1 or 7:1 compaction ratio depending on linker length. Arrays of nucleosomes are further condensed by Ϸ6-fold into a higherorder structure, the so-called 30-nm fiber, whose in vivo architecture is unresolved. Several models have been proposed for this structure (ref. 1 and reviewed in ref. 2), yet species' specific variation in linker histones and nucleosome repeat lengths may lead to variation in fiber characteristics. How interphase Chrs fold beyond this level of organization is even less well understood, although in addition to fiber compaction, local looping and͞or anchoring to subnuclear elements may influence chromatin conformation (3, 4).To analyze chromatin compaction ratios in interphase nuclei, laboratories have generally applied fluorescence in situ hybridization (FISH), using differentially derivatized probes. These data were interpreted as identifying mega bp-sized loops (averaging 3,000 kb) with the bases of the loops being distributed in a random walk throughout the nucleoplasm (5-7) or as a chain of chromosomal subcompartments each comprising Ϸ10-20 loops of Ϸ120 kb (8). The random distribution of the chain may reflect local chromatin dynamics, which have been recently well documented in living cells by rapi...

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.

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.

Bulk chromatin motion has not been analyzed at high resolution. We present HiD , a method to quantitatively map dynamics of chromatin and abundant nuclear proteins for every pixel simultaneously over the entire nucleus from fluorescence image series. HiD 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-μm domains in a mosaic-like pattern, uncoupled from chromatin compaction. This pattern was remodeled in response to transcriptional activity. HiD can be applied to any dense and bulk structures opening new perspectives towards understanding motion of nuclear molecules.

Chromosome breakage is a major threat to genome integrity. The most accurate way to repair DNA double strand breaks (DSB) is homologous recombination (HR) with an intact copy of the broken locus. Mobility of the broken DNA has been seen to increase during the search for a donor copy. Observing chromosome dynamics during the earlier steps of HR, mainly the resection from DSB ends that generates recombinogenic single strands, requires a visualization system that does not interfere with the process, and is small relative to the few kilobases of DNA that undergo processing. Current visualization tools, based on binding of fluorescent repressor proteins to arrays of specific binding sites, have the major drawback that highly-repeated DNA and lengthy stretches of strongly bound protein can obstruct chromatin function. We have developed a new, non-intrusive method which uses protein oligomerization rather than operator multiplicity to form visible foci. By applying it to HO cleavage of the MAT locus on Saccharomyces cerevisiae chromosome III, we provide the first real-time analysis of resection in single living cells. Monitoring the dynamics of a chromatin locus next to a DSB revealed transient confinement of the damaged chromatin region during the very early steps of resection, consistent with the need to keep DNA ends in contact. Resection in a yku70 mutant began ∼10 min earlier than in wild type, defining this as the period of commitment to homology-dependent repair. Beyond the insights into the dynamics and mechanism of resection, our new DNA-labelling and -targeting method will be widely applicable to fine-scale analysis of genome organization, dynamics and function in normal and pathological contexts.

SummaryA key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response—in the absence of any amplification step—remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or “assisted” loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down.

Intrinsic dynamics of chromatin contribute to gene regulation. How chromatin mobility responds to genomic processes, and whether this response relies on coordinated chromatin 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 reconstructs dense global flow fields of fluorescent images acquired in real-time. 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 μm 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 s points to clear-cut boundaries between distinct regions, while smooth transitions in small (<1 μm) neighborhoods dominate for short time intervals. Rough 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 nuclear activity.