Compartmentalization of Interphase Chromosomes Observed in Simulation and Experiment

141

No methods proposed thus far have the capability to measure overall molecular flow in the nucleus of living cells. Here, we apply the pair correlation function analysis (pCF) to measure molecular anisotropic diffusion in the interphase nucleus of live cells. In the pCF method, we cross-correlate fluctuations at several distances and locations within the nucleus, enabling us to define migration paths and barriers to diffusion. We use monomeric EGFP as a prototypical inert molecule and measure flow in and between different nuclear environments. Our results suggest that there are two disconnect molecular flows throughout the nucleus associated with high and low DNA density regions. We show that different density regions of DNA form a networked channel that allows EGFP to diffuse freely throughout, however with restricted ability to traverse the channel. We also observe rare and sudden bursts of molecules traveling across DNA density regions with characteristic time of ≈300 ms, suggesting intrinsic localized change in chromatin structure. This is a unique in vivo demonstration of the intricate chromatin network showing channel directed diffusion of an inert molecule with high spatial and temporal resolution.chromatin organization | fluctuation spectroscopy | nucleus structure N uclear architecture is fundamental to the manner in which molecules traverse the nucleus (1). The cell nucleus is a functionally and spatially structured organelle (2) in which diffusion is the mode of motion for inert molecules (3-5). The diffusion of molecules within the nucleus is obstructed by the steric constraints imposed by structural components, such as chromatin (6, 7). Proposed models for 3D arrangement of chromatin vary from defined regions of chromatin compartmentalisation (8) to intermingled chromatin fibers and loops (9). The requirement for biologically significant molecules to reach different destinations within the cell nucleus raises the question, how is the diffusive route directed? Given that diffusion cannot be regulated because it is essentially a default mechanism of motion (10), it has been postulated that structural features of the nucleus must impart retention at particular sites and control flux of movement between compartments (1, 11).Insights into intranuclear trafficking are predominantly derived from measurement of the accessibility of the nuclear landscape and the effect it has on diffusion of biologically active and inert molecules. Current approaches commonly used for such investigations are fluorescence recovery after photo bleaching (FRAP) (12, 13), single particle tracking (SPT) (14), and fluorescence correlation spectroscopy (FCS) (15, 16). FRAP, however, is invasive because it requires high illumination power on the sample to induce photobleaching, and SPT requires the observation of isolated particles for a long time, which yields poor statistics. FCS, in contrast, provides information at the single molecule level with good statistics by averaging the behavior of many molecules (10), and this technique ...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

In vivo pair correlation analysis of EGFP intranuclear diffusion reveals DNA-dependent molecular flow

Hinde

Cardarelli

Digman

et al. 2010

141

“…Thus, although H2AX represents 2.4% of the total H2A in HeLa cells (3), the fractional amount of H2AX within a cluster would be significantly higher. These observations could be accommodated by higher-order chromatin structures proposed to contain on the order of 1-2 Mbp DNA in which chromatin loops emanate from a central region (27,28) where H2AX would be concentrated. We propose that the H2AX clusters observed here by using 4Pi microscopy may correspond to the central region of these higher-order structures.…”

Section: Discussionmentioning

confidence: 99%

H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy

Bewersdorf

Bennett

Knight

2006

115

DNA double-strand breaks (DSBs) caused by cellular exposure to genotoxic agents or produced by inherent metabolic processes initiate a rapid and highly coordinated series of molecular events resulting in DNA damage signaling and repair. Phosphorylation of histone H2AX to form ␥-H2AX is one of the earliest of these events and is important for coordination of signaling and repair activities. An intriguing aspect of H2AX phosphorylation is that ␥-H2AX spreads a limited distance up to 1-2 Mbp from the site of a DNA break in mammalian cells. However, neither the distribution of H2AX throughout the genome nor the mechanism that defines the boundary of ␥-H2AX spreading have yet been described. Here, we report the identification of previously undescribed H2AX chromatin structures by successfully applying 4Pi microscopy to visualize endogenous nuclear proteins. Our observations suggest that H2AX is not distributed randomly throughout bulk chromatin, rather it exists in distinct clusters that themselves are uniformly distributed within the nuclear volume. These data support a model in which the size and distribution of H2AX clusters define the boundaries of ␥-H2AX spreading and also may provide a platform for the immediate and robust response observed after DNA damage.␥-H2AX ͉ H2AX chromatin clusters ͉ super-resolution 4Pi microscopy ͉ chromatin response to DNA damage ͉ 3D quantification of chromatin structures M aintenance of genome integrity is critical for organism development and survival, and higher organisms have evolved sophisticated mechanisms for detection and repair of chromosome breaks. DNA damage results in the rapid and coordinated action of various pathways, including activation of cell cycle checkpoints (1, 2), histone modification near the site of the break (3, 4), and recruitment of chromatin remodeling enzymes (4-6), cohesins (7-9), and DNA repair proteins (1, 2, 10). The significance of these molecular processes is highlighted by the fact that defects in many are associated with an increased risk of cancer and developmental and immunologic abnormalities (1). Important insights into the positioning of nuclear signaling and repair proteins and their response to various types and levels of genomic insults have been achieved by using immunofluorescence methods (10-12). However, it has been impossible to distinguish f luorescent signals in a threedimensional (3D) environment that are closer together than 500-800 nm in distance given the depth resolution limits of current light microscopes. With Ϸ100-nm resolution along the optic axis (z axis), 4Pi microscopy (13, 14) provides a significant increase in resolution and has allowed more defined images of cellular structures such as microtubules, mitochondria, or the Golgi apparatus (15). However, until now, imaging of endogenous nuclear proteins had not been achieved (a comparison of 4Pi vs. confocal is described in Materials and Methods). In this study, we describe the successful use of 4Pi microscopy to visualize endogenous nuclear proteins. By applying previo...

“…Previous polymer models that consider the effects of both specified chromatin loops at fixed lengths of 120 kb and 3 Mb (12,36), and the possibility of chromatin loops of all sizes (37), also produce constant K values. Furthermore, although these models explain the plateau of the mean-square spatial distance, R 2 ðsÞ, at large s, they do not reproduce the behavior of the contact probability, P c ðsÞ, observed in Hi-C data, as does the SBS model.…”

Section: Comparison Of the Sbs And Other Models Against Experimental mentioning

confidence: 99%

“…Imaging of single loci by FISH and genome-wide mapping of chromatin interactions by chromosome conformation capture (3C) approaches revealed a variety of chromatin architectures across genomic regions and cell types, and upon environmental cues (9)(10)(11)(12)(13)(14) (Fig. S1A).…”

mentioning

confidence: 99%

Complexity of chromatin folding is captured by the strings and binders switch model

Barbieri

Chotalia

Fraser

et al. 2012

552

688

Chromatin has a complex spatial organization in the cell nucleus that serves vital functional purposes. A variety of chromatin folding conformations has been detected by single-cell imaging and chromosome conformation capture-based approaches. However, a unified quantitative framework describing spatial chromatin organization is still lacking. Here, we explore the "strings and binders switch" model to explain the origin and variety of chromatin behaviors that coexist and dynamically change within living cells. This simple polymer model recapitulates the scaling properties of chromatin folding reported experimentally in different cellular systems, the fractal state of chromatin, the processes of domain formation, and looping out. Additionally, the strings and binders switch model reproduces the recently proposed "fractal-globule" model, but only as one of many possible transient conformations.genome organization | genome architecture | long-range chromatin interactions | fluorescence in situ hybridization | Monte Carlo simulations U nderstanding the interplay between genome architecture and gene regulation is one of the most challenging problems in biology. During mitosis, chromosomes are found in a condensed state, but decondense during interphase, when highly coordinated cellular processes such as transcription, DNA repair, and replication take place, creating cell-type-specific chromatin folding (1-3).Chromosome organization occurs at different scales of genomic length to yield variable degrees of compaction (4). Linear nucleosome arrays fold into higher-order structures, first through local chromatin interactions, such as between promoters and enhancers, and then eventually giving rise to discrete chromosome territories (1).Spatial genome organization is guided by intra-and interchromosomal interactions mediated by nuclear components that include transcription factors, transcription and replication factories, Polycomb bodies, and contacts with the lamina (5-8). However, how binding of diffusible factors to specific genomic regions drives chromatin folding remains poorly understood.Imaging of single loci by FISH and genome-wide mapping of chromatin interactions by chromosome conformation capture (3C) approaches revealed a variety of chromatin architectures across genomic regions and cell types, and upon environmental cues (9-14) (Fig. S1A). In FISH experiments, chromatin folding is often measured by the mean-square spatial distance, R 2 ðsÞ, between two genomic regions as a function of their linear genomic distance, s (Fig. S1B), which usually exhibits scaling properties R 2 ðsÞ ∼ s 2v . Although the behavior of R 2 ðsÞ appears to depend on the genomic regions and cell types assessed (Fig. S1A), in general, at large genomic distances, R 2 ðsÞ reaches a plateau (i.e., v ¼ 0) that reflects the folding of chromosomes into territories (15).A global analysis of genome-wide 3C (Hi-C) ligation products in human cells averaged across all chromosomes has been used to estimate the "contact probability," P c ðsÞ (13). Th...