Complex multi-enhancer contacts captured by genome architecture mapping.

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. Here we report a genome-wide method, genome architecture mapping (GAM), for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify enrichment for specific interactions between active genes and enhancers across very large genomic distances using a mathematical model termed SLICE (statistical inference of co-segregation). GAM also reveals an abundance of three-way contacts across the genome, especially between regions that are highly transcribed or contain super-enhancers, providing a level of insight into genome architecture that, owing to the technical limitations of current technologies, has previously remained unattainable. Furthermore, GAM highlights a role for gene-expression-specific contacts in organizing the genome in mammalian nuclei.

A polymer model explains the complexity of large-scale chromatin folding.

The underlying global organization of chromatin within the cell nucleus has been the focus of intense recent research. Hi-C methods have allowed for the detection of genome-wide chromatin interactions, revealing a complex large-scale organization where chromosomes tend to partition into megabase-sized "topological domains" of local chromatin interactions and intra-chromosomal contacts extends over much longer scales, in a cell-type and chromosome specific manner. Until recently, the distinct chromatin folding properties observed experimentally have been difficult to explain in a single conceptual framework. We reported that a simple polymer-physics model of chromatin, the strings and binders switch (SBS) model, succeeds in describing the full range of chromatin configurations observed in vivo. The SBS model simulates the interactions between randomly diffusing binding molecules and binding sites on a polymer chain. It explains how polymer architectural patterns can be established, how different stable conformations can be produced and how conformational changes can be reliably regulated by simple strategies, such as protein upregulation or epigenetic modifications, via fundamental thermodynamics mechanisms.

A model of the large-scale organization of chromatin.

In the cell nucleus, chromosomes have a complex spatial organization, spanning several length scales, which serves vital functional purposes. It is unknown, however, how their three-dimensional architecture is orchestrated. In the present article, we review the application of a model based on classical polymer physics, the strings and binders switch model, to explain the molecular mechanisms of chromatin self-organization. We explore the scenario where chromatin architecture is shaped and regulated by the interactions of chromosomes with diffusing DNA-binding factors via thermodynamics mechanisms and compare it with available experimental data.

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

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.

CryoFISH: fluorescence in situ hybridization on ultrathin cryosections.

The visualization of cellular structures and components has become an invaluable tool in biological and medical sciences. Imaging subcellular compartments and single molecules within a cell has prompted the development of a wide range of sample preparation techniques as well as various microscope devices to obtain images with increased spatial resolution. Here, we present cryoFISH, a method for fluorescence in situ hybridization (FISH) on thin ( approximately 150 nm thick) cryosections from sucrose-embedded fixed cells or tissues. CryoFISH can be used in combination with immunodetection (IF) of other cellular components. The main advantages of cryoFISH and cryoIF over whole-cell labeling methods are increased spatial resolution with confocal microscopy, greater sensitivity of detection due to increased probe accessibility, and better image contrast. CryoFISH and cryoIF methods typically used on samples fixed in conditions that preserve ultrastructure, are compatible with the labeling of cells in their tissue context and are ideal for correlative studies that compare fluorescence with electron microscopy.