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.

Gene positioning.

Eukaryotic gene expression is an intricate multistep process, regulated within the cell nucleus through the activation or repression of RNA synthesis, processing, cytoplasmic export, and translation into protein. The major regulators of gene expression are chromatin remodeling and transcription machineries that are locally recruited to genes. However, enzymatic activities that act on genes are not ubiquitously distributed throughout the nucleoplasm, but limited to specific and spatially defined foci that promote preferred higher-order chromatin arrangements. The positioning of genes within the nuclear landscape relative to specific functional landmarks plays an important role in gene regulation and disease.

Multiple roles for neurofibromin in skeletal development and growth.

Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder primarily characterized by the formation of neurofibromas, café-au-lait spots and freckling. Skeletal abnormalities such as short stature or bowing/pseudarthrosis of the tibia are relatively common. To investigate the role of the neurofibromin in skeletal development, we crossed Nf1flox mice with Prx1Cre mice to inactivate Nf1 in undifferentiated mesenchymal cells of the developing limbs. Similar to NF1 affected individuals, Nf1(Prx1) mice show bowing of the tibia and diminished growth. Tibial bowing is caused by decreased stability of the cortical bone due to a high degree of porosity, decreased stiffness and reduction in the mineral content as well as hyperosteoidosis. Accordingly, osteoblasts show an increase in proliferation and a decreased ability to differentiate and mineralize in vitro. The reduction in growth is due to lower proliferation rates and a differentiation defect of chondrocytes. Abnormal vascularization of skeletal tissues is likely to contribute to this pathology as it exerts a negative effect on cortical bone stability. Furthermore, Nf1 has an important role in the development of joints, as shown by fusion of the hip joints and other joint abnormalities, which are not observed in neurofibromatosis type I. Thus, neurofibromin has multiple essential roles in skeletal development and growth.

Essential role for IkappaB kinase beta in remodeling Carma1-Bcl10-Malt1 complexes upon T cell activation.

T cell receptor (TCR) signaling to IkappaB kinase (IKK)/NF-kappaB is controlled by PKCtheta-dependent activation of the Carma1, Bcl10, and Malt1 (CBM) complex. Antigen-induced phosphorylation of Bcl10 has been reported, but its physiological function is unknown. Here we show that the putative downstream kinase IKKbeta is required for initial CBM complex formation. Further, upon engagement of IKKbeta/Malt1/Bcl10 with Carma1, IKKbeta phosphorylates Bcl10 in the C terminus and thereby interferes with Bcl10/Malt1 association and Bcl10-mediated IKKgamma ubiquitination. Mutation of the IKKbeta phosphorylation sites on Bcl10 enhances expression of NF-kappaB target genes IL-2 and TNFalpha after activation of primary T cells. Thus, our data provide evidence that IKKbeta serves a dual role upstream of its classical substrates, the IkappaB proteins. While being essential for triggering initial CBM complex formation, IKKbeta-dependent phosphorylation of Bcl10 exhibits a negative regulatory role in T cell activation.