Leonid A. Mirny

Comprehensive mapping of long range interactions reveals folding principles of the human genome

Chromosomal Mapping The conformation of the genome in the nucleus and contacts between both proximal and distal loci influence gene expression. In order to map genomic contacts, Lieberman-Aiden et al. (p. 289, see the cover) developed a technique to allow the detection of all interactions between genomic loci in the eukaryotic nucleus followed by deep sequencing. This technology was used to map the organization of the human genome and to examine the spatial proximity of chromosomal loci at one megabase resolution. The map suggests that the genome is partitioned into two spatial compartments that are related to local chromatin state and whose remodeling correlates with changes in the chromatin state. Chromosomes are organized in a fractal knot-free conformation that is densely packed while easily folded and unfolded. We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1 megabase. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes. We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free, polymer conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.

Protein complexes and functional modules in molecular networks

Proteins, nucleic acids, and small molecules form a dense network of molecular interactions in a cell. Molecules are nodes of this network, and the interactions between them are edges. The architecture of molecular networks can reveal important principles of cellular organization and function, similarly to the way that protein structure tells us about the function and organization of a protein. Computational analysis of molecular networks has been primarily concerned with node degree [Wagner, A. & Fell, D. A. (2001) Proc. R. Soc. London Ser. B 268, 1803–1810; Jeong, H., Tombor, B., Albert, R., Oltvai, Z. N. & Barabasi, A. L. (2000) Nature 407, 651–654] or degree correlation [Maslov, S. & Sneppen, K. (2002) Science 296, 910–913], and hence focused on single/two-body properties of these networks. Here, by analyzing the multibody structure of the network of protein–protein interactions, we discovered molecular modules that are densely connected within themselves but sparsely connected with the rest of the network. Comparison with experimental data and functional annotation of genes showed two types of modules: (i) protein complexes (splicing machinery, transcription factors, etc.) and (ii) dynamic functional units (signaling cascades, cell-cycle regulation, etc.). Discovered modules are highly statistically significant, as is evident from comparison with random graphs, and are robust to noise in the data. Our results provide strong support for the network modularity principle introduced by Hartwell et al. [Hartwell, L. H., Hopfield, J. J., Leibler, S. & Murray, A. W. (1999) Nature 402, C47–C52], suggesting that found modules constitute the “building blocks” of molecular networks.

Organization of the Mitotic Chromosome

Chromosome Conundrum The three-dimensional organization of chromosomal DNA within the cell nucleus plays an important role in gene regulation. Naumova et al. (p. 948, published online 7 November; see the Perspective by Kleckner et al.) used chromosome conformation capture-based methods in human tissue culture cells to analyze the higher order folding of human chromosomes across the cell cycle. During interphase the chromosomes showed locus-specific compart-mentalization. In mitotic cells, on the other hand, the chromosome organization was more linear, consistent with arrays of consecutive chromatin loops. Chromosome conformation changes dramatically during the cell cycle and is unlikely to carry epigenetic information. [Also see Perspective by Kleckner et al.] Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two distinct three-dimensional folding states of the human genome. We show that the highly compartmentalized and cell type–specific organization described previously for nonsynchronous cells is restricted to interphase. In metaphase, we identified a homogenous folding state that is locus-independent, common to all chromosomes, and consistent among cell types, suggesting a general principle of metaphase chromosome organization. Using polymer simulations, we found that metaphase Hi-C data are inconsistent with classic hierarchical models and are instead best described by a linearly organized longitudinally compressed array of consecutive chromatin loops.

Iterative correction of Hi-C data reveals hallmarks of chromosome organization.

Extracting biologically meaningful information from chromosomal interactions obtained with genome-wide chromosome conformation capture (3C) analyses requires the elimination of systematic biases. We present a computational pipeline that integrates a strategy to map sequencing reads with a data-driven method for iterative correction of biases, yielding genome-wide maps of relative contact probabilities. We validate this ICE (iterative correction and eigenvector decomposition) technique on published data obtained by the high-throughput 3C method Hi-C, and we demonstrate that eigenvector decomposition of the obtained maps provides insights into local chromatin states, global patterns of chromosomal interactions, and the conserved organization of human and mouse chromosomes.

Kinetics of Protein-DNA Interaction: Facilitated Target Location in Sequence-Dependent Potential

Recognition and binding of specific sites on DNA by proteins is central for many cellular functions such as transcription, replication, and recombination. In the process of recognition, a protein rapidly searches for its specific site on a long DNA molecule and then strongly binds this site. Here we aim to find a mechanism that can provide both a fast search (1-10 s) and high stability of the specific protein-DNA complex (Kd=10(-15)-10(-8) M). Earlier studies have suggested that rapid search involves sliding of the protein along the DNA. Here we consider sliding as a one-dimensional diffusion in a sequence-dependent rough energy landscape. We demonstrate that, despite the landscapes roughness, rapid search can be achieved if one-dimensional sliding is accompanied by three-dimensional diffusion. We estimate the range of the specific and nonspecific DNA-binding energy required for rapid search and suggest experiments that can test our mechanism. We show that optimal search requires a protein to spend half of its time sliding along the DNA and the other half diffusing in three dimensions. We also establish that, paradoxically, realistic energy functions cannot provide both rapid search and strong binding of a rigid protein. To reconcile these two fundamental requirements we propose a search-and-fold mechanism that involves the coupling of protein binding and partial protein folding. The proposed mechanism has several important biological implications for search in the presence of other proteins and nucleosomes, simultaneous search by several proteins, etc. The proposed mechanism also provides a new framework for interpretation of experimental and structural data on protein-DNA interactions.

Formation of Chromosomal Domains by Loop Extrusion

Topologically associating domains (TADs) are fundamental structural and functional building blocks of human interphase chromosomes, yet the mechanisms of TAD formation remain unclear. Here, we propose that loop extrusion underlies TAD formation. In this process, cis-acting loop-extruding factors, likely cohesins, form progressively larger loops but stall at TAD boundaries due to interactions with boundary proteins, including CTCF. Using polymer simulations, we show that this model produces TADs and finer-scale features of Hi-C data. Each TAD emerges from multiple loops dynamically formed through extrusion, contrary to typical illustrations of single static loops. Loop extrusion both explains diverse experimental observations-including the preferential orientation of CTCF motifs, enrichments of architectural proteins at TAD boundaries, and boundary deletion experiments-and makes specific predictions for the depletion of CTCF versus cohesin. Finally, loop extrusion has potentially far-ranging consequences for processes such as enhancer-promoter interactions, orientation-specific chromosomal looping, and compaction of mitotic chromosomes.

High-Resolution Mapping of the Spatial Organization of a Bacterial Chromosome

Caulobacter Chromosome Chromosomal DNA must be highly compacted to fit within the tiny volume of the cell, while at the same time it must maintain a conformation that allows critical cellular processes access to the genome. Le et al. (p. 731, published online 24 October) analyzed the structure of the circular chromosome in the prokaryote Caulobacter crescentus by using chromosome conformation capture and deep-sequencing. Highly self-interacting regions (chromosomal interaction domains, or CIDs) were observed—similar to the topologically associated domains previously seen in eukaryotes. Supercoiling helped to establish CIDs, and CID boundaries were defined by highly expressed genes. CIDs appeared to be established during or shortly after DNA replication, and could potentially facilitate chromosomal segregation by preventing newly replicated chromosomes from becoming entangled. A bacterial chromosome is organized into self-interacting regions delimited by highly expressed genes. Chromosomes must be highly compacted and organized within cells, but how this is achieved in vivo remains poorly understood. We report the use of chromosome conformation capture coupled with deep sequencing (Hi-C) to map the structure of bacterial chromosomes. Analysis of Hi-C data and polymer modeling indicates that the Caulobacter crescentus chromosome consists of multiple, largely independent spatial domains that are probably composed of supercoiled plectonemes arrayed into a bottle brush–like fiber. These domains are stable throughout the cell cycle and are reestablished concomitantly with DNA replication. We provide evidence that domain boundaries are established by highly expressed genes and the formation of plectoneme-free regions, whereas the histone-like protein HU and SMC (structural maintenance of chromosomes) promote short-range compaction and the colinearity of chromosomal arms, respectively. Collectively, our results reveal general principles for the organization and structure of chromosomes in vivo.

Virus shapes and buckling transitions in spherical shells.

We show that the icosahedral packings of protein capsomeres proposed by Caspar and Klug for spherical viruses become unstable to faceting for sufficiently large virus size, in analogy with the buckling instability of disclinations in two-dimensional crystals. Our model, based on the nonlinear physics of thin elastic shells, produces excellent one-parameter fits in real space to the full three-dimensional shape of large spherical viruses. The faceted shape depends only on the dimensionless Foppl-von Kármán number gamma=YR(2)/kappa, where Y is the two-dimensional Youngs modulus of the protein shell, kappa is its bending rigidity, and R is the mean virus radius. The shape can be parametrized more quantitatively in terms of a spherical harmonic expansion. We also investigate elastic shell theory for extremely large gamma, 10(3)<gamma<10(8), and find results applicable to icosahedral shapes of large vesicles studied with freeze fracture and electron microscopy.

Zfx controls the self-renewal of embryonic and hematopoietic stem cells

Stem cells (SC) exhibit a unique capacity for self-renewal in an undifferentiated state. It is unclear whether the self-renewal of pluripotent embryonic SC (ESC) and of tissue-specific adult SC such as hematopoietic SC (HSC) is controlled by common mechanisms. The deletion of transcription factor Zfx impaired the self-renewal but not the differentiation capacity of murine ESC; conversely, Zfx overexpression facilitated ESC self-renewal by opposing differentiation. Furthermore, Zfx deletion abolished the maintenance of adult HSC but did not affect erythromyeloid progenitors or fetal HSC. Zfx-deficient ESC and HSC showed increased apoptosis and SC-specific upregulation of stress-inducible genes. Zfx directly activated common target genes in ESC and HSC, as well as ESC-specific target genes including ESC self-renewal regulators Tbx3 and Tcl1. These studies identify Zfx as a shared transcriptional regulator of ESC and HSC, suggesting a common genetic basis of self-renewal in embryonic and adult SC.

Intricate Knots in Proteins: Function and Evolution

Our investigation of knotted structures in the Protein Data Bank reveals the most complicated knot discovered to date. We suggest that the occurrence of this knot in a human ubiquitin hydrolase might be related to the role of the enzyme in protein degradation. While knots are usually preserved among homologues, we also identify an exception in a transcarbamylase. This allows us to exemplify the function of knots in proteins and to suggest how they may have been created.