Search

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs) 1-3 , which contribute to transcription, recombination and genomic stability 4,5 . Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered 6-12 . Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are 'active' barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome., (© 2022. The Author(s).)

Proper timing of gene expression requires that transcription factors (TFs) efficiently locate and bind their target sites within a genome. Theoretical studies have long proposed that one-dimensional sliding along DNA while simultaneously reading its sequence can accelerate TF's location of target sites. Sliding by prokaryotic and eukaryotic TFs were subsequently observed. More recent theoretical investigations have argued that simultaneous reading and sliding is not possible for TFs without their possessing at least two DNA-binding modes. The tumor suppressor p53 has been shown to slide on DNA, and recent experiments have offered structural and single molecule support for a two-mode model for the protein. If the model is applicable to p53, then the requirement that TFs be able to read while sliding implies that noncognate sites will affect p53's mobility on DNA, which will thus be generally sequence-dependent. Here, we confirm this prediction with single-molecule microscopy measurements of p53's local diffusivity on noncognate DNA. We show how a two-mode model accurately predicts the variation in local diffusivity, while a single-mode model does not. We further determine that the best model of sequence-specific binding energy includes terms for "hemi-specific" binding, with one dimer of tetrameric p53 binding specifically to a half-site and the other binding nonspecifically to noncognate DNA. Our work provides evidence that the recognition by p53 of its targets and the timing thereof can depend on its noncognate binding properties and its ability to change between multiple modes of binding, in addition to the much better-studied effects of cognate-site binding.

Recognition and binding of specific sites on DNA by proteins is central for many cellular functions such as transcription, replication, and recombination. In the search for its target site, the DNA-associated protein is facing both thermodynamic and kinetic difficulties. The thermodynamic challenge lies in recognizing and tightly binding a cognate (specific) site among the billions of other (non-specific) sequences on the DNA. The kinetic difficulty lies in finding a cognate site in mere seconds amidst the crowded cellular environment that is filled with other DNA sequences and proteins. Herein, we discuss the history of the DNA search problem, the theoretical background and the various experimental methods used to study the kinetics of proteins searching for target sites on DNA., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity (2-6). Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible (7-10). We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments. Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

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.

The p53 protein, a transcription factor of key importance in tumorigenesis, is suggested to diffuse one-dimensionally along DNA via its C-terminal domain, a process that is proposed to regulate gene activation both positively and negatively. There has been no direct observation of p53 moving along DNA, however, and little is known about the mechanism and rate of its translocation. Here, we use single-molecule techniques to visualize, in real time, the one-dimensional diffusion of p53 along DNA. The one-dimensional diffusion coefficient is measured to be close to the theoretical limit, indicative of movement along a free energy landscape with low activation barriers. We further investigate the mechanism of translocation and determine that p53 is capable of sliding--moving along DNA while in continuous contact with the duplex, rather than through a series of hops between nearby bases.

What are the forces that shape the structure of prokaryotic genomes: the order of genes, their proximity, and their orientation? Coregulation and coordinated horizontal gene transfer are believed to promote the proximity of functionally related genes and the formation of operons. However, forces that influence the structure of the genome beyond the level of a single operon remain unknown. Here, we show that the biophysical mechanism by which regulatory proteins search for their sites on DNA can impose constraints on genome structure. Using simulations, we demonstrate that rapid and reliable gene regulation requires that the transcription factor (TF) gene be close to the site on DNA the TF has to bind, thus promoting the colocalization of TF genes and their targets on the genome. We use parameters that have been measured in recent experiments to estimate the relevant length and times scales of this process and demonstrate that the search for a cognate site may be prohibitively slow if a TF has a low copy number and is not colocalized. We also analyze TFs and their sites in a number of bacterial genomes, confirm that they are colocalized significantly more often than expected, and show that this observation cannot be attributed to the pressure for coregulation or formation of selfish gene clusters, thus supporting the role of the biophysical constraint in shaping the structure of prokaryotic genomes. Our results demonstrate how spatial organization can influence timing and noise in gene expression.

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 landscape's 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.

Many biological processes involve one-dimensional diffusion over a correlated inhomogeneous energy landscape with a correlation length xi(c). Typical examples are specific protein target location on DNA, nucleosome repositioning, or DNA translocation through a nanopore, in all cases with xi(c) approximately 10 nm. We investigate such transport processes by the mean first passage time (MFPT) formalism, and find diffusion times which exhibit strong sample to sample fluctuations. For a displacement N, the average MFPT is diffusive, while its standard deviation over the ensemble of energy profiles scales as N(3/2) with a large prefactor. Fluctuations are thus dominant for displacements smaller than a characteristic N(c) >> xi(c) : typical values are much less than the mean, and governed by an anomalous diffusion rule. Potential biological consequences of such random walks, composed of rapid scans in the vicinity of favorable energy valleys and occasional jumps to further valleys, is discussed.

Understanding of protein-DNA interactions is crucial for prediction of DNA-binding specificity of transcription factors and design of novel DNA-binding proteins. In this paper we develop a novel approach to analysis of protein-DNA interactions. We bring together two sources of information: (i) structures of protein-DNA complexes (PDB/NDB database) and (ii) experimentally obtained sites recognized by DNA-binding proteins. Sites are used to compute conservation (information content) of each base pair, which indicates relative importance of the base pair in specific recognition. The main result of this study is that conservation of base pairs in a site exhibits significant correlation with the number of contacts the base pairs have with the protein. In particular, base pairs that have more contacts with the protein are more conserved in evolution. As natural as it is, this result has never been reported before. We also observe that for most of the studied proteins, hydrogen bonds and hydrophobic interactions alone cannot explain the pattern of evolutionary conservation in the binding site suggesting cumulative contribution of different types of interactions to specific recognition. Implications for prediction of the DNA-binding specificity are discussed.

Strong experimental and theoretical evidence shows that transcription factors (TFs) and other specific DNA-binding proteins find their sites using a two-mode search: alternating between three-dimensional (3D) diffusion through the cell and one-dimensional (1D) sliding along the DNA. We show that, due to the 1D component of the search process, the search time of a TF can depend on the initial position of the TF. We formalize this effect by discriminating between two types of searches: global and local. Using analytical calculations and simulations, we estimate how close a TF and binding site need to be to make a local search likely. We then use our model to interpret the wide range of experimental measurements of this parameter. We also show that local and global searches differ significantly in average search time and the variability of search time. These results lead to a number of biological implications, including suggestions of how prokaryotes achieve rapid gene regulation and the relationship between the search mechanism and noise in gene expression. Lastly, we propose a number of experiments to verify the existence and quantify the extent of spatial effects on the TF search process in prokaryotes.

Chromosome compaction is essential for reliable transmission of genetic information. Experiments suggest that ~1000-fold compaction is driven by condensin complexes that extrude chromatin loops, by progressively collecting chromatin fiber from one or both sides of the complex to form a growing loop. Theory indicates that symmetric two-sided loop extrusion can achieve such compaction, but recent single-molecule studies (Golfier et al., 2020) observed diverse dynamics of condensins that perform one-sided, symmetric two-sided, and asymmetric two-sided extrusion. We use simulations and theory to determine how these molecular properties lead to chromosome compaction. High compaction can be achieved if even a small fraction of condensins have two essential properties: a long residence time and the ability to perform two-sided (not necessarily symmetric) extrusion. In mixtures of condensins I and II, coupling two-sided extrusion and stable chromatin binding by condensin II promotes compaction. These results provide missing connections between single-molecule observations and chromosome-scale organization. [ABSTRACT FROM AUTHOR]

How DNA is organized in three dimensions inside the cell nucleus and how this affects the ways in which cells access, read and interpret genetic information are among the longest standing questions in cell biology. Using newly developed molecular, genomic and computational approaches based on the chromosome conformation capture technology (such as 3C, 4C, 5C and Hi-C), the spatial organization of genomes is being explored at unprecedented resolution. Interpreting the increasingly large chromatin interaction data sets is now posing novel challenges. Here we describe several types of statistical and computational approaches that have recently been developed to analyse chromatin interaction data. [ABSTRACT FROM AUTHOR]

Coordinated regulation of gene expression relies on transcription factors (TFs) binding to specific DNA sites. Our large-scale information–theoretical analysis of >950 TF-binding motifs demonstrates that prokaryotes and eukaryotes use strikingly different strategies to target TFs to specific genome locations. Although bacterial TFs can recognize a specific DNA site in the genomic background, eukaryotic TFs exhibit widespread, nonfunctional binding and require clustering of sites to achieve specificity. We find support for this mechanism in a range of experimental studies and in our evolutionary analysis of DNA-binding domains. Our systematic characterization of binding motifs provides a quantitative assessment of the differences in transcription regulation in prokaryotes and eukaryotes. [Copyright &y& Elsevier]