Protein-mediated Loops Research Articles

CharID: a two-step model for universal prediction of interactions between chromatin accessible regions.

Open chromatin regions (OCRs) allow direct interaction between cis-regulatory elements and trans-acting factors. Therefore, predicting all potential OCR-mediated loops is essential for deciphering the regulation mechanism of gene expression. However, existing loop prediction tools are restricted to specific anchor types. Here, we present CharID (Chromatin Accessible Region Interaction Detector), a two-step model that combines neural network and ensemble learning to predict OCR-mediated loops. In the first step, CharID-Anchor, an attention-based hybrid CNN-BiGRU network is constructed to discriminate between the anchor and nonanchor OCRs. In the second step, CharID-Loop uses gradient boosting decision tree with chromosome-split strategy to predict the interactions between anchor OCRs. The performance was assessed in three human cell lines, and CharID showed superior prediction performance compared with other algorithms. In contrast to the methods designed to predict a particular type of loops, CharID can detect varieties of chromatin loops not limited to enhancer-promoter loops or architectural protein-mediated loops. We constructed the OCR-mediated interaction network using the predicted loops and identified hub anchors, which are highlighted by their proximity to housekeeping genes. By analyzing loops containing SNPs associated with cardiovascular disease, we identified an SNP-gene loop indicating the regulation mechanism of the GFOD1. Taken together, CharID universally predicts diverse chromatin loops beyond other state-of-the-art methods, which are limited by anchor types, and experimental techniques, which are limited by sensitivities drastically decaying with the genomic distance of anchors. Finally, we hosted Peaksniffer, a user-friendly web server that provides online prediction, query and visualization of OCRs and associated loops.

Negative DNA supercoiling makes protein-mediated looping deterministic and ergodic within the bacterial doubling time.

Protein-mediated DNA looping is fundamental to gene regulation and such loops occur stochastically in purified systems. Additional proteins increase the probability of looping, but these probabilities maintain a broad distribution. For example, the probability of lac repressor-mediated looping in individual molecules ranged 0–100%, and individual molecules exhibited representative behavior only in observations lasting an hour or more. Titrating with HU protein progressively compacted the DNA without narrowing the 0–100% distribution. Increased negative supercoiling produced an ensemble of molecules in which all individual molecules more closely resembled the average. Furthermore, in only 12 min of observation, well within the doubling time of the bacterium, most molecules exhibited the looping probability of the ensemble. DNA supercoiling, an inherent feature of all genomes, appears to impose time-constrained, emergent behavior on otherwise random molecular activity.

Positive Supercoiling Ahead of RNA Polymerase Aids Exit from Protein-Mediated Loops

Positive Supercoiling Ahead of RNA Polymerase Aids Exit from Protein-Mediated Loops

Supercoiling Makes Protein-Mediated Looping of DNA Tethers Deterministic

Supercoiling Makes Protein-Mediated Looping of DNA Tethers Deterministic

Protein-mediated loops in supercoiled DNA create large topological domains.

Supercoiling can alter the form and base pairing of the double helix and directly impact protein binding. More indirectly, changes in protein binding and the stress of supercoiling also influence the thermodynamic stability of regulatory, protein-mediated loops and shift the equilibria of fundamental DNA/chromatin transactions. For example, supercoiling affects the hierarchical organization and function of chromatin in topologically associating domains (TADs) in both eukaryotes and bacteria. On the other hand, a protein-mediated loop in DNA can constrain supercoiling within a plectonemic structure. To characterize the extent of constrained supercoiling, 400 bp, lac repressor-secured loops were formed in extensively over- or under-wound DNA under gentle tension in a magnetic tweezer. The protein-mediated loops constrained variable amounts of supercoiling that often exceeded the maximum writhe expected for a 400 bp plectoneme. Loops with such high levels of supercoiling appear to be entangled with flanking domains. Thus, loop-mediating proteins operating on supercoiled substrates can establish topological domains that may coordinate gene regulation and other DNA transactions across spans in the genome that are larger than the separation between the binding sites.

Protein-Mediated Loops in Supercoiled DNA Generate Large, Dynamic Topological Domains

Proteins that act at a distance along DNA by binding at one site and contacting another create loops that form topological domains and influence regulation. Such loops are affected by the torsional state of DNA, which dramatically modulates topology, driving the DNA from extended and accessible, to more compact and genetically secured forms. Furthermore, the response of the DNA filament to supercoiling is biased by accessory factors. For example, small molecules like polyamines, which neutralize the negative charge repulsions along the phosphate backbone, enhance flexibility and promote writhe over twist in response to torsion. On the other hand, stiffer DNA antagonizes looping and bending. Recent experiments quantitatively reveal the extent to which negatively supercoiling DNA lowers the free energy of looping, and could therefore bias the operation of genetic switches. A new role for DNA supercoiling has emerged; it synergizes with protein-mediated looping to create large, dynamic topological domains that extend beyond the length of the loop. Such domains may coordinate gene regulation and other DNA transactions across spans in the genome that exceed the separation between the protein binding sites.

Protein-mediated looping of DNA under tension requires supercoiling

Protein-mediated DNA looping is ubiquitous in chromatin organization and gene regulation, but to what extent supercoiling or nucleoid associated proteins promote looping is poorly understood. Using the lac repressor (LacI), a paradigmatic loop-mediating protein, we measured LacI-induced looping as a function of either supercoiling or the concentration of the HU protein, an abundant nucleoid protein in Escherichia coli. Negative supercoiling to physiological levels with magnetic tweezers easily drove the looping probability from 0 to 100% in single DNA molecules under slight tension that likely exists in vivo. In contrast, even saturating (micromolar) concentrations of HU could not raise the looping probability above 30% in similarly stretched DNA or 80% in DNA without tension. Negative supercoiling is required to induce significant looping of DNA under any appreciable tension.

Protein-Mediated Loops in Supercoiled DNA Create Large Topological Domains

Supercoiling can alter the form and base pairing of the double helix and directly impact protein binding. More indirectly, changes in protein binding and the stress of supercoiling also influence the thermodynamic stability of regulatory, protein-mediated loops and shift the equilibria of fundamental DNA/chromatin transactions. For example, supercoiling affects the hierarchical organization and function of chromatin in topologically associating domains (TADs) in both eukaryotes and bacteria. On the other hand, a protein-mediated loop in DNA can constrain supercoiling within a plectonemic structure. To characterize the extent of constrained supercoiling, 400 bp, lac repressor-secured loops were formed in extensively over- or under-wound DNA under gentle tension in a magnetic tweezer. The protein-mediated loops constrained variable amounts of supercoiling that often exceeded the maximum writhe expected for a 400 bp plectoneme. Loops with such high levels of supercoiling appear to be entangled with flanking domains. Thus, loop-mediating proteins operating on supercoiled substrates can establish topological domains that may coordinate gene regulation and other DNA transactions across spans in the genome that are larger than the separation between the binding sites.

DNA supercoiling: a regulatory signal for the λ repressor.

Topoisomerases, polymerases, and the chirality introduced by the binding of histones or nucleoid-associated proteins affect DNA supercoiling in vivo. However, supercoiling is not just a by-product of DNA metabolism. Supercoiling is an indicator of cell health, it modifies the accessibility of chromatin, and coordinates the transcription of genes. This suggests that regulatory, protein-mediated loops in DNA may sense supercoiling of the genome in which they are embedded. The λ repressor (CI) maintains the quiescent (lysogenic) transcriptome of bacteriophage λ in infected Escherichia coli. CI-mediated looping prevents overexpression of the repressor protein to preserve sensitivity to conditions that trigger virulence (lysis). Experiments were performed to assess how well the CI-mediated DNA loop traps superhelicity and determine whether supercoiling enhances CI-mediated DNA looping. CI oligomers partitioned plasmids into topological domains and prevented the passage of supercoiling between them. Furthermore, in single DNA molecules stretched and twisted with magnetic tweezers, levels of superhelical density confined in CI-mediated DNA loops ranged from -15% or +11%. Finally, in DNA under tensions that may occur in vivo, supercoiling lowered the free energy of loop formation and was essential for DNA looping. Supercoiling-enhanced looping can influence the maintenance of lysogeny in the λ repressor system; it can encode sensitivity to the energy level of the cell and creates independent topological domains of distinct superhelical density.

Single Molecule Studies of Sequence Dependence Elasticity in DNA

DNA looping is a vital regulatory process in the cell to maintain proper function and viability. In protein-mediated looping, the DNA's sequence dictates both protein binding sites and the local mechanical properties of the DNA. These mechanical properties, notably its elasticity and intrinsic curvature, govern the ease at which loops can form. To probe the effects of sequence dependence on elasticity and the loop formation process, single molecule experiments were performed on short segments (∼150bp) of DNA with varying amounts of AT and GC content between the protein-binding operators. Tethered particle motion (TPM) microscopy was employed to observe protein-mediated DNA loop formation in this system, and an analytical model of the loop formation process was used to calculate the elasticity of the DNA from the observed loop formation rates. For comparison, axial constant-force optical tweezers were used to directly stretch the same DNA molecules mechanically to determine their persistence length as a measure for their elasticity. Our results indicate that the intraoperator sequence has a larger effect on elasticity in the loop formation experiments than in the stretching experiments, which we attribute to different elasticity regimes when the DNA is strongly bent as in a DNA loop, compared to the thermally induced small curvature fluctuations in stretched DNA.

How Do Nucleosomes Bundle DNA into Chromatin?

The binding of histones and the formation of nucleosomes play major roles in bundling DNA into chromatin in eukaryotic cells. This process not only facilitates the packaging of DNA in the cell nucleus but also influences the communication between distant genomic sites, such as enhancers and promoters found at the ends of protein-mediated loops. The details of chromatin organization, however, remain unclear. In particular, the effects of nucleosome positioning (i.e., the sequential locations of nucleosomes along DNA) on chromatin architecture are still poorly understood. With a view toward understanding the interplay between nucleosomes and DNA in chromatin, we have developed a mesoscale model of nucleosome-decorated DNA at a resolution of a single base pair. We use Monte Carlo numerical strategies to unravel how nucleosomes shape DNA into chromatin and we validate our model by successfully reproducing various experimental measurements. With the help of a cloud computing approach we have been able to perform simulations for a wide range of nucleosome positionings, that is, for a wide range of DNA linker lengths. Our results show a broad variety of chromatin organization. This structural diversity, which extends beyond the conventional solenoid and zigzag models of chromatin, comes along with very different physical and mechanical properties, such as apparent bending and twisting stiffnesses and degrees of compaction. We have also studied how fluctuations in nucleosome positioning can affect chromatin. We have found that minor changes of the order of a few base pairs in the locations of nucleosomes along DNA can greatly alter the local and global properties of chromatin. Our work shows that the synergy between DNA and nucleosomes in chromatin gives rise to a highly versatile biomolecule, which is able to accommodate tight packing while maintaining the accessibility necessary for specific expression.

The Synergy Between DNA and Nucleosomes in Chromatin

Although it is now common to look at the information carried by the genome as a linear sequence of nucleotides, such representation does not say much about the organization of the genetic information within the cell nucleus. How the genome accommodates the tight packing needed to fit in the cell nucleus and at the same time maintains the accessibility necessary for specific expression is one of the open questions in modern biology. In eukaryotic cells, DNA is wrapped and packaged into chromatin through the binding of histones assembled into nucleosomes. In addition to bundling DNA, the nucleosomes also facilitate communication between distant genomic sites, such as enhancers and promoters found at the ends of protein-mediated loops. In order to understand the physical and chemical basis of such processes, we have begun to investigate chromatin organization and looping. We have developed a mesoscale model of chromatin at a resolution of a single base pair and used Monte Carlo numerical strategies to unders...

59 The synergy between DNA and nucleosomes in chromatin

Although it is now common to look at the information carried by the genome as a linear sequence of nucleotides, such representation does not say much about the organization of the genetic information within the cell nucleus. How the genome accommodates the tight packing needed to fit in the cell nucleus and at the same time maintains the accessibility necessary for specific expression is one of the open questions in modern biology. In eukaryotic cells, DNA is wrapped and packaged into chromatin through the binding of histones assembled into nucleosomes. In addition to bundling DNA, the nucleosomes also facilitate communication between distant genomic sites, such as enhancers and promoters found at the ends of protein-mediated loops. In order to understand the physical and chemical basis of such processes, we have begun to investigate chromatin organization and looping. We have developed a mesoscale model of chromatin at a resolution of a single base pair and used Monte Carlo numerical strategies to understand how the presence of nucleosomes on DNA can influence and possibly control chromatin looping. We have validated this model by successfully reproducing experimental measurements of gene expression on nucleosomal arrays (Kulaeva et al., 2012). Our results show a wide variety of chromatin organization depending on the way nucleosomes are positioned on DNA (i.e. on the spacing between successive nucleosomes as illustrated in the pictures below) and also on chemical details at the histone level, such as modifications of the N-terminal tails. This diversity in chromatin organization, which extends beyond the conventional solenoid and zigzag models, comes along with very different physical and mechanical properties and looping propensities. Furthermore, our simulations reveal some surprising properties of chromatin: for example, we found that tightly packed chromatin fragments are highly flexible, a remarkable feature for a material that needs to fit inside a cell nucleus. In conclusion, our work uncovers parts of a rich and dynamic picture of chromatin where the DNA sequence influences the positioning of nucleosomes, which in turn shape the genomic material and have an impact on the accessibility and expression of the genetic message.

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs. The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension. Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces. We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs. The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension. Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces. We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

Dual-Color Fluorescence Microscopy for Imaging DNA Loops in Living Cells

The formation and breakdown of protein-mediated loops in DNA is an important regulatory motif in gene expression. Unfortunately, direct microscopic observations of this mechanism within living cells have remained elusive because of a lack of in vivo techniques with sufficient spatial and temporal resolution. To address this deficiency, we have developed a two-color fluorescence microscopy technique designed to witness DNA looping in living cells. Our method involves the simultaneous tracking of two different colored quantum dot fluorophores conjugated to opposite ends of a strand of dsDNA. By analyzing the spatio-temporal correlations of the motion of the two fluorophores, we are able to determine when a loop has formed. We illustrate the technique by analyzing the motion of model constructs of varying lengths that are transfected into Dictyostelium discoideum cells.

Protein-mediated loops and phase transition in nonthermal denaturation of DNA

We use a statistical mechanical model to study nonthermal denaturation of DNA in thepresence of protein-mediated loops. We find that looping proteins which randomly linkDNA bases located at a distance along the chain could cause a first-order phase transition.We estimate the denaturation transition time near the phase transition, which can becompared with experimental data. The model describes the formation of multiple loops viadynamical (fluctuational) linking between looping proteins, which is essential in manycellular biological processes.

Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI

Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investigated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA containing two endonuclease recognition sites spaced a few 100 bp apart connect small polystyrene beads to a glass surface. The position of a bead is tracked through video microscopy. Protein-mediated looping and unlooping is then observed as a sudden specific change in Brownian motion of the bead. With this method we are able to directly follow DNA looping kinetics of single protein–DNA complexes to obtain loop stability and loop formation times. We show that, in the absence of divalent cations, NaeI induces DNA loops of specific size. In contrast, under these conditions NarI mainly creates non-specific loops, resulting in effective DNA compaction for higher enzyme concentrations. Addition of Ca2+ increases the NaeI-DNA loop lifetime by two orders of magnitude and stimulates specific binding by NarI. Finally, for both enzymes we observe exponentially distributed loop formation times, indicating that looping is dominated by (re)binding the second recognition site.

Collagen II Promoter and Enhancer Interact Synergistically Through Sp1 and Distinct Nuclear Factors

The collagen II gene is expressed primarily in chondrocytes. Its transcription is activated through the interaction of cell type-specific regulatory elements located in the promoter region and in the first intron. In this study, we found that a short promoter sequence including two GC boxes was required for efficient enhancer-mediated transcription. Gel-shift analysis, site mutations, and footprint analysis showed that one of the GC boxes bound functionally to an Sp1-related factor and that an oligonucleotide containing this GC box did interact with an enhancer-nuclear factor complex. Additionally, an enhancer-derived oligonucleotide was found to complex CIIZFP, a zinc-finger protein that binds to the enhancer within the first intron and Sp1, but only in presence of CIIZFP. Antibodies against Sp1 specifically inhibited the formation of this complex. Western/Southwestern analysis also showed that a protein complex including Sp1 was able to bind the enhancer and the promoter regions in non-denaturing conditions. This complex was dissociated by denaturation. These results suggest that the formation of a nuclear protein-mediated loop structure between the promoter and enhancer may regulate transcription of the collagen II gene transcription.

20] Topological approaches to studies of protein-mediated looping of DNA in vivo

This chapter presents topological approaches for studying the protein-mediated looping of DNA in vivo. It describes methods for studying protein-mediated looping of DNA in vivo using the lac repressor–operator complex as a model system. The tetrameric lac repressor has been shown to bind simultaneously to two operators both in vitro and in vivo . The simultaneous binding of the lac repressor to two operators on the same DNA molecule causes looping of the intervening DNA. Such lac repressor-mediated looping of DNA interferes with template supercoiling during RNA transcription and can cause changes in the supercoiled state of plasmid DNAs. The looping of DNA due to lac repressor binding requires the presence of at least two lac repressor-binding sites on the same molecule. For plasmid DNAs containing only a single lac repressor binding site, only the multimeric forms of the plasmid DNAs can be efficiently looped by the tetrameric lac repressor complex. The preferential supercoiling of the multimeric forms of the plasmid DNAs can, therefore, be a good indication of protein-mediated looping of DNA in vivo .