Histone H1 compacts DNA under force and during chromatin assembly

Histone H1 binds to linker DNA between nucleosomes, but the dynamics and biological ramifications of this interaction remain poorly understood. We performed single-molecule experiments using magnetic tweezers to determine the effects of H1 on naked DNA in buffer or during chromatin assembly in Xenopus egg extracts. In buffer, nanomolar concentrations of H1 induce bending and looping of naked DNA at stretching forces below 0.6 pN, effects that can be reversed with 2.7-pN force or in 200 mM monovalent salt concentrations. Consecutive tens-of-nanometer bending events suggest that H1 binds to naked DNA in buffer at high stoichiometries. In egg extracts, single DNA molecules assemble into nucleosomes and undergo rapid compaction. Histone H1 at endogenous physiological concentrations increases the DNA compaction rate during chromatin assembly under 2-pN force and decreases it during disassembly under 5-pN force. In egg cytoplasm, histone H1 protects sperm nuclei undergoing genome-wide decondensation and chromatin assembly from becoming abnormally stretched or fragmented due to astral microtubule pulling forces. These results reveal functional ramifications of H1 binding to DNA at the single-molecule level and suggest an important physiological role for H1 in compacting DNA under force and during chromatin assembly.     

Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening-closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension.     

Single chromatin fiber stretching reveals physically distinct populations of disassembly events

Eukaryotic DNA is packaged into the cell nucleus as a nucleoprotein complex, chromatin. Despite this condensed state, access to the DNA sequence must occur during gene expression and other essential genetic events. Here we employ optical tweezers stretching of reconstituted chromatin fibers to investigate the release of DNA from its protein-bound structure. Analysis of fiber length increase per unbinding event revealed discrete values of approximately 30 and approximately 60 nm. Furthermore, a loading rate analysis of the disruption forces revealed three individual energy barriers. The heights of these barriers were found to be approximately 20 k(B)T, approximately 25 k(B)T, and approximately 28 k(B)T. For subsequent stretches of the fiber it was found that events corresponding to the approximately 28 k(B)T energy barrier were significantly reduced. No correlation between energy barrier crossed and DNA length release was found. These studies clearly demonstrate that optical tweezers stretching of chromatin provides insight into the energetic penalties imposed by chromatin structure. Furthermore these studies reveal possible pathways via which chromatin may be disrupted during genetic code access.     

Measuring intermolecular rupture forces with a combined TIRF-optical trap microscope and DNA curtains

We report a new approach to probing DNA-protein interactions by combining optical tweezers with a high-throughput DNA curtains technique. Here we determine the forces required to remove the individual lipid-anchored DNA molecules from the bilayer. We demonstrate that DNA anchored to the bilayer through a single biotin-streptavidin linkage withstands ∼20 pN before being pulled free from the bilayer, whereas molecules anchored to the bilayer through multiple attachment points can withstand ?65 pN; access to this higher force regime is sufficient to probe the responses of protein-DNA interactions to force changes. As a proof-of-principle, we concurrently visualized DNA-bound fluorescently-tagged RNA polymerase while simultaneously stretching the DNA molecules. This work presents a step towards a powerful experimental platform that will enable concurrent visualization of DNA curtains while applying defined forces through optical tweezers.     

Entropy and heat capacity of DNA melting from temperature dependence of single molecule stretching

When a single molecule of double-stranded DNA is stretched beyond its B-form contour length, the measured force shows a highly cooperative overstretching transition. We have measured the force at which this transition occurs as a function of temperature. To do this, single molecules of DNA were captured between two polystyrene beads in an optical tweezers apparatus. As the temperature of the solution surrounding a captured molecule was increased from 11 degrees C to 52 degrees C in 500 mM NaCl, the overstretching transition force decreased from 69 pN to 50 pN. This reduction is attributed to a decrease in the stability of the DNA double helix with increasing temperature. These results quantitatively agree with a model that asserts that DNA melting occurs during the overstretching transition. With this model, the data may be analyzed to obtain the change in the melting entropy DeltaS of DNA with temperature. The observed nonlinear temperature dependence of DeltaS is a result of the positive change in heat capacity of DNA upon melting, which we determine from our stretching measurements to be DeltaC(p) = 60 +/- 10 cal/mol K bp, in agreement with calorimetric measurements.     

Electrostatic background of chromatin fiber stretching

We have carried out an investigation of the electrostatic forces involved in gradual removal of the DNA from the histone proteins in chromatin. Two simple models of DNA-histone core dissociation were considered. Calculations of the electrostatic free energy within the Poisson-Boltzmann theory gave similar results for the both models, which turned out to be in a qualitative agreement with recent optical tweezers stretching experiments measuring the force necessary to unwrap DNA from the histone core. Our analysis shows that the electrostatic interactions between the highly negatively charged polymeric DNA and the positively charged histones play a determining role in stabilizing the nucleosomes at physiological conditions.     

30 nm chromatin fibre decompaction requires both H4-K16 acetylation and linker histone eviction

The mechanism by which chromatin is decondensed to permit access to DNA is largely unknown. Here, using a model nucleosome array reconstituted from recombinant histone octamers, we have defined the relative contribution of the individual histone octamer N-terminal tails as well as the effect of a targeted histone tail acetylation on the compaction state of the 30 nm chromatin fiber. This study goes beyond previous studies as it is based on a nucleosome array that is very long (61 nucleosomes) and contains a stoichiometric concentration of bound linker histone, which is essential for the formation of the 30 nm chromatin fiber. We find that compaction is regulated in two steps: Introduction of H4 acetylated to 30% on K16 inhibits compaction to a greater degree than deletion of the H4 N-terminal tail. Further decompaction is achieved by removal of the linker histone.     

Stability of the conservative mode of nucleosome assembly.

The conservative assembly of nucleosome histone octamer cores has been confirmed by electrophoretic analysis of density labeled histones following equilibrium buoyant density centrifugation. After normal replication, crosslinked octamers are shown not to contain a mixture of new and old core histones. Moreover, when DNA synthesis is inhibited by ara-C nucleosome cores are still assembled exclusively from nascent histone. Similarly, after release from cycloheximide inhibition newly synthesized core histone is conservatively deposited. Thus, a conservative mechanism of histone octamer assembly occurs when nascent histone is present in the normal stoichiometry to nascent DNA and when chromatin is assembled in nascent histone or nascent DNA excess.     

Behavior of supercoiled DNA.

We study DNA supercoiling in a quantitative fashion by micromanipulating single linear DNA molecules with a magnetic field gradient. By anchoring one end of the DNA to multiple sites on a magnetic bead and the other end to multiple sites on a glass surface, we were able to exert torsional control on the DNA. A rotating magnetic field was used to induce rotation of the magnetic bead, and reversibly over- and underwind the molecule. The magnetic field was also used to increase or decrease the stretching force exerted by the magnetic bead on the DNA. The molecule's degree of supercoiling could therefore be quantitatively controlled and monitored, and tethered-particle motion analysis allowed us to measure the stretching force acting on the DNA. Experimental results indicate that this is a very powerful technique for measuring forces at the picoscale. We studied the effect of stretching forces ranging from 0.01 pN to 100 pN on supercoiled DNA (-0.1 < sigma < 0.2) in a variety of ionic conditions. Other effects, such as stretching-relaxing hysteresis and the braiding of two DNA molecules, are discussed.     

The core histone N termini function independently of linker histones during chromatin condensation

The relationships between the core histone N termini and linker histones during chromatin assembly and salt-dependent chromatin condensation were investigated using defined chromatin model systems reconstituted from tandemly repeated 5 S rDNA, histone H5, and either native "intact" core histone octamers or "tailless" histone octamers lacking their N-terminal domains. Nuclease digestion and sedimentation studies indicate that H5 binding and the resulting constraint of entering and exiting nucleosomal DNA occur to the same extent in both tailless and intact chromatin arrays. However, despite possessing a normal chromatosomal structure, tailless chromatin arrays can neither condense into extensively folded structures nor cooperatively oligomerize in MgCl(2). Tailless nucleosomal arrays lacking linker histones also are unable to either fold extensively or oligomerize, demonstrating that the core histone N termini perform the same functions during salt-dependent condensation regardless of whether linker histones are components of the array. Our results further indicate that disruption of core histone N termini function in vitro allows a linker histone-containing chromatin fiber to exist in a decondensed state under conditions that normally would promote extensive fiber condensation. These findings have key implications for both the mechanism of chromatin condensation, and the regulation of genomic function by chromatin.     

Torque-limited RecA polymerization on dsDNA

The assembly of RecA onto a torsionally constrained double-stranded DNA molecule was followed in real time using magnetic tweezers. Formation of a RecA–DNA filament on the DNA tether was stalled owing to different physical processes depending on the applied stretching force. For forces up to 3.6 pN, the reaction stalled owing to the formation of positive plectonemes in the remaining DNA molecule. Release of these plectonemes by rotation of the magnets led to full coverage of the DNA molecule by RecA. At stretching forces larger than 3.6 pN, the twist induced during filament formation caused the reaction to stall before positive supercoils were generated. We deduce a maximum built-up torsion of 10.1 ± 0.7 k_bT. In vivo this built-up torsion may be used to favor regression of a stalled replication fork or to free the chromosomal DNA in E.coli from its condensing proteins.     

Histone Octamer Helical Tubes Suggest that an Internucleosomal Four-Helix Bundle Stabilizes the Chromatin Fiber

A major question in chromatin involves the exact organization of nucleosomes within the 30-nm chromatin fiber and its structural determinants of assembly. Here we investigate the structure of histone octamer helical tubes via the method of iterative helical real-space reconstruction. Accurate placement of the x-ray structure of the histone octamer within the reconstructed density yields a pseudoatomic model for the entire helix, and allows precise identification of molecular interactions between neighboring octamers. One such interaction that would not be obscured by DNA in the nucleosome consists of a twofold symmetric four-helix bundle formed between pairs of H2B-α3 and H2B-αC helices of neighboring octamers. We believe that this interface can act as an internucleosomal four-helix bundle within the context of the chromatin fiber. The potential relevance of this interface in the folding of the 30-nm chromatin fiber is discussed.     

Sequence-dependent mechanics of single DNA molecules

Atomic force microscope-based single-molecule force spectroscopy was employed to measure sequence-dependent mechanical properties of DNA by stretching individual DNA double strands attached between a gold surface and an AFM tip. We discovered that in lambda-phage DNA the previously reported B-S transition, where 'S' represents an overstretched conformation, at 65 pN is followed by a nonequilibrium melting transition at 150 pN. During this transition the DNA is split into single strands that fully recombine upon relaxation. The sequence dependence was investigated in comparative studies with poly(dG-dC) and poly(dA-dT) DNA. Both the B-S and the melting transition occur at significantly lower forces in poly(dA-dT) compared to poly(dG-dC). We made use of the melting transition to prepare single poly(dG-dC) and poly(dA-dT) DNA strands that upon relaxation reannealed into hairpins as a result of their self-complementary sequence. The unzipping of these hairpins directly revealed the base pair-unbinding forces for G-C to be 20 +/- 3 pN and for A-T to be 9 +/- 3 pN.     

Single-molecule adhesion forces and attachment lifetimes of myosin-I phosphoinositide interactions

Phosphoinositides regulate the activities and localization of many cytoskeletal proteins involved in crucial biological processes, including membrane-cytoskeleton adhesion. Yet little is known about the mechanics of protein-phosphoinositide interactions, or about the membrane-attachment mechanics of any peripheral membrane proteins. Myosin-Ic (myo1c) is a molecular motor that links membranes to the cytoskeleton via phosphoinositide binding, so it is particularly important to understand the mechanics of its membrane attachment. We used optical tweezers to measure the strength and attachment lifetime of single myo1c molecules as they bind beads coated with a bilayer of 2% phosphatidylinositol 4,5-bisphosphate and 98% phosphatidylcholine. Adhesion forces measured under ramp-load ranged between 5.5 and 16 pN at loading rates between 250 and 1800 pN/s. Dissociation rates increased linearly with constant force (0.3-2.5 pN), with rates exceeding 360 s⁻¹ at 2.5 pN. Attachment lifetimes calculated from adhesion force measurements were loading-rate-dependent, suggesting nonadiabatic behavior during pulling. The adhesion forces of myo1c with phosphoinositides are greater than the motors stall forces and are within twofold of the force required to extract a lipid molecule from the membrane. However, attachment durations are short-lived, suggesting that phosphoinositides alone do not provide the mechanical stability required to anchor myo1c to membranes during multiple ATPase cycles.