Pulling a single chromatin fiber reveals the forces that maintain its higher-order structure

Abstract

Single chicken erythrocyte chromatin fibers were stretched and released at room temperature with force-measuring laser tweezers. In low ionic strength, the stretch-release curves reveal a process of continuous deformation with little or no internucleosomal attraction. A persistence length of 30 nm and a stretch modulus of Ϸ5 pN is determined for the fibers. At forces of 20 pN and higher, the fibers are modified irreversibly, probably through the mechanical removal of the histone cores from native chromatin. In 40 -150 mM NaCl, a distinctive condensation-decondensation transition appears between 5 and 6 pN, corresponding to an internucleosomal attraction energy of Ϸ2.0 kcal͞mol per nucleosome. Thus, in physiological ionic strength the fibers possess a dynamic structure in which the fiber locally interconverting between ''open'' and ''closed'' states because of thermal fluctuations. The DNA of all eukaryotic cells is organized in the form of chromatin and its structure has been the subject of intense research during the last 25 years. These studies have shown that the basic structural unit of chromatin is the nucleosome comprising the core particle and linker DNA. The core particle contains two of each of four core histones H2A, H2B, H3, and H4, and 146 bp of DNA wrapped around this core. The chromatosome (1, 2) includes the core particle and an additional 20 bp of linker DNA associated to a linker histone (H1 or H5). Although there is still some controversy about the position of the linker histone (3, 4) and the location of H3 and H4 histone tails in the chromatosome, many details have been revealed by the crystal structure of the nucleosome core particle (5-7).Much less is known about the next level of chromatin structure, i.e., the spatial organization of chromatosomes interspersed by linker DNA (8, 9). Many models involving the regular, three-dimensional organization of nucleosomes into chromatin fibers have been proposed (10). Recently, new methods of direct visualization such as scanning force microscopy (11-13) and cryo-electron microscopy (14-16), as well as reinterpretation of older data, indicate that, at low ionic strength at least, nucleosomes in the so-called 30-nm fiber are organized in an irregular three-dimensional zigzag.Chromatin undergoes a process of condensation and decondensation during the cell cycle in vivo. Higher-order structures occur in transcriptionally inactive regions, whereas regions of decondensed nucleosomal arrays often are associated with active chromatin (10). Because in most cell types only a small percentage of the total chromatin content is active at any given time, dynamic changes in the folding state of local chromatin domains must occur to modify the accessibility of the transcription machinery to these domains. These structural transitions may involve H1 removal, histone modifications (acetylation, phosphorylation, or methylation), and changes in the nonhistone protein complement (10,17). Despite the role played by changes in fiber compaction in the regulation of gene ex...

Wavelet Analysis of DNA Bending Profiles reveals Structural Constraints on the Evolution of Genomic Sequences

“…At first level of organization, the chromatin fiber is built from a linear array of nucleosomes [7,8]. In the nucleus, this fiber is further packed into a higher order structure known as the 30 nm fiber [7,9,10,11,12,13,14]. This hierarchical folding of the DNA molecule is likely to imply constraints on the molecule bending and flexibility properties and on the capability of interacting with and of being anchored to protein matrix and scaffold.…”

Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics

“…Laser tweezer experiments generally impose a time-varying extension and measure the resulting tension. In the experiments of Cui and Bustamante (2000) and Bennink et al (2001), the fiber pulls were done in Ͻ1 min; relatively large nucleosome removal forces were likely observed simply because the pulling rates were far greater than that at which nucleosomes could be removed by near-to-equilibrium fluctuations.…”

“…A complementary approach is to study individual chromatin fibers by using micromanipulation (Cui and Bustamante, 2000;Ladoux et al, 2000;Bennink et al, 2001;Brower-Toland et al, 2002;Leuba et al, 2003;Claudet et al, 2005;Gemmen et al, 2005;Bancaud et al, 2006). A major objective of such experiments has been the study of mechanically triggered changes in protein-DNA contacts, with an emphasis on force-driven opening of nucleosomes.…”

“…Laser tweezer experiments generally impose a time-varying extension and measure the resulting tension. In the experiments of Cui and Bustamante (2000) and Bennink et al (2001), the fiber pulls were done in Ͻ1 min; relatively large nucleosome removal forces were likely observed simply because the pulling rates were far greater than that at which nucleosomes could be removed by near-to-equilibrium fluctuations.Thus, the high nucleosome-removal forces observed by Cui and Bustamante (2000), Bennink et al (2001), andBrower-Toland et al (2002) may be in part a result of the high strain rates imposed. The effect of an increase in bond failure force with increasing strain rate has been well documented theoretically, mainly in connection with probing of receptor-ligand interactions (Evans, 2001).…”

“…The same principle will apply for other proteins involved in folding of DNA into chromatin, although their disruption would likely yield a different DNA length per complex than the ϳ50 nm expected for removal of one nucleosome. The earliest force-extension experiments on chromatin used fibers isolated from cells (Cui and Bustamante, 2000), and found irreversible changes in fiber length to result from tensions of roughly 20 pN (1 pN ϭ 10 Ϫ12 newtons). The experimenters attributed this to force-driven disruption of histone-DNA interactions.…”