A Method for the In Vitro Reconstitution of a Defined “30nm” Chromatin Fibre Containing Stoichiometric Amounts of the Linker Histone

Huynh, Van An; Robinson, Philip J.; Rhodes, Daniela

doi:10.1016/j.jmb.2004.10.075

Cited by 168 publications

(219 citation statements)

References 51 publications

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“…11. Our reconstitution protocol produces nucleosome arrays with regularly spaced nucleosomes and containing one histone octamer and one linker histone H5 per 200 bp of 601 DNA repeat (11). Titrations with increasing concentrations of histone octamer and linker histone H5 show that in each case a clear point of saturation in binding is reached (Fig.…”

Section: Resultsmentioning

confidence: 98%

“…Importantly, our nucleosome arrays contain a native-like composition of both the histone octamer and linker histone and consequently reach a level of compaction equivalent to native chromatin at physiological ionic strength (11). Here, we have extended our in vitro reconstitution method to produce much longer model 30-nm chromatin fibers that are less affected by destabilizing end effects and to which it is much easier to assign directionality.…”

mentioning

confidence: 99%

“…In nucleosome arrays, adjacent nucleosomes are separated by linker DNA, varying in length between 0 and 80 bp in a tissue-and species-specific manner (9,10). In vitro, linear nucleosome arrays fold into the ''30-nm'' fiber upon increasing ionic strength (11) in a process that depends on both the integrity of the core histone N-terminal tails (12,13) and the presence of the linker histone (14,15).…”

mentioning

confidence: 99%

“…To provide a model system for the analysis of the 30-nm chromatin fiber, we have developed an in vitro reconstitution system that generates regularly spaced nucleosome arrays from purified histones and tandem arrays of a strong nucleosome positioning sequence (11). Importantly, our nucleosome arrays contain a native-like composition of both the histone octamer and linker histone and consequently reach a level of compaction equivalent to native chromatin at physiological ionic strength (11).…”

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confidence: 99%

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EM measurements define the dimensions of the “30-nm” chromatin fiber: Evidence for a compact, interdigitated structure

Robinson

Fairall

Huynh

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Chromatin structure plays a fundamental role in the regulation of nuclear processes such as DNA transcription, replication, recombination, and repair. Despite considerable efforts during three decades, the structure of the 30-nm chromatin fiber remains controversial. To define fiber dimensions accurately, we have produced very long and regularly folded 30-nm fibers from in vitro reconstituted nucleosome arrays containing the linker histone and with increasing nucleosome repeat lengths (10 to 70 bp of linker DNA). EM measurements show that the dimensions of these fully folded fibers do not increase linearly with increasing linker length, a finding that is inconsistent with two-start helix models. Instead, we find that there are two distinct classes of fiber structure, both with unexpectedly high nucleosome density: arrays with 10 to 40 bp of linker DNA all produce fibers with a diameter of 33 nm and 11 nucleosomes per 11 nm, whereas arrays with 50 to 70 bp of linker DNA all produce 44-nm-wide fibers with 15 nucleosomes per 11 nm. Using the physical constraints imposed by these measurements, we have built a model in which tight nucleosome packing is achieved through the interdigitation of nucleosomes from adjacent helical gyres. Importantly, the model closely matches raw image projections of folded chromatin arrays recorded in the solution state by using electron cryo-microscopy.chromatin structure ͉ electron microscopy ͉ linker histone ͉ reconstitution E ukaryotic chromosomes have a compact structure in which linear nucleosome arrays are first folded into a fiber of around 30-nm diameter (1, 2). The fundamental repeating unit of chromatin, the nucleosome core particle, organizes 147 bp of DNA in 1.7 left-handed superhelical turns around an octamer of the four core histones (H2A, H2B, H3, and H4) (3-5). Linker histone (H1͞H5) binding organizes an additional 20 bp of DNA to complete the nucleosome containing 167 bp of DNA (6, 7). Such binding determines the geometry of the DNA entering and exiting the nucleosome core particle (8). In nucleosome arrays, adjacent nucleosomes are separated by linker DNA, varying in length between 0 and 80 bp in a tissue-and species-specific manner (9, 10). In vitro, linear nucleosome arrays fold into the ''30-nm'' fiber upon increasing ionic strength (11) in a process that depends on both the integrity of the core histone N-terminal tails (12, 13) and the presence of the linker histone (14,15).During the past three decades evidence from EM (14-23), x-ray and neutron scattering (24-27), electric and photochemical dichroism (28-31), sedimentation analysis (32-35), nuclease digestion (6, 9, 36), and x-ray crystallography (4, 5, 37, 38) has led to the proposal of a number of different models for the 30-nm fiber. These models fall into two main classes: the one-start helix or solenoid models, and the two-start helix models. The solenoid models are comprised of simple one-start helices in which successive nucleosomes are adjacent in the filament and connected by linker DNA that bends into t...

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Section: Resultsmentioning

confidence: 98%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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EM measurements define the dimensions of the “30-nm” chromatin fiber: Evidence for a compact, interdigitated structure

Robinson

Fairall

Huynh

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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452

512

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“…These proteins, along with the wrapped DNA, form the basic building block of chromatin, the nucleosome [5]. Additional, linker-histone proteins are needed for compacting the chromatin fiber into condensed states [6,7,8,9,10,11]. The diameter of the chromatin fiber -30nm -is widely quoted as the dimension at physiological ionic environments, but its detailed structure is unknown.…”

Section: Chromatin Structurementioning

confidence: 99%

From Macroscopic to Mesoscopic Models of Chromatin Folding

Schlick¹

2009

Multiscale Methods

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An overview of the evolution of computer models for simulation of chromatin folding is presented. Chromatin is the protein/nucleic acid fiber that stores the genetic material in higher organisms. Many biological questions concerning the fiber structure and its dependence on internal and external factors remain a puzzle. Modeling and simulation can in theory provide molecular view for analysis, but the sheer size and range of spatial and temporal scales involved require tailored multiscale models. Our first-generation, macroscopic models ignored histone tail flexibility but generated insights into preferred zigzag configurations and folding/unfolding dynamics at univalent salt. The second-generation mesoscale models incorporated histone tail flexibility, linker histones, and the presence of divalent ions. Recent results reveal the profound compaction induced by linker histones and the polymorphic fiber structure at divalent salt environments, with a small fraction of the linker DNAs bent rather than straight for ultimate compaction. Our chromatin model can be extended further to study many important questions dealing with histone tail post-translational modifications, the effects of variations in linker DNA length and of histone variants on chromatin structure, and the nature of higher-order fiber structures. IntroductionOne of the current challenges in scientific computing is model development that entails bridging the resolution among different spatial and temporal scales. In biological applications, a wide range of spatial scales defines systems, from the quantum particles to atomic, molecular, cellular, organ, system and genome entities. Temporal scales range from sub-femtosecond for electronic motion to billions of years of evolutionary changes. As our computing power and algorithms have improved, problems of greater scientific significance can be addressed with enhanced confidence and accuracy. However, developing appropriate molecular models and simulation algorithms to answer specific biological questions that require bridging all-atom details with the macroscopic view of activity on the cellular level remains an ad-hoc endeavor which requires as much art as science.As a special volume of SIAM's journal on Multiscale Modeling and Simulation illustrated [1], multiscale biology is being developed by many varied techniques and applied to a variety of problems, such as involving protein and RNA threedimensional structures, DNA supercoiling, ribosomal motions, DNA packaging in viruses, heart muscle motion, RNA translation, or fruit fly circadian rhythm. For these applications, techniques involve hierarchical methods that transform fast, low-resolution to slower, higher-resolution models; dynamics propagation using projection of standard molecular dynamics to longer timescales using master-equation methods; rigid-body dynamics; elastic or normal-mode models; and coarse-grained studies of slow, large-scale motions and features using differential equations for global properties or statistical methods.Thi...

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Single‐molecule nucleosome remodeling by INO80 and effects of histone tails

et al. 2018

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Genome maintenance and integrity requires continuous alterations of the compaction state of the chromatin structure. Chromatin remodelers, among others the INO80 complex, help organize chromatin by repositioning, reshaping, or evicting nucleosomes. We report on INO80 nucleosome remodeling, assayed by single-molecule Foerster resonance energy transfer on canonical nucleosomes as well as nucleosomes assembled from tailless histones. Nucleosome repositioning by INO80 is a processively catalyzed reaction. During the initiation of remodeling, probed by the INO80 bound state, the nucleosome reveals structurally heterogeneous states for tailless nucleosomes (in contrast to wild-type nucleosomes). We, therefore, propose an altered energy landscape for the INO80-mediated nucleosome sliding reaction in the absence of histone tails.

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A Method for the In Vitro Reconstitution of a Defined “30nm” Chromatin Fibre Containing Stoichiometric Amounts of the Linker Histone

Cited by 168 publications

References 51 publications

EM measurements define the dimensions of the “30-nm” chromatin fiber: Evidence for a compact, interdigitated structure

EM measurements define the dimensions of the “30-nm” chromatin fiber: Evidence for a compact, interdigitated structure

From Macroscopic to Mesoscopic Models of Chromatin Folding

Single‐molecule nucleosome remodeling by INO80 and effects of histone tails

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