Orientation of nucleosomes and linker DNA in calf thymus chromatin determined by photochemical dichroism

Mitra, Sekhar; Sen, Dipankar; Crothers, Donald M.

doi:10.1038/308247a0

Cited by 43 publications

(28 citation statements)

References 27 publications

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“…To avoid steric clashes the model also incorporates a nucleosome tilt of 17°in relation to the fiber axis. This value corresponds well to the range (13°to 38°) measured for chromatin isolated from a number of different species by using both electric (28,29) and photochemical dichroism (30,31). The proposed nucleosome packing arrangement can easily be extended to the 44-nm fiber because the concomitant increase in volume and mass per unit length would result in an essentially constant nucleosome density.…”

Section: Discussion Chromatin Fibers Form Two Discrete Structural Clasupporting

confidence: 83%

“…During the past three decades evidence from EM (14-23), x-ray and neutron scattering (24)(25)(26)(27), electric and photochemical dichroism (28)(29)(30)(31), sedimentation analysis (32)(33)(34)(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.…”

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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.

449

512

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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: Discussion Chromatin Fibers Form Two Discrete Structural Clasupporting

confidence: 83%

mentioning

confidence: 99%

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.

449

512

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“…These values are substantially greater than the average curvature of DNA wrapped around the histone octamer (54,55) and indicate that HMG-D bound DNA is not smoothly curved. In the context of linker DNA, such a state would be consistent with both the lack of UV-induced thymine dimer formation in the linker (56) and also, with evidence from electric dichroism studies, that the trajectory of linker DNA differs from that of DNA bound to the core histones (57). Of particular relevance are the observations of Hamiche et al (58), who showed that, in the presence of histone H1 derivatives containing a major proportion of the basic C-terminal domain, the linker DNA enters and leaves a single chromatosome as a straight rod approximately perpendicular to the superhelical axis.…”

Section: A Role For Hmg-d As a Linkersupporting

confidence: 64%

HMG-D and Histone H1 Interplay during Chromatin Assembly and Early Embryogenesis

Ner¹,

Blank²,

Pérez‐Parallé³

et al. 2001

Journal of Biological Chemistry

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HMG-D is an abundant chromosomal protein associated with condensed chromatin during the first nuclear cleavage cycles of the developing Drosophila embryo. We previously suggested that HMG-D might substitute for the linker histone H1 in the preblastoderm embryo and that this substitution might result in the characteristic less compacted chromatin. We have now studied the association of HMG-D with chromatin using a cellfree system for chromatin reconstitution derived from The vertebrate high mobility group proteins of the HMGB class (formerly termed HMG1/2) (1) are relatively abundant nuclear DNA-binding proteins that bend DNA substantially and appear to act primarily as architectural facilitators in the assembly of nucleoprotein complexes (recently reviewed in Refs. 2-4). There is direct evidence that these proteins facilitate the binding of transcription factors to their cognate sites both in vitro and in vivo, but any involvement in the assembly of histone-DNA complexes is less well documented. Circumstantial evidence indicates that certain functions of the vertebrate HMGB proteins (5-7) may parallel those of the linker histone H1 (8). Both have been shown to bind to linker DNA sequences (7, 9, 10), and both stabilize and bind to four-way junctions (11)(12)(13)(14). HMG-D is one of two Drosophila proteins closely related to the vertebrate HMGB proteins but in contrast to these proteins contains only a single HMG DNA-binding domain (6,15,16). The HMG domain is followed by a region that has basic sequences similar to the C-terminal domain of histone H1 and a short C-terminal acidic stretch also seen in HMGB proteins. The NMR structure of the HMG domain from HMG-D is very similar to the previously determined structure of the B domain of HMG1, which revealed the characteristic L-shaped fold formed by three ␣-helices (17-21).High mobility group proteins of the HMGB family bind DNA with little sequence specificity but recognize structural features in DNA (22-24), including cruciforms, kinks, DNA bulges, and bends (22,23,(25)(26)(27)(28). DNA footprint analysis and mutagenesis experiments suggest that HMG domain proteins bind in the minor groove (29). In addition, both crystallization and NMR studies show that HMG-D bending is achieved by the intercalation of hydrophobic residues at two different base steps (21, 30).The addition of histone H1 protein to an extended array of nucleosome core particles promotes its folding into a fiber with an approximate width of 30 nm (31-33). During early Drosophila development, histone H1 is not detectable (34 -36) until nuclear cycles 7/8 (37). Therefore, H1 is not involved in chromatin condensation in these earliest phases of Drosophila development characterized by rapid condensation-decondensation cycles. We have previously suggested that HMG-D could function as a linker protein in the absence of histone H1 in Drosophila (37). In support of this hypothesis, Wolffe and colleagues (10, 38) have postulated a similar role for the Xenopus HMG B1 protein. Thus, as the maternal pool of H...

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“…One parameter of isolated compact fibers that has been estimated from electric dichroism measurements is the tilt angle of the nucleosomal disk with respect to the fiber axis (24)(25)(26). In the modeled system, there is a simple linear relationship between ( and nucleosome tilt, values of 00 (and 3600) for , ( resulting in nucleosomes oriented perpendicular to the fiber axis, whereas for (3= 1800, the nucleosome disks are parallel to the axis (see Fig.…”

Section: Resultsmentioning

confidence: 99%

A chromatin folding model that incorporates linker variability generates fibers resembling the native structures.

Woodcock

Grigoryev

Horowitz

et al. 1993

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

241

230

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The "30-nm" chromatin fibers, as observed in eukaryotic nuclei, are considered a discrete level in a hierarchy of DNA folding. At present, there is considerable debate as to how the nudeosomes and linker DNA are organized within chromatin fibers, and a number of models have been proposed, many of which are based on helical symmetry and imply specific contacts between nucleosomes. However, when observed in nuclei or after isolation, chromatin fibers show considerable structural irregularity. In the present study, chromatin folding is considered solely in terms of the known properties of the nudeosome-linker unit, taking into account the relative rotation between consecutive nucleosomes that results from the helical twist of DNA. Model building based on this premise, and with a constant length of linker DNA between consecutive nucleosomes, results in a family of fiber-and ribbon-like structure. When the linker length between nucleosomes is aflowed to vary, as occurs in nature, fibers showing the types of irregulaity observed in nuclei and in isolated chromatin are created. The potential application of the model in determining the three-dimensional organization of chromatin in which nucleosome positions are known is discussed.The genome of most eukaryotes is complexed with proteins to form chromatin (1). Under low salt conditions in vitro, the complex assumes its simplest conformation and is seen as a beaded chain of 11-nm (diameter) nucleosomes (e.g., ref.2). As the ionic strength of the medium is raised, the nucleosomal chain condenses, eventually forming a compact fiber 30-40 nm in diameter (1-3). These compact fibers observed in isolated chromatin in vitro are presumed to be related to similar structures seen in thin sections of certain types of nuclei such as nucleated erythrocytes (e.g., refs. 4 and 5). X-ray scattering studies of whole cells and nuclei often show 30-to 45-nm reflections that have been interpreted as arising from the center-to-center spacing of compact chromatin fibers (6). These results suggest that the compact chromatin fiber constitutes a distinct level of chromatin organization, especially for transcriptionally inactive chromatin. In this context, the architecture of the fiber would implicitly define the substrate for the regulatory events that lead to chromatin unfolding, a prerequisite for transcription.Attempts to deduce the structure of compact chromatin fibers have resulted in a number of proposals that include both symmetrical and nonsymmetrical arrangements of nucleosomes (reviewed in ref. 7). There is evidence suggesting some degree of symmetry in fiber structure: an intermediate conformation between the nucleosome chain and the compact chromatin fiber is a "zig-zag ribbon" (2) that can display considerable regularity (8), and isolated fibers prepared for electron microscopy contain regions with a limited amount of internal order (2, 3, 9-11). All proposed model structuresThe publication costs of this article were defrayed in part by page charge payment. This article must ...

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Orientation of nucleosomes and linker DNA in calf thymus chromatin determined by photochemical dichroism

Cited by 43 publications

References 27 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

HMG-D and Histone H1 Interplay during Chromatin Assembly and Early Embryogenesis

A chromatin folding model that incorporates linker variability generates fibers resembling the native structures.

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