The Core Histone N Termini Function Independently of Linker Histones during Chromatin Condensation

Carruthers, Lenny M.; Hansen, Jeffrey C.

doi:10.1074/jbc.m006801200

Cited by 114 publications

(85 citation statements)

References 66 publications

(91 reference statements)

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“…The production of very homogeneous and tightly folded 30-nm chromatin fibers from long nucleosome arrays with different nucleosome repeat lengths has permitted us to obtain accurate measurements of fiber dimensions and hence nucleosome packing ratios. Importantly, unlike previous chromatin reconstitution experiments (35,38,44), our nucleosome arrays contain a native-like complement of linker histone (11), which is critical for obtaining correct folding and maximal compaction (15,32,45). We find that over the range of nucleosome repeat lengths analyzed, there are two discrete classes of fiber structure, one 33 nm in diameter and with Ϸ11 nucleosomes per 11 nm, and the other Ϸ44 nm in diameter and with Ϸ15 nucleosomes per 11 nm.…”

Section: Discussion Chromatin Fibers Form Two Discrete Structural Clamentioning

confidence: 77%

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.

450

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 Clamentioning

confidence: 77%

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.

450

512

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“…This result indicates that Sir3p binding to nucleosomal substrates and naked DNA was not DNA sequence-dependent. Importantly, rSir3p-dependent assembly of supershifted species also occurred in 50-150 mM NaCl and 2 mM MgCl 2 (data not shown), demonstrating that Sir3p can bind to nucleosomal arrays under ionic conditions where the arrays are extensively folded (24)(25)(26)(27).…”

Section: Resultsmentioning

confidence: 93%

“…The 12-mer nucleosomal arrays (208-12 NA) used in these experiments were obtained by reconstituting chicken erythrocyte histone octamers onto a DNA template (208-12 DNA) consisting of 12 tandem repeats of 208-1 DNA (23). The intrinsic structural dynamics of 208-12 NA have been characterized extensively both in the absence (24,25) and presence (26,27) of linker histones, providing a firm foundation for interpreting the effects of Sir3p binding on higher order chromatin structure. Incubation of Sir3p with 208-12 NA at r Sir3p ϭ 0.1-25 is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Sir3-dependent assembly of supramolecular chromatin structuresin vitro

Georgel

DeBeer

Pietz

et al. 2001

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

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Baculovirus-expressed recombinant Sir3p (rSir3p) has been purified to near homogeneity, and its binding to naked DNA, mononucleosomes, and nucleosomal arrays has been characterized in vitro. At stoichiometric levels rSir3p interacts with intact nucleosomal arrays, mononucleosomes, and naked DNA, as evidenced by formation of supershifted species on native agarose gels. Proteolytic removal of the core histone tail domains inhibits but does not completely abolish rSir3p binding to nucleosomal arrays. The linker DNA in the supershifted complexes remains freely accessible to restriction endonuclease digestion, suggesting that both the tail domains and nucleosomal DNA contribute to rSir3p-chromatin interactions. Together these data indicate that rSir3p cross-links individual nucleosomal arrays into supramolecular assemblies whose physical properties transcend those of typical 10-nm and 30-nm fibers. Based on these data we hypothesize that Sir3p functions, at least in part, by mediating reorganization of the canonical chromatin fiber into functionally specialized higher order chromosomal domains.silencing ͉ nucleosome

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“…It has been shown that higher-order folding of nucleosomal arrays is dependent on the core histone Ntermini rather than on linker histones (17)(18)(19)(20)(21). Chromatin condensation has been observed in vitro in the absence of linker histones (22), and it has been hypothesized that linker histone binding does not direct the formation of folded chromatin structure (23,24). Linker histone knockout experiments carried out in the protozoan Tetrahymena showed that histone H1 is not essential for nuclear assembly and cell survival, but it seems to be involved in the regulation of specific gene expression (25)(26)(27).…”

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confidence: 99%

Histone H1 and chromatin interactions in human fibroblast nuclei after H1 depletion and reconstitution with H1 subfractions

et al. 2004

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Background:Linker histones constitute a family of lysinerich proteins associated with nucleosome core particles and linker DNA in eukaryotic chromatin. In permeabilized cells, they can be extracted from nuclei by using salt concentration in the range of 0.3 to 0.7 M. Although other nuclear proteins are also extracted at 0.7 M salt, the remaining nucleus represents a template that is relatively intact. Methods: A cytochemical method was used to study the affinity of reconstituted linker histones for chromatin in situ in cultured human fibroblasts. We also investigated their ability to condense chromatin by using DNA-specific osmium ammine staining for electron microscopy. Results: Permeabilized and H1-depleted fibroblast nuclei were suitable for the study of linker histone-chromatin

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The Core Histone N Termini Function Independently of Linker Histones during Chromatin Condensation

Cited by 114 publications

References 66 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

Sir3-dependent assembly of supramolecular chromatin structuresin vitro

Histone H1 and chromatin interactions in human fibroblast nuclei after H1 depletion and reconstitution with H1 subfractions

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