Nucleosome Repeat Length Research Articles

Acetylation Mimics within Individual Core Histone Tail Domains Indicate Distinct Roles in Regulating the Stability of Higher-Order Chromatin Structure

Nucleosome arrays undergo salt-dependent self-association into large oligomers in a process thought to recapitulate essential aspects of higher-order tertiary chromatin structure formation. Lysine acetylation within the core histone tail domains inhibits self-association, an effect likely related to its role in facilitating transcription. As acetylation of specific tail domains may encode distinct functions, we investigated biochemical and self-association properties of model nucleosome arrays containing combinations of native and mutant core histones with lysine-to-glutamine substitutions to mimic acetylation. Acetylation mimics within the tail domains of H2B and H4 caused the largest inhibition of array self-association, while modification of the H3 tail uniquely affected the stability of DNA wrapping within individual nucleosomes. In addition, the effect of acetylation mimics on array self-association is inconsistent with a simple charge neutralization mechanism. For example, acetylation mimics within the H2A tail can have either a positive or negative effect on self-association, dependent upon the acetylation state of the other tails and nucleosomal repeat length. Finally, we demonstrate that glutamine substitutions and lysine acetylation within the H4 tail domain have identical effects on nucleosome array self-association. Our results indicate that acetylation of specific tail domains plays distinct roles in the regulation of chromatin structure.

An All-Atom Model of the Chromatin Fiber Containing Linker Histones Reveals a Versatile Structure Tuned by the Nucleosomal Repeat Length

In the nucleus of eukaryotic cells, histone proteins organize the linear genome into a functional and hierarchical architecture. In this paper, we use the crystal structures of the nucleosome core particle, B-DNA and the globular domain of H5 linker histone to build the first all-atom model of compact chromatin fibers. In this 3D jigsaw puzzle, DNA bending is achieved by solving an inverse kinematics problem. Our model is based on recent electron microscopy measurements of reconstituted fiber dimensions. Strikingly, we find that the chromatin fiber containing linker histones is a polymorphic structure. We show that different fiber conformations are obtained by tuning the linker histone orientation at the nucleosomes entry/exit according to the nucleosomal repeat length. We propose that the observed in vivo quantization of nucleosomal repeat length could reflect nature's ability to use the DNA molecule's helical geometry in order to give chromatin versatile topological and mechanical properties.

Nucleosome positioning in relation to nucleosome spacing and DNA sequence‐specific binding of a protein

Nucleosome positioning is an important mechanism for the regulation of eukaryotic gene expression. Folding of the chromatin fiber can influence nucleosome positioning, whereas similar electrostatic mechanisms govern the nucleosome repeat length and chromatin fiber folding in vitro. The position of the nucleosomes is directed either by the DNA sequence or by the boundaries created due to the binding of certain trans-acting factors to their target sites in the DNA. Increasing ionic strength results in an increase in nucleosome spacing on the chromatin assembled by the S-190 extract of Drosophila embryos. In this study, a mutant lac repressor protein R3 was used to find the mechanisms of nucleosome positioning on a plasmid with three R3-binding sites. With increasing ionic strength in the presence of R3, the number of positioned nucleosomes in the chromatin decreased, whereas the internucleosomal spacings of the positioned nucleosomes in a single register did not change. The number of the positioned nucleosomes in the chromatin assembled in vitro over different plasmid DNAs with 1-3 lac operators changed with the relative position and number of the R3-binding sites. We found that in the presence of R3, nucleosomes were positioned in the salt gradient method of the chromatin assembly, even in the absence of a nucleosome-positioning sequence. Our results show that nucleosome-positioning mechanisms are dominant, as the nucleosomes can be positioned even in the absence of regular spacing mechanisms. The protein-generated boundaries are more effective when more than one binding site is present with a minimum distance of approximately 165 bp, greater than the nucleosome core DNA length, between them.

Mechanism of Histone H1-Stimulated Glucocorticoid Receptor DNA Binding In Vivo

Xenopus oocytes lack somatic linker histone H1 but contain an oocyte-specific variant, B4. The glucocorticoid receptor (GR) inducible mouse mammary tumor virus (MMTV) promoter was reconstituted in Xenopus oocytes to address the effects of histone H1. The expression of Xenopus H1o [corrected] (H1) via cytoplasmic mRNA injection resulted in H1 incorporation into in vivo assembled chromatin based on (i) the appearance of a chromatosome stop, (ii) the increased nucleosome repeat length (NRL), and (iii) H1-DNA binding assayed by chromatin immunoprecipitation (ChIP). The H1 effect on the NRL was saturable and hence represents H1-binding to a specific site. A subsaturating level of H1 enhanced the hormone-dependent binding of GR to the glucocorticoid response elements (GREs) and the hormone-dependent MMTV transcription while it reduced the access to DNA as revealed by micrococcal nuclease (MNase) analysis. These H1 effects were lost at higher levels of H1. ChIP and MNase analysis revealed a hormone-dependent dissociation of H1 from the activated chromatin domain. The proposed mechanism of H1-induced GR binding is based on two effects: (i) a GR-induced asymmetric distribution of H1 in favor of inactive chromatin and (ii) an H1-induced reduction in DNA access. These effects results in increased concentration of free GR and, hence, in increased GR-GRE binding.

Chromatin dynamics of unfolding and refolding controlled by the nucleosome repeat length and the linker and core histones

Chromatin is composed of genomic DNA and histones, forming a hierarchical architecture in the nucleus. The chromatin hierarchy is common among eukaryotes despite different intrinsic properties of the genome. To investigate an effect of the differences in genome organization, chromatin unfolding processes were comparatively analyzed using Schizosaccaromyces pombe, Saccharomyces cerevisiae, and chicken erythrocyte. NaCl titration showed dynamic changes of the chromatin. 400-1000 mM NaCl facilitated beads with approximately 115 nm in diameter in S. pombe chromatin. A similar transition was also observed in S. cerevisiae chromatin. This process did not involve core histone dissociation from the chromatin, and the persistence length after the transition was approximately 26 nm for S. pombe and approximately 28 nm for S. cerevisiae, indicating a salt-induced unfolding to "beads-on-a-string" fibers. Reduced salt concentration recovered the original structure, suggesting that electrostatic interaction would regulate this discrete folding-unfolding process. On the other hand, the linker histone was extracted from chicken chromatin at 400 mM NaCl, and AFM observed the "beads-on-a-string" fibers around a nucleus. Unlike yeast chromatin, therefore, this unfolding was irreversible because of linker histone dissociation. These results indicate that the chromatin unfolding and refolding depend on the presence and absence of the linker histone, and the length of the linker DNA.

In Silico Approaches Reveal the Potential for DNA Sequence-dependent Histone Octamer Affinity to Influence Chromatin Structure in Vivo

Nucleosome positioning signals embedded within the DNA sequence have the potential to influence the detailed structure of the higher-order chromatin fibre. In two previous studies of long stretches of DNA, encompassing the chicken beta-globin and ovine beta-lactoglobulin genes, respectively, we mapped the relative affinity of every site for the core histone octamer. In both cases a periodic arrangement of the in vitro positioning sites suggests that they might influence the folding of a nucleosome chain into higher-order structure; this hypothesis was borne out in the case of the beta-lactoglobulin gene, where the distribution of the in vitro positioning sites is related to the positions nucleosomes actually occupy in sheep liver cells. Here, we have exploited the in vitro nucleosome positioning datasets to simulate nucleosomal organisation using in silico approaches. We use the high-resolution, quantitative positioning maps to define a one-dimensional positioning energy lattice, which can be populated with a defined number of nucleosomes. Monte Carlo techniques are employed to simulate the behaviour of the model at equilibrium to produce a set of configurations, which provide a probability-based occupancy map. Employing a variety of techniques we show that the occupancy maps are a sensitive function of the histone octamer density (nucleosome repeat length) and find that a minimal change in this property can produce dramatic localised changes in structure. Although simulations generally give rise to regular periodic nucleosomal arrangements, they often show octamer density-dependent discontinuities, which tend to co-localise with sequences that adopt distinctive chromatin structure in vivo. Furthermore, the overall organisation of simulated chromatin structures are more closely related to the situation in vivo than is the original in vitro positioning data, particularly at a nucleosome density corresponding to the in vivo state. Although our model is simplified, we argue that it provides a unique insight into the influence that DNA sequence can have in determining chromatin structure and could serve as a useful basis for the incorporation of other parameters.

Topological Analysis of Plasmid Chromatin from Yeast and Mammalian Cells

Yeast has proven to be a powerful system for investigation of chromatin structure. However, the extent to which yeast chromatin can serve as a model for mammalian chromatin is limited by the significant number of differences that have been reported. To further investigate the structural relationship between the two chromatins, we have performed a DNA topological analysis of pRSSVO, a 5889 base-pair plasmid that can replicate in either yeast or mammalian cells. When grown in mammalian cells, pRSSVO contains an average of 33 negative supercoils, consistent with one nucleosome per 181 bp. This is close to the measured nucleosome repeat length of 190 bp. However, when grown in yeast cells, pRSSVO contains an average of only 23 negative supercoils, which is indicative of only one nucleosome per 256 bp. This is dramatically different from the measured nucleosome repeat length of 165 bp. To account for these observations, we suggest that yeast chromatin is composed of relatively short ordered arrays of nucleosomes with a repeat of 165 bp, separated by substantial gaps, possibly corresponding to regulatory regions.

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

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.

Aspects of large-scale chromatin structures in mouse liver nuclei can be predicted from the DNA sequence

The large amount of non-coding DNA present in mammalian genomes suggests that some of it may play a structural or functional role. We provide evidence that it is possible to predict computationally, from the DNA sequence, loci in mouse liver nuclei that possess distinctive nucleosome arrays. We tested the hypothesis that a 100 kb region of DNA possessing a strong, in-phase, dinucleosome period oscillation in the motif period-10 non-T, A/T, G, should generate a nucleosome array with a nucleosome repeat that is one-half of the dinucleosome oscillation period value, as computed by Fourier analysis of the sequence. Ten loci with short repeats, that would be readily distinguishable from the pervasive bulk repeat, were predicted computationally and then tested experimentally. We estimated experimentally that less than 20% of the chromatin in mouse liver nuclei has a nucleosome repeat length that is 15 bp, or more, shorter than the bulk repeat value of 195 ± bp. All 10 computational predictions were confirmed experimentally with high statistical significance. Nucleosome repeats as short as 172 ± 5 bp were observed for the first time in mouse liver chromatin. These findings may be useful for identifying distinctive chromatin structures computationally from the DNA sequence.

Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length

Despite a great deal of attention over many years, the structural and functional roles of the linker histone H1 remain enigmatic. The earlier concepts of H1 as a general transcriptional inhibitor have had to be reconsidered in the light of experiments demonstrating a minor effect of H1 deletion in unicellular organisms. More recent work analysing the results of depleting H1 in mammals through genetic knockouts of selected H1 subtypes in the mouse has shown that cells and tissues can tolerate a surprisingly low H1 content. One common feature of H1-depleted nuclei is a reduction in nucleosome repeat length (NRL). Moreover, there is a robust linear relationship between H1 stoichiometry and NRL, suggesting an inherent homeostatic mechanism that maintains intranuclear electrostatic balance. It is also clear that the 1 H1 per nucleosome paradigm for higher eukaryotes is the exception rather than the rule. This, together with the high mobility of H1 within the nucleus, prompts a reappraisal of the role of linker histone as an obligatory chromatin architectural protein.

Analysis of Chromatin Structure in the Control Regions of the Chlamydomonas HSP70A and RBCS2 Genes

We have used DNaseI and micrococcal nuclease sensitivity assays to determine the chromatin structures in the control regions of the Chlamydomonas reinhardtii HSP70A and RBCS2 genes. Both genes appear to be organized into nucleosome arrays, which exhibit shorter nucleosome repeat lengths than bulk chromatin. In HSP70A we have identified up to four confined DNaseI hypersensitive sites, three of them localize to the promoter region, a fourth one to the fourth intron. Three hypersensitive sites map close to putative heat shock elements, one close to a CCAAT-box. All hypersensitive sites are located to internucleosomal linkers. Alternative nucleosome positions at half-nucleosomal phasing were constitutively detected in the HSP70A promoter region, indicating local chromatin remodelling. Upon heat shock, dramatic changes in the nucleosome structure of HSP70A were detected that particularly affected the promoter, but also a region within the fourth intron. In contrast, light induction entailed no change in HSP70A chromatin. In the RBCS2 control region we identified a strong DNaseI hypersensitive site that maps close to a CCAAT-box. This site forms the boundary of a nucleosome array with a region of approximately 700 bp apparently devoid of nucleosomes. This study demonstrates that chromatin structure may be determined readily at fairly high resolution in Chlamydomonas, suggesting this organism as a well-suited model for studying the role of chromatin structure on gene expression in photosynthetic eukaryotes.

Distinct activities of CHD1 and ACF in ATP-dependent chromatin assembly

CHD1 is a chromodomain-containing protein in the SNF2-like family of ATPases. Here we show that CHD1 exists predominantly as a monomer and functions as an ATP-utilizing chromatin assembly factor. This reaction involves purified CHD1, NAP1 chaperone, core histones and relaxed DNA. CHD1 catalyzes the ATP-dependent transfer of histones from the NAP1 chaperone to the DNA by a processive mechanism that yields regularly spaced nucleosomes. The comparative analysis of CHD1 and ACF revealed that CHD1 assembles chromatin with a shorter nucleosome repeat length than ACF. In addition, ACF, but not CHD1, can assemble chromatin containing histone H1, which is involved in the formation of higher-order chromatin structure and transcriptional repression. These results suggest a role for CHD1 in the assembly of active chromatin and a function of ACF in the assembly of repressive chromatin.

Granulocyte-macrophage colony-stimulating factor enhancer activation requires cooperation between NFAT and AP-1 elements and is associated with extensive nucleosome reorganization.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is activated by an NFAT-dependent enhancer forming an inducible DNase I hypersensitive (DH) site. The enhancer core comprising the DH site contains the GM330 and GM420 elements that bind NFAT and AP-1 cooperatively. Here we demonstrate that both elements are essential for enhancer activity and that Sp1 and AML1 sites in the enhancer become occupied in vivo only after activation. Chromatin structure analysis revealed that the GM-CSF enhancer core elements are divided between two adjacent nucleosomes that become destabilized and highly accessible after activation. Inducible chromatin reorganization was not restricted to the enhancer core but extended across a 3-kb domain of mobilized nucleosomes, within which the nucleosome repeat length was compressed from approximately 185 to 150 bp. The GM420 element is a high-affinity site that binds NFAT independently of AP-1 but depends on the linked AP-1 site for enhancer function. Nevertheless, just the NFAT motif from the GM420 element was sufficient to form a DH site within chromatin even in the absence of the AP-1 site. Hence, NFAT has the potential to cooperate with other transcription factors by promoting chromatin remodelling and increasing accessibility at inducible regulatory elements.

Loopy for telomeres

On page 161, Nikitina and Woodcock present the first isolation of telomeres complete with their chromatin protein complement. Figure TRF1-labeled chromatin reveals that telomeres form loops. Telomeres protect chromosome ends from degradation and shortening, but they must themselves be protected from repair systems that recognize free DNA ends. Images of telomere DNA, lacking its chromatin components, suggested that its protection might be enhanced by looping and insertion of the telomere's single-stranded overhang into the double-stranded telomeric repeats. Wrapping this loop up in chromatin proteins probably hides the overhang from agents that detect free ends, but because telomere chromatin is difficult to extract from the nuclear matrix, whether chromatin looping occurs was unknown. The authors have conquered the chromatin extraction problem by using two types of blood cells that have fewer proteins gluing the chromatin to the matrix. After digesting away nontelomeric DNA, the authors labeled the remainder with biotinylated TRF1, a telomere-binding protein, thus identifying the telomeric chromatin. Like naked telomeric DNA, many of the TRF1-labeled structures formed loops. As expected, the chromatin loop is smaller than that of the naked DNA due to nucleosomal packing. Nucleosomal repeat length is shorter on telomeres than on other DNA, but the authors did not note any changes in fiber morphology resulting from the difference. Future 3-D reconstructions will address this problem. An up-close look is also needed to determine how the overhang associates with the double-stranded DNA and how the nucleosome arrangement allows the loop to open and close during replication. ▪

Acf1 confers unique activities to ACF/CHRAC and promotes the formation rather than disruption of chromatin in vivo.

Chromatin assembly is required for the duplication of chromosomes. ACF (ATP-utilizing chromatin assembly and remodeling factor) catalyzes the ATP-dependent assembly of periodic nucleosome arrays in vitro, and consists of Acf1 and the ISWI ATPase. Acf1 and ISWI are also subunits of CHRAC (chromatin accessibility complex), whose biochemical activities are similar to those of ACF. Here we investigate the in vivo function of the Acf1 subunit of ACF/CHRAC in Drosophila. Although most Acf1 null animals die during the larval-pupal transition, Acf1 is not absolutely required for viability. The loss of Acf1 results in a decrease in the periodicity of nucleosome arrays as well as a shorter nucleosomal repeat length in bulk chromatin in embryos. Biochemical experiments with Acf1-deficient embryo extracts further indicate that ACF/CHRAC is a major chromatin assembly factor in Drosophila. The phenotypes of flies lacking Acf1 suggest that ACF/CHRAC promotes the formation of repressive chromatin. The acf1 gene is involved in the establishment and/or maintenance of transcriptional silencing in pericentric heterochromatin and in the chromatin-dependent repression by Polycomb group genes. Moreover, cells in animals lacking Acf1 exhibit an acceleration of progression through S phase, which is consistent with a decrease in chromatin-mediated repression of DNA replication. In addition, acf1 genetically interacts with nap1, which encodes the NAP-1 nucleosome assembly protein. These findings collectively indicate that ACF/CHRAC functions in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus.

Counting Nucleosomes in Living Cells with a Combination of Fluorescence Correlation Spectroscopy and Confocal Imaging

Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total H2Bs carry the fluorescent tag and an increased nucleosome repeat length of 204 bp for the fluorescent cells. In vivo, the mobility and distribution of H2B-EYFP were studied with a combination of FCS and confocal imaging. With FCS, concentration and brightness of nascent molecules were measured in the cytoplasm, while in the nucleoplasm a background of mobile fluorescent histones was determined by continuous photobleaching. Combining these results allows converting confocal fluorescence images of nuclei into calibrated nucleosome density maps. Absolute nucleosome concentrations in interphase amount up to 250 μM locally, with mean values of 140(±28)μM, suggesting that a condensation-controlled regulation of site accessibility takes place at length scales well below 200 nm.

DNase I digestion reveals alternating asymmetrical protection of the nucleosome by the higher order chromatin structure.

DNase I was used to probe the higher order chromatin structure in whole nuclei. The digestion profiles obtained were the result of single-stranded cuts and were independent of pH, type of divalent ion and chromatin repeat length. Furthermore, the protection from digestion of the DNA at the entry/exit points on the nucleosome was found to be caused not by the H1/H5 histone tails, but by the compact structure that these proteins support. In order to resolve symmetry ambiguities, DNase I digestion fragments over several nucleosome repeat lengths were analysed quantitatively and compared with computer simulations using combinations of the experimentally obtained rate constants (some of which were converted to 0 to simulate steric protection from DNase I digestion). A clear picture of precisely defined, alternating, asymmetrically protected nucleosomes emerged. The linker DNA is inside the fibre, while the nucleosomes are positioned above and below a helical path and/or with alternating orientation towards the dyad axis. The dinucleosomal modulation of the digestion patterns comes from alternate protection of cutting sites inside the nucleosome and not from alternating exposure to the enzyme of the linker DNA.

Hormone activation induces nucleosome positioning in vivo.

The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone- and receptor-dependent activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the transition in chromatin structure following hormone activation. This revealed two novel findings: hormone activation led to the establishment of specific translational positioning of nucleosomes despite the lack of significant positioning in the inactive state; and, in the active promoter, a subnucleosomal particle encompassing the glucocorticoid receptor (GR)-binding region was detected. The presence of only a single GR-binding site was sufficient for the structural transition to occur. Both basal promoter elements and ongoing transcription were dispensable. These data reveal a stepwise process in the transcriptional activation by glucocorticoid hormone.

Deletion of the unique gene encoding a typical histone H1 has no apparent phenotype in Aspergillus nidulans.

We have cloned the H1 histone gene (hhoA) of Aspergillus nidulans. This single-copy gene codes for a typical linker histone with one central globular domain. The open reading frame is interrupted by six introns. The position of the first intron is identical to that of introns found in some plant histones. An H1-GFP fusion shows exclusive nuclear localization, whereas chromosomal localization can be observed during condensation at mitosis. Surprisingly, the deletion of hhoA results in no obvious phenotype. The nucleosomal repeat length and susceptibility to micrococcal nuclease digestion of A. nidulans chromatin are unchanged in the deleted strain. The nucleosomal organization of a number of promoters, including in particular the strictly regulated niiA-niaD bidirectional promoter is not affected.

Identification of DNaseI hypersensitive sites within nuclei.

AbstractThe vast bulk of the genome of eukaryotic cells exists as a highly regular array of nucleosomes. A single nucleosome comprises 146 bp of DNA wrapped around a histone core particle. Arrays of nucleosomes usually exist as condensed chromatin fibers with a nucleosome repeat length of about 200 bp. The DNA within condensed chromatin is for the most part relatively inaccessible to proteins, such as nucleases. However, about 1% of the genome exists as discrete regions of decondensed chromatin termed DNaseI hypersensitive (DH) sites (1) which provide greatly increased access to factors that interact with DNA. DH sites represent local perturbations of the nucleosome array and probably arise via the reorganization of single nucleosomes within the chromatin fiber. Wherease DH sites exist as open regions of chromatin in nuclei, they are not typically hypersensitive to DNaseI within protein-free DNA.KeywordsSodium Dodecyl SulfateRestriction Enzyme SiteChromatin FiberRestriction Enzyme FragmentNucleus IsolationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.