The effect of internucleosomal interaction on folding of the chromatin fiber

The folding of the nucleosome chain into a chromatin fiber modulates DNA accessibility and is therefore an important factor for the control of gene expression. The fiber conformation depends crucially on the interaction between individual nucleosomes. However, this parameter has not been accurately determined experimentally, and it is affected by posttranslational histone modifications and binding of chromosomal proteins. Here, the effect of different internucleosomal interaction strengths on the fiber conformation was investigated by Monte Carlo computer simulations. The fiber geometry was modeled to fit that of chicken erythrocyte chromatin, which has been examined in numerous experimental studies. In the Monte Carlo simulation, the nucleosome shape was described as an oblate spherocylinder, and a replica exchange protocol was developed to reach thermal equilibrium for a broad range of internucleosomal interaction energies. The simulations revealed the large impact of the nucleosome geometry and the nucleosome repeat length on the compaction of the chromatin fiber. At high internucleosomal interaction energies, a lateral self-association of distant fiber parts and an interdigitation of nucleosomes were apparent. These results identify key factors for the control of the compaction and higher order folding of the chromatin fiber.     

Nucleosome gaping supports a functional structure for the 30nm chromatin fiber

The biological functions played by the nucleus of eukaryotic cells and especially those involved in cellular differentiation not only depend on the genomic sequence but also on all the proteins which form the nucleo-protein complex named chromatin. The tridimensional organization of this huge polymer involves many structural levels, the most basic one being the nucleosome. Nucleosomes further organize into the so-called 30nm fiber, which, according to recent works, is likely to be the main functional level of chromatin. We wish here to propose a plausible structure for the 30nm chromatin fiber that could explain its functional role. In our model, silenced chromatin is locked by nucleosome stacking interactions. This is achieved by a conformational transition within the nucleosome core particle (NCP) which allows nucleosomes to stack along two helices without bending the DNA linkers. We used molecular modeling to check that this conformational transition was plausible. Then we proposed to modify the well-known two-angle model according to these atomic level results. The emerging picture is an allosteric behavior of the nucleosomes induced by their collective organization within the 30nm chromatin fiber.     

Nucleosome conformation: pH and organic solvent effects.

Monomer nucleosomes (nu 1) from chicken erythrocyte nuclei were examined in aqueous buffers (8 greater than pH greater than 3) and in solvent mixtures (i.e., water and ethanol, ethylene glycol, dioxane, dimethyl sulfoxide, 2-methyl-2,4-pentanediol, polyethylene glycol, sucrose, or urea). Circular dichroism, laser Raman spectroscopy of nu 1, and the fluorescence of nu 1 labeled with N-(3-pyrene) maleimide on thiol groups of H3 histone were employed to detect conformational transitions in nu 1. The results of pH studies were as follows: 5.5 greater than pH greater than 4.8, suppression of DNA ellipticity and no change of histone alpha-helix; 4.6 greater than pH greater than 4.2 an irreversible increase in the B character of DNA, a slight loss of histone alpha-helix, and a parallel loss of pyrene excimer fluorescence; 4 greater than pH, aggregation of nu 1 and protonation of the DNA bases C and A. Results obtained in the studies of nu 1 in solvent mixtures included the following: sharp conformational transitions that variously involved an increase in the B character of DNA, a slight loss of histone alpha-helix, and a loss of pyrene excimer. Different solvents required different concentrations to effect these conformational changes.     

Nucleosome acetylation sequencing to study the establishment of chromatin acetylation

The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array.     

Nucleosome segregation in chromatin replicated in the presence of cycloheximide

Ehrlich ascites tumour cells and L cells were grown in the presence of [¹⁴C]thymidine to label DNA replicated under normal conditions and were then cultured in the presence of cycloheximide and [³H]thymidine to label DNA replicated in the absence of histone synthesis, Chromatin from these cells was digested with micrococcal nuclease and with restriction endonuclease BspRI (an isoschizomer of HaeIII). The rates of digestion of the ¹⁴C-labelled and of the ³H-labelled DNA, and the size and buoyant density of the BspRI-generated chromatin fragments showed that: (1) chromatin replicated in the presence of cycloheximide contained half the normal amount of histones; (2) it did not contain long stretches of naked DNA; and (3) it was organized in nucleosomes distributed along DNA in groups of several particles separated by relatively short stretches of histone-free DNA. Control experiments showed that this could not be the result of a long-distance sliding of nucleosomes.These data suggest a bilateral mode of nucleosome segregation during DNA replication.     

The salt dependence of chicken and yeast chromatin structure. Effects on internucleosomal organization and relation to active chromatin

The ionic strength dependences of yeast and chicken erythrocyte chromatin structure have been examined by analysis of nuclear DNase I and Staphylococcal nuclease digestions done under various salt and divalent cation concentrations. The basic features of yeast DNase I profiles (intracore/intercore patterns and their 5-base pair offset) remain present under all conditions tested. However, there are changes in specific parts of the patterns. In very low salt, the intercore DNase I pattern is enhanced; even very small intercore bands can be detected. Staphylococcal nuclease intracore cleavage becomes prominent. Increasing salt enhances the large DNase I intracore bands and the frequency of spacer cleavage for both nucleases. Thus, yeast has a salt-dependent higher order structure: a chromatin fiber with a prominent spacer/core distinction in (physiological) salt; a fiber with a decreased distinction between spacer and core, i.e. a more uniform fiber, in very low salt. The salt-dependent bulk changes resemble single gene chromatin changes during gene expression and may provide a model for that process. Above bands 16.5-17.5, chicken and yeast intercore patterns are coincident. Thus, at least a fraction of chicken chromatin has discrete length spacers like yeast does. This fraction may be significant, for the prominence of the intercore pattern, and hence the apparent abundance of discrete spacers, can be significantly enhanced by digestion in very low salt. The major differences between the two chromatins are in the intracore/intercore transition region: the region is larger and more complex in chicken; ionic strength changes affect the chicken transition region more strongly. Since this region of the profile corresponds to digestion near the ends of the core, that part of the nucleosome must differ in structure and in conformational flexibility in the two chromatins.     

Nucleosome Positioning,Nucleosome Spacing and the Nucleosome Code

Abstract

Nucleosome positioning has been the subject of intense study for many years. The properties of micrococcal nuclease, the enzyme central to these studies, are discussed. The various methods used to determine nucleosome positions in vitro and in vivo are reviewed critically. These include the traditional low resolution method of indirect end-labelling, high resolution methods such as primer extension, monomer extension and nucleosome sequencing, and the high throughput methods for genome-wide analysis (microarray hybridisation and parallel sequencing). It is established that low resolution mapping yields an averaged chromatin structure, whereas high resolution mapping reveals the weighted superposition of all the chromatin states in a cell population. Mapping studies suggest that yeast DNA contains information specifying the positions of nucleosomes and that this code is made use of by the cell. It is proposed that the positioning code facilitates nucleosome spacing by encoding information for multiple alternative overlapping nucleosomal arrays. Such a code might facilitate the shunting of nucleosomes from one array to another by ATP-dependent chromatin remodelling machines.     

Nucleosome recognition and spacing by chromatin remodelling factor ISW1a

Nucleosomes are actively positioned along DNA by ATP-dependent, chromatin remodelling factors. A structural model for the ISW1a chromatin remodelling factor from Saccharomyces cerevisiae in complex with a dinucleosome substrate was constructed from the X-ray structures of ISW1a (ΔATPase) with and without DNA bound, two different cryo-EM (cryo-electron microscopy) structures of ISW1a (ΔATPase) bound to a nucleosome, and site-directed photo-cross-linking analyses in solution. The X-ray structure of ISW1a (ΔATPase) with DNA bound suggests that DNA sequence may be involved in nucleosome recognition and thereby specificity of promoter interaction. The model suggests how the highly ordered nucleosome arrays observed by mapping nucleosomes in genes and their promoter regions could be generated by a chromatin remodelling factor.     

Local geometry and elasticity in compact chromatin structure

The hierarchical packaging of DNA into chromatin within a eukaryotic nucleus plays a pivotal role in both the accessibility of genomic information and the dynamics of replication. Our work addresses the role of nanoscale physical and geometric properties in determining the structure of chromatin at the mesoscale level. We study the packaging of DNA in chromatin fibers by optimization of regular helical morphologies, considering the elasticity of the linker DNA as well as steric packing of the nucleosomes and linkers. Our model predicts a broad range of preferred helix structures for a fixed linker length of DNA; changing the linker length alters the predicted ensemble. Specifically, we find that the twist registry of the nucleosomes, as set by the internucleosome repeat length, determines the preferred angle between the nucleosomes and the fiber axis. For moderate to long linker lengths, we find a number of energetically comparable configurations with different nucleosome-nucleosome interaction patterns, indicating a potential role for kinetic trapping in chromatin fiber formation. Our results highlight the key role played by DNA elasticity and local geometry in regulating the hierarchical packaging of the genome.     

Nucleosome DNA repeat length and histone complement in a fungus exhibiting condensed chromatin

Fungal chromatins are reported to exhibit unusually short nucleosomal DNA repeat lengths. To test whether this is a phylogenetic feature of fungi or rather is correlated with an apparent absence of condensed chromatin in the organisms studied, we have examined the chromatin organization and the complement of basic nuclear proteins in the fungus Entomophthora, an organism which exhibits marked chromatin condensation. Micrococcal nuclease digestion of Entomophthora chromatin revealed a nucleosomal DNA repeat length of 197 +/- 1.2 base pairs (bp). This repeat length is 20-40 bp longer than that reported for any fungus. Entomophthora nucleosomes exhibited an HI-like protein which was much less basic than the HI histones reported for higher eukaryotes but which was similar in basicity to the HI histone reported for the fungus Neurospora. However, the nucleosomal DNA repeat length of Neurospora chromatin is reported to be unusually short, whereas that of Entomophthora was found to be typical of the repeat lengths observed for chromatins of higher eukaryotes. Thus, repeat length, at least in fungi, would not appear to be directly determined by the basicity of the fungal cognate of histone HI.