Histone variants and nucleosome deposition pathways

In this issue of Molecular Cell, Ahmad and Henikoff show that the replication-independent pathway of chromatin assembly in vivo can discriminate between different histone variants on the basis of their primary amino acid sequences. These results have important implications for chromatin remodeling and epigenetic imprinting.     

Histone proteomics and the epigenetic regulation of nucleosome mobility

Chromatin structure plays a vital role in the transmission of heritable gene expression patterns. The recent application of mass spectrometry to histone biology provides several striking insights into chromatin regulation. The continuing identification of new histone post-translational modifications is revolutionizing the ways in which we think about how access to genomic DNA is controlled. While post-translational modifications of the flexible histone tails continue to be an active area of investigation, the recent discovery of multiple modifications in the structured globular domains of histones provides new insights into how the nucleosome works. Recent experiments underscore the importance of a subgroup of these modifications: those that regulate histone-DNA interactions on the lateral surface of the nucleosome. This information highlights an emerging new paradigm in chromatin control, that of the epigenetic regulation of nucleosome mobility.     

Histone hyperacetylation: its effects on nucleosome conformation and stability

We have prepared nucleosome particles from HeLa cells that have been subjected to butyrate treatment. Fractions containing different levels of acetylation have been obtained within the range 7-17 acetyl groups per nucleosome. We have put special emphasis in the characterization of the particles with the highest level of histone acetylation. At low to physiological ionic strengths, these nucleosomes exhibit only small differences in hydrodynamic behavior and circular dichroism from control particles with minimal acetylation. There are, however, significant differences in thermal denaturation and nuclease sensitivity. In terms of stability toward high salt, the hyperacetylated and control particles behave identically. A model that reconciles these results is proposed. The major conclusion from our results, however, is that, at physiological ionic strength and in the absence of factors other than acetylation, the highly hyperacetylated nucleosomes remain essentially folded.     

Histone acetylation status and DNA sequence modulate ATP-dependent nucleosome repositioning

A cell-free system derived from Drosophila embryos was used to investigate positioning of nucleosomes on specific DNA sequences. This system can be used to reconstitute differently acetylated nucleosome arrays possessing ATP-dependent dynamic properties that are not observed with chromatin assembled from pure components. Nucleosome positioning on different DNA sequences was studied by restriction endonuclease assay. The sequence of DNA and the acetylation status of histones had profound effects on the distribution of nucleosomes, suggesting their cooperative effect on nucleosome repositioning.     

Histone acetylation reduces nucleosome core particle linking number change

Nucleosome core particles differing in their levels of histone acetylation have been formed on a closed circular DNA that contains a tandemly repeated 207 bp nucleosome positioning sequence. The effect of acetylation on the linking number per nucleosome particle has been determined. With increasing levels of acetylation, the negative linking number change per nucleosome decreases from -1.04 +/- 0.08 for control to -0.82 +/- 0.05 for highly acetylated nucleosomes. These results indicate that histone acetylation has the ability to release negative supercoils previously constrained by nucleosomes into a closed chromatin loop and in effect function as a eukaryotic gyrase.     

Histone H1, polylysine and spermine--factors of nucleosome assembly in vitro

We studied nucleosome assemble in vitro in a system containing the relaxed DNA CoIE1, core histones and a crude extract Drosophila embryos. Supercoiling is a criterium for making conclusions about forming nucleosomes. Supercoiling raises more if nucleosome assemble takes place in the presence of histone H1, polylysine of the 20 000 molecular weight or spermine. These agents do not stimulate the relaxation, and they are more effective when they are added earlier. Thus histone H1, spermine and polylysine can facilitate nucleosome assemble in vitro and with two former agents it may be possibly that the same process takes place in vivo.     

Histone protein surface accessibility dictates direction of RSC-dependent nucleosome mobilization

Chromatin remodeling enzymes use energy derived from ATP hydrolysis to mobilize nucleosomes and alter their structure to facilitate DNA access. The Remodels the Structure of Chromatin (RSC) complex has been extensively studied, yet aspects of how this complex functionally interacts with nucleosomes remain unclear. We introduce a steric mapping approach to determine how RSC activity depends on interaction with specific surfaces within the nucleosome. We find that blocking SHL + 4.5/–4.5 via streptavidin binding to the H2A N-terminal tail domains results in inhibition of RSC nucleosome mobilization. However, restriction enzyme assays indicate that remodeling-dependent exposure of an internal DNA site near the nucleosome dyad is not affected. In contrast, occlusion of both protein faces of the nucleosome by streptavidin attachment near the acidic patch completely blocks both remodeling-dependent nucleosome mobilization and internal DNA site exposure. However, we observed partial inhibition when only one protein surface is occluded, consistent with abrogation of one of two productive RSC binding orientations. Our results indicate that nucleosome mobilization requires RSC access to the trailing but not the leading protein surface, and reveals a mechanism by which RSC and related complexes may drive unidirectional movement of nucleosomes to regulate cis-acting DNA sequences in vivo.     

The core histone tail domains contribute to sequence-dependent nucleosome positioning

The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin.     

Histone variant H2ABbd confers lower stability to the nucleosome

The histone H2ABbd is a novel histone variant of H2A with a totally unknown function. We have investigated the behaviour of the H2ABbd nucleosomes. Nucleosomes were reconstituted with recombinant histone H2ABbd and changes in their conformations at different salt concentrations were studied by analytical centrifugation. The data are in agreement with H2ABbd being less tightly bound compared with conventional H2A in the nucleosome. In addition, stable cell lines expressing either green fluorescent protein (GFP)-H2A or GFP-H2ABbd were established and the mobility of both fusions was measured by fluorescence recovery after photobleaching. We show that GFP-H2ABbd exchanges much more rapidly than GFP-H2A within the nucleosome. The reported data are compatible with a lower stability of the variant H2ABbd nucleosome compared with the conventional H2A particle.     

Histone hyperacetylation. Its effects on nucleosome core particle transitions.

Effects of histone hyperacetylation on transitions of HeLa cell nucleosome core particles were studied. The transitions examined were induced by low salt concentrations, pH, temperature, and nondissociating high salt. Effects of salt dissociation were also examined. The low-salt transition was found to shift to higher ionic strength by approximately three fold for hyperacetylated particles, a change which may be due simply to the increased overall negative charge on the particles caused by acetylation of lysine residues. Some differences were also seen in the way in which core particles refold after exposure to very low salt (which induces a nonreversible change in the particles). Otherwise no significant effects of hyperacetylation were observed.     

Histone acetylation reduces H1-mediated nucleosome interactions during chromatin assembly

During chromatin replication and nucleosome assembly, newly synthesized histone H4 is acetylated before it is deposited onto DNA, then deacetylated as assembly proceeds. In a previous study (Perry and Annunziato, Nucleic Acids Res. 17, 4275 [1989]) it was shown that when replication occurs in the presence of sodium butyrate (thereby inhibiting histone deacetylation), nascent chromatin fails to mature fully and instead remains preferentially sensitive to DNaseI, more soluble in magnesium, and depleted of histone H1 (relative to mature chromatin). In the following report the relationships between chromatin replication, histone acetylation, and H1-mediated nucleosome aggregation were further investigated. Chromatin was replicated in the presence or absence of sodium butyrate; isolated nucleosomes were stripped of linker histone, reconstituted with H1, and treated to produce Mg(2+)-soluble and Mg(2+)-insoluble chromatin fractions. Following the removal of H1, all solubility differences between chromatin replicated in sodium butyrate for 30 min (bu-chromatin) and control chromatin were lost. Reconstitution with H1 completely restored the preferential Mg(2+)-solubility of bu-chromatin, demonstrating that a reduced capacity for aggregation/condensation is an inherent feature of acetylated nascent nucleosomes; however, titration with excess H1 caused the solubility differences to be lost again. Moreover, when the core histone N-terminal "tails" (the sites of acetylation) were removed by trypsinization prior to reconstitution, H1 was unable to reestablish the altered solubility of chromatin replicated in butyrate. Thus, the core histone "tails," and the acetylation thereof, not only modulate H1-mediated nucleosome interactions in vitro, but also strongly influence the ability of H1 to differentiate between new and old nucleosomes. The data suggest a possible mechanism for the control of H1 deposition and/or chromatin folding during nucleosome assembly.