Properties of nucleosomes in acetylated mouse mammary tumor virus versus 5S arrays

Acetylation is one of the most abundant histone modifications found in nucleosomes. Although such modifications are thought to function mainly in recognition, acetylation is known to produce nucleosome structural alterations. These could be of functional significance in vivo. Here, the basic features of mouse mammary tumor virus (MMTV) promoter nucleosomal arrays reconstituted with highly acetylated histones prepared from butyrate-treated HeLa cells are characterized by atomic force microscopy. Results are compared to previous results obtained with hypoacetylated MMTV and hyper- or hypoacetylated 5S rDNA arrays. MMTV arrays containing highly acetylated histones show diminished intramolecular compaction compared to hypoacetylated MMTV arrays and no tendency for cooperativity in nucleosome occupation. Both features have been suggested to reflect histone tail-mediated internucleosomal interactions; these observations are consistent with that suggestion. 5S arrays show qualitatively similar behavior. Two other effects of acetylation show stronger DNA template dependence. Nucleosome salt stability is diminished in highly acetylated compared to hypoacetylated MMTV arrays, but nucleosome (histone) loading tendencies are unaffected by acetylation. However, highly acetylated histones show reduced loading tendencies on 5S templates (vs hypoacetylated), but 5S nucleosome salt stabilities are unaffected by acetylation. ATP-dependent nucleosome remodeling by human Swi-Snf is similar on hyper- and hypoacetylated MMTV arrays.     

Histone hyperacetylation can induce unfolding of the nucleosome core particle.

A direct correlation exists between the level of histone H4 hyperacetylation induced by sodium butyrate and the extent to which nucleosomes lose their compact shape and become elongated (62.0% of the particles have a length/width ratio over 1.6; overall mean in the length/width ratio = 1.83 +/- 0.48) when bound to electron microscope specimen grids at low ionic strength (1mM EDTA, 10mM Tris, pH 8.0). A marked proportion of elongated core particles is also observed in the naturally occurring hyperacetylated chicken testis chromatin undergoing spermatogenesis when analyzed at low ionic strength (36.8% of the particles have a length/width ratio over 1.6). Core particles of elongated shape (length/width ratio over 1.6) generated under low ionic strength conditions are absent in the hypoacetylated chicken erythrocyte chromatin and represent only 2.3% of the untreated Hela S3 cell core particles containing a low proportion of hyperacetylated histones. The marked differences between control and hyperacetylated core particles are absent if the particles are bound to the carbon support film in the presence of 0.2 M NaCl, 6mM MgCl2 and 10mM Tris pH 8.0, conditions known to stabilize nucleosomes. A survey of the published work on histone hyperacetylation together with the present results indicate that histone hyperacetylation does not produce any marked disruption of the core particle 'per se', but that it decreases intranucleosomal stabilizing forces as judged by the lowered stability of the hyperacetylated core particle under conditions of shearing stress such as cationic competition by the carbon support film of the EM grid for DNA binding.     

DNA associated with hyperacetylated histone is preferentially digested by DNase I.

Butyrate-treated cells give rise to massive hyperacetylation of histones and have been used to test the idea that regions of DNA in association with hyperacetylated histones are preferentially solubilized upon digestion with DNase I. Such hyperacetylated histones can be derived from both pre-existing histones or from histone newly synthesized in the presence of butyrate which leads to extreme modification. The DNA in association with both types of hypermodified histone is equally and selectively digested.     

Histones are incorporated in trans during reassembly of the yeast PHO5 promoter

In yeast, remodeling of PHO5 promoter chromatin upon activation is accompanied by transient hyperacetylation and subsequent eviction of histones from the promoter in trans. In the course of rerepression, nucleosomes have to be reassembled on the promoter. We have analyzed where the histones for reassembly of the inactive promoter chromatin come from. The use of a strain with two differently tagged and differently regulated versions of histone H3 allowed us to discriminate between histones originating from the chromatin fraction and histones arising from the soluble histone pool. In this way, we show that the incorporated histones originate from a source in trans. Promoter closure occurs very rapidly, and the histone chaperones Asf1 and Hir1 as well as the SWI/SNF nucleosome remodeling complex appear to be important for rapid reassembly of nucleosomes at the PHO5 promoter.     

Mapping nucleosome locations on the 208-12 by AFM provides clear evidence for cooperativity in array occupation

Concatameric sea urchin 5S rDNA templates reconstituted with histones provide very popular chromatin models for many kinds of in vitro studies. We have used AFM to characterize the locational aspects of nucleosome occupation on one such array, the 208-12, by determining the internucleosomal- and end-distance distributions for arrays reconstituted to various subsaturating levels with nonacetylated or hyperacetylated HeLa histones. A simulation analysis of the experimental distributions confirms the qualitative conclusions and provides quantitative parameter values for the identified features. For nonacetylated arrays, the end-distance data demonstrate the nucleosome positioning ability of the 5S sequence and detect an enhanced preference for nucleosomes to bind at DNA termini. The internucleosomal-distance data provide clear evidence for cooperativity in nucleosome location on these templates, detectable even at subsaturated loading levels. Hyperacetylated arrays show no change in the preference of nucleosomes to bind at termini and a slight change in nucleosome positioning behavior but, most strikingly, little or no evidence for cooperativity in nucleosome location. Thus, acetylation of the N-terminal histone tails abolishes the cooperativity.     

Hyperacetylation of histone H4 promotes chromatin decondensation prior to histone replacement by protamines during spermatogenesis in rainbow trout

During the final stages of spermatogenesis in rainbow trout a dramatic increase in the level of histone H4 hyperacetylation is observed which is closely correlated with the replacement of histones by protamines. In order to understand further how H4 hyperacetylation might assist in protamine replacement of the histones, we have investigated the effect of H4 hyperacetylation on chromatin structure in trout testes actively undergoing the replacement process. Long chromatin fragments enriched in hyperacetylated H4 have been isolated and characterized. Evidence is presented that hyperacetylated H4 is clustered in certain regions (domains) of late stage testis chromatin and within these domains the chromatin exhibits an altered, highly relaxed structure which is believed to be the result of the extensive hyperacetylation. These domains, which are nearly devoid of protamine, are postulated to represent an initial structural transition which is necessary for the proper histone removal and protamine replacement process to take place.     

Chicken erythrocyte beta-globin chromatin: enhanced solubility is a direct consequence of induced histone hyperacetylation.

Chicken immature red blood cells were incubated for 1 hour in Swim's medium containing 3H-acetate and 10 mM n-butyrate. During the incubation period, the small percentage of dynamically acetylated and deacetylated histone is radiolabeled and hyperacetylated. A second effect of the n-butyrate incubation is to shift a small subset of nucleohistone into a soluble form. This chromatin is predominantly polynucleosome size (approximately dimer to pentamer) and can be separated from soluble mononucleosomes by 5-30% sucrose gradient centrifugation. The soluble polynucleosomes are 25-30 fold enriched for adult beta-globin (beta A) DNA and contain the hyperacetylated histones. We have tested whether histone hyperacetylation is responsible for the enhanced beta-globin chromatin solubility by in vitro deacetylation of the soluble chromatin histones. This procedure converts the beta-globin polynucleosomes to an insoluble form, demonstrating that histone hyperacetylation is in fact directly responsible for the increased solubility of the beta A chromatin.     

Changes in the structural organization of chromatin during spermatogenesis in the rat

In this study, histone H4 was shown to be extensively hyperacetylated in mid-spermatids of the rat during the time period when the entire complement of histones is replaced by basic spermatidal transition proteins. The degree of hyperacetylation of histone H4 was minimal in pachytene spermatocytes. Therefore, the hyperacetylation appears to be directly involved in the histone replacement process late in spermatogenesis in mid-spermatids. In order to investigate further the possible effects of histone H4 hyperacetylation and the other dramatic changes in the nuclear proteins on the structure of chromatin in germinal cells, we examined the thermal denaturation profiles of chromatin from various purified germinal cell types. Our analyses revealed that chromatins from pachytene spermatocytes and early spermatids have similar thermal denaturation profiles, with their major thermal transitions slightly lower than those for rat liver. However, the major thermal transitions for chromatin from mid-spermatids are much lower than those from pachytene spermatocytes and early-spermatids. We propose that the greatly lowered thermal stability of mid-spermatid chromatin represents a dramatic relaxation or decondensation of the chromatin in this cell type in preparation for the replacement of histone by the basic spermatidal transition proteins and that the decondensation is due in large part to the extensive histones hyperacetylation which occurs in these cells.