Jean O. Thomas

Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain

Heterochromatin protein 1 (HP1) is localized at heterochromatin sites where it mediates gene silencing. The chromo domain of HP1 is necessary for both targeting and transcriptional repression. In the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homologue of the mammalian SUV39H1 histone methylase. Both Clr4 and SUV39H1 methylate specifically lysine 9 of histone H3 (ref. 6). Here we show that HP1 can bind with high affinity to histone H3 methylated at lysine 9 but not at lysine 4. The chromo domain of HP1 is identified as its methyl-lysine-binding domain. A point mutation in the chromo domain, which destroys the gene silencing activity of HP1 in Drosophila, abolishes methyl-lysine-binding activity. Genetic and biochemical analysis in S. pombe shows that the methylase activity of Clr4 is necessary for the correct localization of Swi6 at centromeric heterochromatin and for gene silencing. These results provide a stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a ‘methyl marker’ on histone H3, which is then recognized by HP1 through its chromo domain. This model may also explain the stable inheritance of the heterochromatic state.

HMG1 and 2, and related ‘architectural’ DNA-binding proteins

The HMG-box proteins, one of the three classes of high mobility group (HMG) chromosomal proteins, bend DNA and bind preferentially to distorted DNA structures. The proteins appear to act primarily as architectural facilitators in the assembly of nucleoprotein complexes; for example, in effecting recombination and in the initiation of transcription. HMG-box proteins might be targeted to particular DNA sites in chromatin by either protein-protein interactions or recognition of specific DNA structures.

Preparation of Native Chromatin and Damage Caused by Shearing

Chromatin prepared by a method involving limited nuclease digestion contains the same repeating structure as chromatin in the nucleus, whereas chromatin prepared by conventional methods involving shear does not.

HISTONE H1 : LOCATION AND ROLE

Recent experiments have shown directly that, in bulk chromatin, the globular domain of histone H1 is positioned close to the dyad axis and is asymmetrically disposed, consistent with a polar arrangement of H1 molecules along the nucleosome filament. In addition to a structural role, H1 may also have gene-specific effects on transcription. The positioning of a core particle relative to a transcription factor binding-site may favour either transcription factor binding or H1 binding, depending on the location of the site.

Changes in chromatin folding in solution.

Abstract The formation of higher order structures by nucleosome oligomers of graded sizes with increasing ionic strength has been studied in solution, by measuring sedimentation coefficients. Nucleosome monomers and dimers show no effect of ionic strength at the concentrations used, while trimers to pentamers show a linear dependence of the logarithm of sedimentation coefficient upon the logarithm of ionic strength between 5 and 25 m m , but no dependence above 25 m m . Between pentamer and hexamer a change occurs and the linear relationship is observed up to ionic strength 125 m m with hexamer and above. The simple power-law dependence of the sedimentation coefficient upon the ionic strength ( s ∝ I n ) is observed up to nucleosome 30mers, but by 60mer a jump in the sedimentation coefficient occurs between ionic strengths 45 and 55 m m , with the power-law applying both above and below the jump. Removal of histone H1 and non-histone proteins lowers the overall sedimentation rate and abolishes the jump. Cross-linking large oligomers at ionic strength 65 m m stabilizes the structure in the conformation found above the jump, leading to a simple power-law dependence throughout the range of ionic strength for cross-linked material. Cleavage of the cross-links restores the jump, presumably by allowing the conformational transition that causes it. Large oligomers are indistinguishable in sedimentation behaviour whether extracted from nuclei at low ionic strength or at 65 m m and maintained in the presence of salt. We interpret these results, together with the detailed electron microscopic studies reported by Thoma et al. (1979) under similar salt conditions, as showing the histone H1-dependent formation of superstructures of nucleosomes in solution induced by increasing ionic strength. The unit of higher order structure probably contains five or six nucleosomes, leading to the change in stability with hexamer. Although this size corresponds to the lower limit of size suggested for “superbeads” (Renz et al. , 1977), we see no evidence that multiples of six nucleosomes have any special significance as might be predicted if superbeads had any structural importance. Rather, our results are compatible with a continuous pattern of condensation, such as a helix of nucleosomes (see e.g. Finch & Klug, 1976). The jump in sedimentation observed between ionic strengths 45 and 55 m m , together with the effect of cross-linking, suggests the co-operative stabilization of this structure at higher ionic strengths. A plausible hypothesis is that the turns of the solenoid are not tightly bonded in the axial direction below 45 m m , but come apart due to the hydrodynamic shearing forces in the larger particles leading to less compact structures with slower sedimentation rates. Above 55 m m the axial bonding is strong enough to give a stable structure of dimensions compatible with the 30 nm structures observed in the cell nucleus.

Assembly of nucleosomes: the reaction involving X. laevis nucleoplasmin

We analyze the nucleosome core assembly reaction which is mediated in vitro by a protein previously purified from Xenopus laevis eggs, now named nucleoplasmin in reference to its occurrence in the soluble phase of the nucleus of a wide range of vertebrate cell types. Nucleoplasmin is present in solution as a pentamer. We use nuclease digestion analysis to show that the protein assembles bona fide nucleosome cores in vitro from purified histones and DNA. Nucleoplasmin itself binds neither to DNA nor to the nucleoprotein particles which it assembles in vitro. However, it interacts with histones in vitro in such a way that histones no longer adhere to negatively charged surfaces. We have found no evidence for sterically specific interactions with particular histones. The initial rate of the nucleosome core assembly reaction mediated by purified nucleoplasmin in vitro is essentially identical with the rate of the nucleosome assembly reaction which occurs in the cell-free extracts of Xenopus eggs from which nucleoplasmin was purified. This rate is sufficient to account for the rate of nucleosome assembly required during the early development of Xenopus embryos.

Exchange of histone H1 between segments of chromatin

Abstract We have asked whether histone H1 is tightly bound in chromatin at relatively low ionic strength (below physiological) or whether it can exchange between binding sites. We have studied this question in chromatin fragments generated by digestion with micrococcal nuclease, by mixing two fragments of known H1 content and different length (either a fragment radioactively labelled in all its histones with an unlabelled fragment, or two labelled fragments, only one of which contains H1) and then separating them again by centrifugation in sucrose gradients in order tom reexamine their H1 contents. At very low ionic strength (5 m m -Tris · HCl (pH 7.1), 1 m m -Na2EDTA, 0.5 m m -phenylmethylsulphonyl fluoride) there was very little (less than 5 to 10%) exchange of H1. In contrast, the presence of 70 m m -NaCl in the buffer caused rapid and complete equilibration of H1 between sites in less (possibly much less) than about one hour. At 30 m m added NaCl, the result was intermediate between those at 0 m m and 70 m m added NaCl, partial exchange occurring with a half-time of one to two hours. The results were essentially the same whether or not both fragments contained H1. We infer from these results that at physiological ionic strength (~150 m m ) there will be rapid and complete equilibration of H1 between sites in chromatin. We do not know whether the exchange of particular H1 subtypes is restricted to a particular class of binding site.

Alpha-helix in the carboxy-terminal domains of histones H1 and H5.

Although the carboxy‐terminal domains of histones H1 and H5 exist as random‐coil in aqueous solution, secondary structure prediction suggests that this region has a high potential for alpha‐helix formation. We have measured CD spectra in various conditions known to stabilize alpha‐helices, to determine whether this potential can be realized in an appropriate environment. Trifluoroethanol increases the helix contents of H1, H5 and their carboxy‐terminal fragments, presumably through promotion of axial hydrogen bonding. Sodium perchlorate is also effective and better than sodium chloride, suggesting stabilization by binding of bulky perchlorate ions rather than simple charge screening. Extrapolating from these measurements in solution, and taking into account the occurrence of proline residues throughout the carboxy‐terminal domain, we propose that binding to DNA stabilizes helical segments in the carboxy‐terminal domains of histones H1 and H5, and that it is this structured form of the domain that is functionally important in chromatin.

Chapter 22 The Study of Histone—Histone Associations by Chemical Cross-Linking

Publisher Summary This chapter presents a study of histone–histone associations by chemical cross-linking. Chemical cross-linking can be used to reveal both the pattern and the degree of association of polypeptides in a multisubunit structure. Limited cross-linking results in dimmers that are formed from neighboring polypeptides. Extensive cross-linking gives a series of higher-molecular-weight products, the largest of which comprises the total number of subunits in the structure. Both types of analysis have been applied to the histones with the use of a variety of cross-linking agents, such as formaldehyde, imidoesters, tetranitromethane, ultraviolet light, and dicyclohexylcarbodiimide. The chapter focuses on the imidoesters, whose reaction with proteins is well understood. The procedures for cross-linking with imidoesters are straightforward, and success in their application to histones and chromatin is largely dependent on the resolving power of the methods used to identify the cross-linked products. Fractionation of the histones and cross-linked products is difficult because of their similar charges and molecular weights. In the chapter, cross-linking of histones in chromatin and in free solution is described as well as examples of cross-linking with dimethyl suberimidate are presented.

Salt-dependent co-operative interaction of histone H1 with linear DNA☆

The nature of the complexes formed between histone H1 and linear double-stranded DNA is dependent on ionic strength and on the H1 : DNA ratio. At an input ratio of less than about 60% (w/w) H1 : DNA, there is a sharp transition from non-co-operative to co-operative binding at a critical salt concentration that depends on the DNA size and is in the range 20 to 50 mM-NaCl. Above this critical ionic strength the H1 binds to only some of the DNA molecules leaving the rest free, as shown by sedimentation analysis. The ionic strength range over which this change in behaviour occurs is also that over which chromatin folding is induced. Above the salt concentration required for co-operative binding of H1 to DNA, but not below it, H1 molecules are in close proximity as shown by the formation of H1 polymers upon chemical cross-linking. The change in binding mode is not driven by the folding of the globular domain of H1, since this is already folded at low salt in the presence of DNA, as indicated by its resistance to tryptic digestion. The H1-DNA complexes at low salt, where H1 is bound distributively to all DNA molecules, contain thickened regions about 6 nm across interspersed with free DNA, as shown by electron microscopy. The complexes formed at higher salt through co-operative interactions are rods of relatively uniform width (11 to 15 nm) whose length is about 1.6 times shorter than that of the input DNA, or are circular if the DNA is long enough. They contain approximately 70% (w/w) H1 : DNA and several DNA molecules. These thick complexes can also be formed at low salt (15 mM-NaCl) when the H1 : DNA input ratio is sufficiently high (approximately 70%).