In vitro reconstitution of Artemia satellite chromatin

We report the characterization of an in vitro chromatin assembly system derived from Artemia embryos and its application to the study of AluI-113 satellite DNA organization in nucleosomes. The system efficiently reconstitutes chromatin templates by associating DNA, core histones, and H1. The polynucleosomal complexes show physiological spacing of repeat length 190 +/- 5 base pairs, and the internucleosomal distances are modulated by energy-using activities that contribute to the dynamics of chromatin conformation. The assembly extract was used to reconstitute tandemly repeated AluI-113 sequences. The establishment of preferred histone octamer/satellite DNA interactions was observed. In vitro, AluI-113 elements dictated the same nucleosome translational localizations as found in vivo. Specific rotational constraints seem to be the central structural requirement for nucleosome association. Satellite dinucleosomes showed decreased translational mobility compared with mononucleosomes. This could be the consequence of interactions between rotationally positioned nucleosomes separated by linker DNA of uniform length. AluI-113 DNA led to weak cooperativity of nucleosome association in the proximal flanking regions, which decreased with distance. Moreover, the structural properties of satellite chromatin can spread, thus leading to a specific organization of adjacent nucleosomes.     

Linker histones stabilize the intrinsic salt-dependent folding of nucleosomal arrays: mechanistic ramifications for higher-order chromatin folding

Defined nucleosomal arrays reconstituted from core histone octamers and twelve 208 bp tandem repeats of Lytechinus 5S rDNA (208-12 nucleosomal arrays) possess the ability to form an unstable folded species in MgCl2 whose extent of compaction equals that of canonical higher-order 30 nm diameter chromatin structures [Schwarz, P. M., and Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. To address the mechanistic functions of linker histones in chromatin condensation, purified histone H5 has been assembled with 208-12 nucleosomal arrays in 50 mM NaCl. Novel purification procedures subsequently were developed that yielded preparations of 208-12 chromatin model systems in which a majority of the sample contained both one histone octamer per 5S rDNA repeat and one molecule of histone H5 per histone octamer. The integrity of the purified 208-12 chromatin has been extensively characterized under low-salt conditions using analytical ultracentrifugation, quantitative agarose gel electrophoresis, electron cryomicroscopy, and nuclease digestion. Results indicate that histone H5 binding to 208-12 nucleosomal arrays constrains the entering and exiting linker DNA in a way that produces structures that are indistinguishable from native chicken erythrocyte chromatin. Folding experiments performed in NaC1 and MgC12 have shown that H5 binding markedly stabilizes both the intermediate and extensively folded states of nucleosomal arrays without fundamentally altering the intrinsic nucleosomal array folding pathway. These results provide new insight into the mechanism of chromatin folding by demonstrating for the first time that distinctly different macromolecular determinants are required for formation and stabilization of higher-order chromatin structures.     

Chromatin assembly in a yeast whole-cell extract

A simple in vitro system that supports chromatin assembly was developed for Saccharomyces cerevisiae. The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes. We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast. This analysis supports the model of sequential recruitment of H3/H4 tetramers and H2A/H2B dimers into nucleosomes. Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly. These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast.     

Linker histone tails and N-tails of histone H3 are redundant: scanning force microscopy studies of reconstituted fibers

The mechanisms responsible for organizing linear arrays of nucleosomes into the three-dimensional structure of chromatin are still largely unknown. In a companion paper (Leuba, S. H., et al. 1998. Biophys. J. 74:2823-2829), we study the contributions of linker histone domains and the N-terminal tail of core histone H3 to extended chromatin fiber structure by scanning force microscopy imaging of mildly trypsinized fibers. Here we complement and extend these studies by scanning force microscopy imaging of selectively reconstituted chromatin fibers, which differ in subtle but distinctive ways in their histone composition. We demonstrate an absolute requirement for the globular domain of the linker histones and a structural redundancy of the tails of linker histones and of histone H3 in determining conformational stability.     

The effects of noise in transient EOAE newborn hearing screening

Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha-amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205-217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed.     

Interactions between arginine-rich histones and deoxyribonucleic acids. II. Circular dichroism

Circular dichroism (CD) was used to investigate the conformations of arginine-rich histones, H3 (III or f3) and H4 (IV or f2a1), and DNA in the complexes prepared by four different methods: (A) NaCl gradient dialysis with urea; (B) NaCl gradient dialysis without urea; (C) direct mixing in 2.5 x 10(-4) M EDTA, pH 8.0; and (D) direct mixing in 0.01 M sodium phosphate, pH 7.0. Using the CD spectrum of native chromatin as a criterion to judge the closeness of a complex to its native state, it was observed that a complex made by direct mixing at low ionic strength (methods C and D) is better than the ones made by NaCl gradient dialysis with or without urea (methods A and B). It is explained as a result of lack of ordered secondary structures in histones due to the presence of urea in method A or due to nonspecific aggregation in NaCl without urea (method B). Compared with all the earlier reports in literature on the CD of histone-DNA complexes, the CD spectra of arginine-rich histone-DNA complexes prepared by methods C and D are closest to that of native chromatin both in shape and in amplitude. These results imply (a) that arginine-rich histones play an important role in maintaining the conformation of chromatin and (b) that the binding of these two histones to DNA prepared by methods C and D are close to that in native chromatin. Noticeable variation in conformation of free and bound histone and histone-bound DNA has also been observed in histone H3 with one or two cysteine residues, and in reduced or oxidized state even when the complexes were prepared and examined in the same condition. CD spectra of arginine-rich histones in 0.01 M phosphates, pH 7.0, indicate the presence of alpha-helix which could be responsible for a favorable binding of the less basic regions of these histones to DNA under this condition as demonstrated by thermal denaturation (Yu, S. .S, Li H. J., and Shih, T. Y. (1976), Bio-chemistry, the preceding paper in this issue). To preserve or generate alpha-helical structures in histones seems to be a critical step in reconstituting good histone-DNA complexes.     

Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I

In vivo, nucleosomes are formed rapidly on newly synthesized DNA after polymerase passage. Previously, a protein complex from human cells, termed chromatin assembly factor-I (CAF-I), was isolated that assembles nucleosomes preferentially onto SV40 DNA templates that undergo replication in vitro. Using a similar assay, we now report the purification of CAF-I from the budding yeast Saccharomyces cerevisiae. Amino acid sequence data from purified yeast CAF-I led to identification of the genes encoding each subunit in the yeast genome data base. The CAC1 and CAC2 (chromatin assembly complex) genes encode proteins similar to the p150 and p60 subunits of human CAF-I, respectively. The gene encoding the p50 subunit of yeast CAF-I (CAC3) is similar to the human p48 CAF-I subunit and was identified previously as MSI1, a member of a highly conserved subfamily of WD repeat proteins implicated in histone function in several organisms. Thus, CAF-I has been conserved functionally and structurally from yeast to human cells. Genes encoding the CAF-I subunits (collectively referred to as CAC genes) are not essential for cell viability. However, deletion of any CAC gene causes an increase in sensitivity to ultraviolet radiation, without significantly increasing sensitivity to gamma rays. This is consistent with previous biochemical data demonstrating the ability of CAF-I to assemble nucleosomes on templates undergoing nucleotide excision repair. Deletion of CAC genes also strongly reduces silencing of genes adjacent to telomeric DNA; the CAC1 gene is identical to RLF2 (Rap1p localization factor-2), a gene required for the normal distribution of the telomere-binding Rap1p protein within the nucleus. Together, these data suggest that CAF-I plays a role in generating chromatin structures in vivo.     

A novel function of the DNA repair gene rhp6 in mating-type silencing by chromatin remodeling in fission yeast

Recent studies have indicated that the DNA replication machinery is coupled to silencing of mating-type loci in the budding yeast Saccharomyces cerevisiae, and a similar silencing mechanism may operate in the distantly related yeast Schizosaccharomyces pombe. Regarding gene regulation, an important function of DNA replication may be in coupling of faithful chromatin assembly to reestablishment of the parental states of gene expression in daughter cells. We have been interested in isolating mutants that are defective in this hypothesized coupling. An S. pombe mutant fortuitously isolated from a screen for temperature-sensitive growth and silencing phenotype exhibited a novel defect in silencing that was dependent on the switching competence of the mating-type loci, a property that differentiates this mutant from other silencing mutants of S. pombe as well as of S. cerevisiae. This unique mutant phenotype defined a locus which we named sng1 (for silencing not governed). Chromatin analysis revealed a switching-dependent unfolding of the donor loci mat2P and mat3M in the sng1(-) mutant, as indicated by increased accessibility to the in vivo-expressed Escherichia coli dam methylase. Unexpectedly, cloning and sequencing identified the gene as the previously isolated DNA repair gene rhp6. RAD6, an rhp6 homolog in S. cerevisiae, is required for postreplication DNA repair and ubiquitination of histones H2A and H2B. This study implicates the Rad6/rhp6 protein in gene regulation and, more importantly, suggests that a transient window of opportunity exists to ensure the remodeling of chromatin structure during chromosome replication and recombination. We propose that the effects of the sng1(-)/rhp6(-) mutation on silencing are indirect consequences of changes in chromatin structure.     

The role of nucleocapsid of HIV-1 in virus assembly

The role of the nucleocapsid protein of HIV-1 Gag in virus assembly was investigated using Gag truncation mutants, a nucleocapsid deletion mutant, and point mutations in the nucleocapsid region of Gag, in transfected COS cells, and in stable T-cell lines. Consistent with previous investigations, a truncation containing only the matrix and capsid regions of Gag was unable to assemble efficiently into particles; also, the pelletable material released was lighter than the density of wild-type HIV-1. A deletion mutant lacking p7 nucleocapsid but containing the C-terminal p6 protein was also inefficient in particle release and released lighter particles, while a truncation containing only the first zinc finger of p7 could assemble more efficiently into virions. These results clearly show that p7 is indispensable for virus assembly and release. Some point mutations in the N-terminal basic domain and in the basic linker region between the two zinc fingers, which had been previously shown to have reduced RNA binding in vitro [Schmalzbauer, E., Strack, B., Dannull, J., Guehmann, S., and Moelling, K. (1996). J. Virol. 70: 771-777], were shown to reduce virus assembly dramatically when expressed in full-length viral clones. A fusion protein consisting of matrix and capsid fused to a heterologous viral protein known to have nonspecific RNA binding activity [Ribas, J. C., Fujimura, T., and Wickner, R. B. (1994) J. Biol. Chem. 269: 28420-28428] released pelletable material slightly more efficiently than matrix and capsid alone, and these particles had density higher than matrix and capsid alone. These results demonstrate the essential role of HIV-1 nucleocapsid in the virus assembly process and show that the positively charged N terminus of p7 is critical for this role.     

Functional beta1-integrins release the suppression of fibronectin matrix assembly by vitronectin

beta1-null GD25 fibroblasts adherent to vitronectin fail to bind the N-terminal 70-kDa matrix assembly domain of fibronectin or to assemble fibronectin (Sakai, T., Zhang, Q., F?ssler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538). We have made four observations that extend this finding. First, the presence of vitronectin on a substrate that otherwise can support fibronectin assembly has a dominant-negative effect on assembly. Second, the dominant-negative effect is lost when active beta1A is expressed. Third, beta1A containing the extracellular D130A inactivating mutation has a dominant-negative effect on fibronectin assembly. Fourth, beta1-null cells adherent to vitronectin are flat and lack filopodia, whereas beta1-null cells adherent to fibronectin or beta1A-expressing cells adherent to either vitronectin or fibronectin are contracted and exhibit numerous filopodia. These results reveal, therefore, that GD25 cells adherent to vitronectin can only assume a shape suitable for assembly of fibronectin when there is a countervailing signal from functional beta1-integrins.     

Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens

Unlike mammals, birds, and most other fishes, winter flounder completes spermatogenesis without replacing its germ cell histones with protamines. Instead, during spermiogenesis, these fish produce a family of high molecular weight (80,000-200,000) basic nuclear proteins (HMrBNPs) that bind to sperm chromatin containing the normal complement of histones. These large, basic proteins are built up of tandem iterations of oligopeptide repeats that contain phosphorylatable DNA-binding motifs. Although the HMrBNPs have no obvious homology to histones, protamines, or other sperm-specific chromatin proteins, we report here the isolation of a clone (2B) from a winter flounder genomic DNA library that establishes a link between the HMrBNPs and histone H1. The 2B sequence contains an open reading frame, which, when conceptually translated, encodes a 265-residue protein. At its N terminus the translation product contains numerous simple repeats that match the oligopeptides contained within the HMrBNPs. Unexpectedly, the C terminus of the putative protein shows 66% identity and 76% conservation to the histone H1 globular domain. This connection suggests that the HMrBNPs may have originated from the extended N-terminal tail region of a testis-specific, H1-like linker histone.     

Folding of the glucocorticoid receptor by the heat shock protein (hsp) 90-based chaperone machinery. The role of p23 is to stabilize receptor.hsp90 heterocomplexes formed by hsp90.p60.hsp70

In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor.hsp90 heterocomplexes. We have reconstituted a minimal receptor.hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833-12839). We have shown that hsp90, p60, and hsp70 are sufficient for carrying out the folding change that converts the glucocorticoid receptor (GR) hormone binding domain (HBD) from a non-steroid binding to a steroid binding conformation, but to form stable GR.hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047-13054). In this work, we show that addition of p23 to native GR.hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR.hsp90 heterocomplexes prepared with the minimal (hsp90.p60.hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR.hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90.p60.hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR.hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90.p60.hsp70 forms a GR.hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR.hsp90 heterocomplex to inactivation and disassembly.     

Association products and conformations of salt-dissociated and acid-extracted histones. A two-phase procedure for isolating salt-dissociated histones

We present an extremely rapid and efficient method for the separation of salt-dissociated histones from DNA in which the macromolecular components of chicken erythrocyte chromatin are partitioned in a two-phase system of the water-soluble, nonionic polymers, poly(ethylene glycol) and dextran. We have compared the association products and conformations of salt-dissociated histones purified with the two-phase procedure and histones that had been extracted with 0.4 M H2SO4. In the gel chromatography system of D. R. vander Westhuyzen and C. von Holt (1971), FEBS Lett. 14, 333-337] the association products of salt-dissociated and acid-extracted histones are indistinguishable. Furthermore, the circular dichroism spectra of histones prepared with the two methods are identical within experimental error. These results indicate that histones extracted, with sulfuric acid can adopt conformations at least very similar to those of salt-dissociated preperties of total erythrocyte histones are the same in 2 M NaCl as those of these histones bound to DNA in chromatin in 1 mM Tris-Cl (pH 7.5). This result and the studies of Weintraub et al. [Weintraub, H., Palter, K., and Van Lente, F. (1975), Cell 6, 68-110] on the patterns of tryptic digest products of histones strongly suggest that in 2 M NaCl the histones exist in conformations very similar to their conformations when bound to DNA. The concept of native histone conformations is discussed in light of our results.     

Histones associated with non-nucleosomal rat ribosomal genes are acetylated while those bound to nucleosome-organized gene copies are not

Acetylation of histones bound to rat rRNA genes has been studied relative to their organization in chromatin, either as canonical nucleosomes, containing the inactive copies, or as anucleosomal nonrepeating structures, corresponding to the transcribed genes (Conconi, A., Widmer, R. M., Koller, T., and Sogo, J. M. (1989) Cell 57, 753-761). Nuclei from butyrate-treated rat tumor cells were irradiated with a UV laser to cross-link proteins to DNA, and the purified covalent complexes were immunofractionated by an antibody that specifically recognized the acetylated histones. Upon probing with sequences coding for mature rat 28 S RNA, DNA of the antibody-bound complexes was 5-20-fold enriched relative to the total rat DNA. Since the laser cross-links histones to DNA in both active and inactive genes, one cannot distinguish which one of them, or both, are bound to acetylated histones. Alternatively, purified mononucleosomes were immunofractionated, but DNA from the antibody-bound monosomes was not enriched in coding rDNA. Taken together, these results suggest that nucleosome-organized rRNA genes are bound to nonmodified histones and that the acetylated histones are associated with the active, anucleosomal gene copies.