Multistep chromatin assembly on supercoiled plasmid DNA by nucleosome assembly protein-1 and ATP-utilizing chromatin assembly and remodeling factor

We examine in vitro nucleosome assembly by nucleosome assembly protein-1 (NAP-1) and ATP-utilizing chromatin assembly and remodeling factor (ACF). In contrast to previous studies that used relaxed, circular plasmids as templates, we have found that negatively supercoiled templates reveal the distinct roles of NAP-1 and ACF in histone deposition and the formation of an ordered nucleosomal array. NAP-1 can efficiently deposit histones onto supercoiled plasmids. Furthermore, NAP-1 exhibits a greater affinity for histones H2A-H2B than does naked DNA, but in the presence of H3-H4, H2A-H2B are transferred from NAP-1 to the plasmid templates. These observations underscore the importance of a high affinity between H2A-H2B and NAP-1 for ordered transfer of core histones onto DNA. In addition, recombinant ACF composed of imitation switch and Acf1 can extend closely packed nucleosomes, which suggests that recombinant ACF can mobilize nucleosomes. In the assembly reaction with a supercoiled template, ACF need not be added simultaneously with NAP-1. Regularly spaced nucleosomes are generated even when recombinant ACF is added after core histones are transferred completely onto the DNA. Atomic force microscopy, however, suggests that NAP-1 alone fails to accomplish the formation of fine nucleosomal core particles, which are only formed in the presence of ACF. These results suggest a model for the ordered deposition of histones and the arrangement of nucleosomes during chromatin assembly in vivo.     

Nuclear reassembly in vitro is independent of nucleosome/chromatin assembly

It was show11 that nuclear reassembly was induced by small pieces of DNA fragments in cell-free extracts ofXenopus. In an attempt to learn the relationship between the nuclear reassembly and nucleosome/chromatin assembly, limited amounts of CM-Cellulose are used to eliminate the capacity of the egg extract S-150 to assemble chromatin. while the forming of nucleosomes is checked with DNA supercoiling by plasmid DNA pBR322 incubated in the extract, and further analysed by micrococcal nuclease digestion. This depleted extract is then used to induce nuclear reassembly around demembraned sperms with membrane vesicles. It is found that CM-Cellulose depletes histones H2A and H2B efficiently and blocks the assembly of nucleosomes, the demembraned sperms are yet reconstituted into nuclei in the treated S-150, although the chromatin in reassembled nuclei does not produce protected DNA fragments when digested with micrococcal nuclease. It suggests that in the cell-free system ofXenopus, DNA can be formed into nuclei without assembly of nucleosomes or chromatin. Projrrt supported by the National Natural Science Foundation of China (Grant No. 39730240)     

Presence of an unusually high concentration of an ubiquitinated histone-like protein in Trypanosoma cruzi

The conjugation of ubiquitin to histones H2A and H2B has been established in higher eukaryotes and has been related to changes in chromatin organization. In Trypanosoma cruzi, no condensation of chromatin occurs during mitosis. In order to determine the presence of histone ubiquitination in T. cruzi epimastigotes, histones were extracted from chromatin and analyzed by three electrophoretic systems: acid-urea, triton-acid-urea and sodium-dodecyl-sulphate polyacrylamide gel. The immunochemical detection of ubiquitin-histone conjugates by Western blotting showed a strong reaction with a slow migrating band of M_r 19 kDa. The high percentage of ubiquitin-histone conjugates present in T. cruzi chromatin may be related to the inability of this parasite to condense chromatin into a 30 nm fiber. J. Cell. Biochem. 66:433–440, 1997. © 1997 Wiley-Liss, Inc.     

The Yeast Histone Chaperone Hif1p Functions with RNA in Nucleosome Assembly

Background

Hif1p is an H3/H4-specific histone chaperone that associates with the nuclear form of the Hat1p/Hat2p complex (NuB4 complex) in the yeast Saccharomyces cerevisiae. While not capable of depositing histones onto DNA on its own, Hif1p can act in conjunction with a yeast cytosolic extract to assemble nucleosomes onto a relaxed circular plasmid.

Results

To identify the factor(s) that function with Hif1p to carry out chromatin assembly, multiple steps of column chromatography were carried out to fractionate the yeast cytosolic extract. Analysis of partially purified fractions indicated that Hif1p-dependent chromatin assembly activity resided in RNA rather than protein. Fractionation of isolated RNA indicated that the chromatin assembly activity did not simply purify with bulk RNA. In addition, the RNA-mediated chromatin assembly activity was blocked by mutations in the human homolog of Hif1p, sNASP, that prevent the association of this histone chaperone with histone H3 and H4 without altering its electrostatic properties.

Conclusions

These results suggest that specific RNA species may function in concert with histone chaperones to assemble chromatin.     

The fate of parental nucleosomes during SV40 DNA replication.

The fate of parental nucleosomes during the replication of chromatin templates was studied using a modification of the cell-free SV40 DNA replication system. Plasmid DNA molecules containing the SV40 origin were assembled into chromatin with purified core histones and fractionated assembly factors derived from HeLa cells. When these templates were replicated in vitro, the resulting progeny retained a nucleosomal organization. To determine whether the nucleosomes associated with the progeny molecules resulted from displacement of parental histones during replication followed by reassembly, the replication reactions were performed in the presence of control templates. It was observed that the progeny genomes resulting from the replication of chromatin templates retained a nucleosomal structure, whereas the progeny of the control DNA molecules were not assembled into chromatin. Additional experiments, involving direct addition of histones to the replication reaction mixtures, confirmed that the control templates were not sequestered in some form which made them unavailable for nucleosome assembly. Thus, our data demonstrate that parental nucleosomes remain associated with the replicating molecules and are transferred to the progeny molecules without displacement into solution. We propose a simple model in which nucleosomes ahead of the fork are transferred intact to the newly synthesized daughter duplexes.     

Assembly of newly replicated chromatin.

Mild staphylococcal nuclease digestions under isotonic conditions release fragments of a 200 Å diameter fiber from nuclei of Drosophila melanogaster tissue culture cells. These soluble fragments have high sedimentation coefficients (30–100S) and show tightly packed nucleosomes in the electron microscope. Under the same conditions, newly replicated chromatin is released as more slowly sedimenting fragments (14S). Within 20 min after DNA replication, the nascent chromatin gradually matures into compact supranucleosomal structures which are indistinguishable from bulk chromatin on the isokinetic sucrose gradients.We have used this fractionation technique to examine the question of the fate and assembly of the new histones. After short pulses with either ³⁵S-methionine or ³H-lysine, the radioactive histones do not co-sediment with the bulk chromatin but appear instead in the fractions where the newly replicated DNA is found. Furthermore, the various nascent histones appear in different fractions on the gradient: histones H3 and H4 in 10–15S structures, histones H2A and H2B in 15–50S structures and histone H1 in 30–100S structures. These results, together with the analysis of pulse and pulse-chase experiments of both nascent DNA and histones, strongly suggest that histones H3 and H4 are deposited first on the nascent DNA (during or slightly after the DNA is replicated), histones H2A and H2B are deposited next (2–10 min later) and histone H1 is deposited last (10–20 min after DNA replication). A high turnover 20,000 dalton protein is also associated with the newly replicated chromatin.     

Nuclear reassemblyin vitro is independent of nucleosome/chromatin assembly

It was show11 that nuclear reassembly was induced by small pieces of DNA fragments in cell-free extracts ofXenopus. In an attempt to learn the relationship between the nuclear reassembly and nucleosome/chromatin assembly, limited amounts of CM-Cellulose are used to eliminate the capacity of the egg extract S-150 to assemble chromatin. while the forming of nucleosomes is checked with DNA supercoiling by plasmid DNA pBR322 incubated in the extract, and further analysed by micrococcal nuclease digestion. This depleted extract is then used to induce nuclear reassembly around demembraned sperms with membrane vesicles. It is found that CM-Cellulose depletes histones H2A and H2B efficiently and blocks the assembly of nucleosomes, the demembraned sperms are yet reconstituted into nuclei in the treated S-150, although the chromatin in reassembled nuclei does not produce protected DNA fragments when digested with micrococcal nuclease. It suggests that in the cell-free system ofXenopus, DNA can be formed into nuclei without assembly of nucleosomes or chromatin.     

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.     

The histone chaperone Asf1 is dispensable for direct de novo histone deposition in <Emphasis Type="Italic">Xenopus</Emphasis> egg extracts

Histone chaperones that escort histones during their overall lifetime from synthesis to sites of usage can participate in various tasks. Their requirement culminates in the dynamic processes of nucleosome assembly and disassembly. In this context, it is important to define the exact role of the histone chaperone Asf1. In mammals, Asf1 interacts with two other chaperones, CAF-1 and HIRA, which are critical in DNA synthesis-coupled and synthesis-uncoupled nucleosome assembly pathways, respectively. A key issue is whether Asf1 is able or not to deposit histones onto DNA by itself in both pathways. Here, to delineate the precise role of Asf1 in chromatin assembly, we used Xenopus egg extracts as a powerful system to assay de novo chromatin assembly pathways in vitro. Following characterization of both Xenopus Asf1 and p60 (CAF-1), we used immunodepletion strategies targeting Asf1, HIRA, or CAF-1. Strikingly, the depletion of Asf1 led to the simultaneous depletion of HIRA and consequently impaired the DNA synthesis-independent nucleosome assembly pathway. The rescue of nucleosome assembly capacity in such extracts was effective when adding HIRA along with H3/H4 histones, yet addition of Asf1 along with H3/H4 histones did not work. Moreover, nucleosome assembly coupled to DNA repair was not affected in these Asf1/HIRA-depleted extracts, a pathway impaired by CAF-1 depletion. Thus, these data show that Asf1 is not directly involved in de novo histone deposition during DNA synthesis-independent and synthesis-dependent pathways in egg extracts. Based on our results, it becomes important to consider the implications for Asf1 function during early development in Xenopus.     

Comparison of replicative and non-replicative chromatin assembly pathways in HeLa cell extracts.

It has been reported that chromatin assembly in mammalian cell extracts depends exclusively or preferentially on ongoing DNA replication (Stillman, B. (1986) Cell 45, 555-565). More recently, this view has been challenged demonstrating that, in the same extracts, chromatin can also be formed efficiently in the absence of DNA replication (Gruss et al. (1990) EMBO J. 9, 2911-2922). The experiments, described in this communication, were performed to resolve this apparent contradiction. We found that there are at least two distinct in vitro pathways for chromatin assembly in HeLa cell extracts. The replicative pathway requires a nuclear protein, most likely identical with the chromatin assembly factor, described by Stillman (1986, Cell 45, 555-565), and the free soluble histones present in the cytosol of S phase cells. In contrast, a non-replicative pathway was identified that depends on isolated nuclear histones. As one component of the non-replicative assembly pathway we identified a cytosolic factor that was purified to apparent homogeneity and shown to be an acidic 50 kDa polypeptide. The isolated cytosolic 50 kDa protein efficiently promoted nucleosome assembly as demonstrated by one- and two-dimensional gel electrophoresis of in vitro packaged plasmid DNA.     

Preliminary study of sperm chromatin characteristics of the brachyuran crab Maja brachydactyla. Histones and nucleosome-like structures in decapod crustacean sperm nuclei previously described without SNBPs

An interesting characteristic of decapod crustacean sperm nuclei is that they do not contain highly packaged chromatin. In the present study we re-examine the presence of DNA-interacting proteins in sperm nuclei of the brachyuran Maja brachydactyla. Although previous reports have indicated that, unlike the majority of sperm cells, DNA of decapod sperm is not organized by basic proteins, in this work we show that: (1) histones are present in sperm of M. brachydactyla; (2) histones are associated with sperm DNA; (3) histone H3 appears in lower proportions than the other core histones, while histone H2B appears in higher proportions; and (4) histone H3 in sperm nuclei is acetylated. This work complements a previous study of sperm histones of Cancer pagurus and supports the suggestion that decapod crustacean sperm chromatin deserves further attention.