Casein kinase 2 binds and phosphorylates the nucleosome assembly protein-1 (NAP1) in Drosophila melanogaster.

The nucleosome assembly protein-1 (NAP1) was originally identified in HeLa cells as a factor facilitating the in vitro assembly of nucleosomes. However, in yeast cells NAP1 is required in the control of mitotic events induced by the Clb2/p34(CDC28). Here, we show that Drosophila NAP1 is a phosphoprotein that is associated with a kinase able to phosphorylate NAP1. By using an in-gel kinase assay we found that this kinase displays a molecular mass of 38 kDa. Following purification and peptide microsequencing, we identified the kinase phosphorylating NAP1 as the alpha subunit of casein kinase 2 (CK2). With the help of a series of NAP1 segments and synthetic peptides, we assigned the CK2 phosphorylation sites to residues Ser118, Thr120, and Ser284. Interestingly, Ser118 and Thr120 are located within a PEST domain, while Ser284 is adjacent to the nuclear localization signal. Substitution of the identified phosphoresidues by alanine was found to reduce considerably the ability of CK2 to phosphorylate NAP1. The enhanced ability of CK2 to phosphorylate phosphatase-treated NAP1 extracted from Drosophila embryos and the similar tryptic phospho-peptide pattern of in vivo labelled NAP1 and in vitro labelled NAP1 with CK2 indicate that NAP1 is a natural substrate of CK2. Further analysis revealed that both CK2alpha and beta subunits are associated with NAP1 but we found that only the catalytic alpha subunit establishes direct contact with NAP1 on two distinct domains of this protein. The location of CK2 phosphorylation sites in NAP1 suggests that their phosphorylation can contribute to a PEST-mediated protein degradation of NAP1 and the translocation of NAP1 between cytoplasm and nucleus.     

Functional analysis of nucleosome assembly protein, NAP-1. The negatively charged COOH-terminal region is not necessary for the intrinsic assembly activity.

A nucleosome assembly protein (NAP-1) of Saccharomyces cerevisiae facilitates the association of histones with DNA to form nucleosomes in vitro at physiological ionic conditions. The cloned gene was expressed in Escherichia coli using a T7 expression system, and the protein (417 amino acid residues) was purified by Mono Q column chromatography. Various deletion fragments of NAP-1 protein were also produced, and their nucleosome assembly activity was examined by supercoiling assay. The internal fragment containing the residues 43-365 was necessary and sufficient for the activity, and a long stretch of negatively charged region near the carboxyl terminus was dispensable. This minimal size fragment could form the 12 S NAP-1-histone complex as the whole protein could, whereas deleted fragments on either side could bind with core histones only to form aggregates.     

Scm3 is a centromeric nucleosome assembly factor

The Cse4 nucleosome at each budding yeast centromere must be faithfully assembled each cell cycle to specify the site of kinetochore assembly and microtubule attachment for chromosome segregation. Although Scm3 is required for the localization of the centromeric H3 histone variant Cse4 to centromeres, its role in nucleosome assembly has not been tested. We demonstrate that Scm3 is able to mediate the assembly of Cse4 nucleosomes in vitro, but not H3 nucleosomes, as measured by a supercoiling assay. Localization of Cse4 to centromeres and the assembly activity depend on an evolutionarily conserved core motif in Scm3, but localization of the CBF3 subunit Ndc10 to centromeres does not depend on this motif. The centromere targeting domain of Cse4 is sufficient for Scm3 nucleosome assembly activity. Assembly does not depend on centromeric sequence. We propose that Scm3 plays an active role in centromeric nucleosome assembly.     

TTLL10 is a protein polyglycylase that can modify nucleosome assembly protein 1

Certain proteins can undergo polyglycylation and polyglutamylation. Polyglutamylases (glutamate ligases) have recently been identified in a family of tubulin tyrosine ligase-like (TTLL) proteins. However, no polyglycylase (glycine ligase) has yet been reported. Here we identify a polyglycylase in the TTLL proteins by using an anti-poly-glycine antibody. The antibody reacted with a cytoplasmic 60-kDa protein that accumulated in elongating spermatids. Using tandem mass spectrometry of trypsinized samples, immunoprecipitated by the antibody from the TTLL10-expressing cells, we identified the 60-kDa protein as nucleosome assembly protein 1 (NAP1). Recombinant TTLL10 incorporated glycine into recombinant NAP1 in vitro. Mutational analyses indicated that Glu residues at 359 and 360 in the C-terminal part of NAP1 are putative sites for the modification. Thus, TTLL10 is a polyglycylase for NAP1.     

Self-association of the yeast nucleosome assembly protein 1

The self-association properties of the yeast nucleosome assembly protein 1 (yNAP1) have been investigated using biochemical and biophysical methods. Protein cross-linking and calibrated gel filtration chromatography of yNAP1 indicate the protein exists as a complex mixture of species at physiologic ionic strength (75-150 mM). Sedimentation velocity reveals a distribution of species of 4.5-12 Svedbergs (S) over a 50-fold range of concentrations. The solution-state complexity is reduced at higher ionic strength, allowing for examination of the fundamental oligomer. Sedimentation equilibrium of a homogeneous 4.5 S population at 500 mM sodium chloride reveals these species to be yNAP1 dimers. These dimers self-associate to form higher order oligomers at more moderate ionic strength. Titration of guanidine hydrochloride converts the higher order oligomers to the homogeneous 4.5 S dimer and then converts the 4.5 S dimers to 2.5 S monomers. Circular dichroism shows that guanidine-mediated dissociation of higher order oligomers into yNAP1 dimers is accompanied by only slight changes in secondary structure. Dissociation of the dimer requires a nearly complete denaturation event.     

The ins and outs of nucleosome assembly

De novo nucleosome assembly coupled to DNA replication and repair in vitro involves the histone chaperone chromatin assembly factor 1 (CAF-1). Recent studies support a model in which CAF-1 can be targeted to newly synthesized DNA through a direct interaction with proliferating cell nuclear antigen (PCNA) and can act synergistically with a newly identified histone chaperone. Insights have also been obtained into mechanisms by which this CAF-1-dependent pathway can establish a repressed chromatin state.     

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.     

Nucleosome assembly proteins NAP1L1 and NAP1L4 modulate p53 acetylation to regulate cell fate

The p53 tumor suppressor regulates expression of genes involved in various stress responses. Upon genotoxic stress, p53 induces target genes regulating cell cycle arrest for survival or apoptosis. Nevertheless, detailed mechanisms of how p53 selectively regulates these opposing outcomes remain unclear. For this study, we investigated p53 regulatory mechanisms exerted by nucleosome assembly protein 1-like 1 (NAP1L1) and NAP1L4, both of which are identified as DGKζ-interacting proteins. Here we demonstrate that, under normal conditions, NAP1L1 knockdown decreases Lys320 acetylation of p53 with attenuated proarrest p21 expression, whereas NAP1L4 knockdown increases Lys320 acetylation with enhanced p21 expression. These conditions lead respectively to facilitation and suppression of cell growth. Under genotoxic stress conditions, NAP1L1 knockdown increases Lys382 acetylation with enhanced proapoptotic Bax levels, thereby facilitating cell death. By contrast, NAP1L4 knockdown decreases Lys382 acetylation with attenuated Bax levels, thereby suppressing apoptosis. These results suggest that NAP1L1 and NAP1L4 regulate cell fate by controlling the expression of p53-responsive proarrest and proapoptotic genes through selective modulation of p53 acetylation at specific sites during normal homeostasis and in stress-induced responses.     

Human SWI/SNF nucleosome remodeling activity is partially inhibited by linker histone H1

The physical structure and the compact nature of the eukaryotic genome present a functional barrier for any cellular process that requires access to the DNA. The linker histone H1 is intrinsically involved in both the determination of and the stability of higher order chromatin structure. Because histone H1 plays a pivotal role in the structure of chromatin, we investigated the effect of histone H1 on the nucleosome remodeling activity of human SWI/SNF, an ATP-dependent chromatin remodeling complex. The results from both DNase I digestion and restriction endonuclease accessibility assays indicate that the presence of H1 partially inhibits the nucleosome remodeling activity of hSWI/SNF. Neither H1 bound to the nucleosome nor free H1 affected the ATPase activity of hSWI/SNF, suggesting that the observed inhibition of hSWI/SNF nucleosome remodeling activity depends on the structure formed by the addition of H1 to nucleosomes.     

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)     

Replication-independent nucleosome exchange is enhanced by local and specific acetylation of histone H4

We used a novel single-cell strategy to examine the fate of histones during G₂-phase. Consistent with previous results, we find that in G₂-phase, the majority of nuclear histones are assembled into chromatin, whereas a small fraction comprises an unassembled pool. Small increases in the amount of histones within the free pool affect the extent of exchange, suggesting that the free pool is in dynamic equilibrium with chromatin proteins. Unexpectedly, acetylated H4 is preferentially partitioned to the unassembled pool. Although an increase in global histone acetylation did not affect overall nucleosome dynamics, an H4 containing lysine to glutamine substitutions as mimics of acetylation significantly increased the rate of exchange, but did not affect the acetylation state of neighbouring nucleosomes. Interestingly, transcribed regions are particularly predisposed to exchange on incorporation of H4 acetylation mimics compared with surrounding regions. Our results support a model whereby histone acetylation on K8 and K16 specifically marks nucleosomes for eviction, with histones being rapidly deacetylated on reassembly.     

Nuclear reassembly in vitro is independent of nucleosome/chromatin assembly

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Polyglutamylation of nucleosome assembly proteins

Polyglutamylation is an original posttranslational modification, discovered on tubulin, consisting in side chains composed of several glutamyl units and leading to a very unusual protein structure. A monoclonal antibody directed against glutamylated tubulin (GT335) was found to react with other proteins present in HeLa cells. After immunopurification on a GT335 affinity column, two prominent proteins of approximately 50 kDa were observed. They were identified by microsequencing and mass spectrometry as NAP-1 and NAP-2, two members of the nucleosome assembly protein family that are implicated in the deposition of core histone complexes onto chromatin. Strikingly, NAP-1 and NAP-2 were found to be substrates of an ATP-dependent glutamylation enzyme co-purifying on the same column. We took advantage of this property to specifically label and purify the polyglutamylated peptides. NAP-1 and NAP-2 are modified in their C-terminal domain by the addition of up to 9 and 10 glutamyl units, respectively. Two putative glutamylation sites were localized for NAP-1 at Glu-356 and Glu-357 and, for NAP-2, at Glu-347 and Glu-348. These results demonstrate for the first time that proteins other than tubulin are polyglutamylated and open new perspectives for studying NAP function.     

HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis

The mammalian HIRA gene encodes a histone-interacting protein whose homolog in Xenopus laevis is characterized here. In vitro, recombinant Xenopus HIRA bound purified core histones and promoted their deposition onto plasmid DNA. The Xenopus HIRA protein, tightly associated with nuclear structures in somatic cells, was found in a soluble maternal pool in early embryos. Xenopus egg extracts, known for their chromatin assembly efficiency, were specifically immunodepleted for HIRA. These depleted extracts were severely impaired in their ability to assemble nucleosomes on nonreplicated DNA, although nucleosome formation associated with DNA synthesis remained efficient. Furthermore, this defect was largely corrected by reintroduction of HIRA along with (H3-H4)(2) tetramers. We thus delineate a nucleosome assembly pathway that depends on HIRA.     

The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly

Two very similar H3 histones-differing at only four amino acid positions-are produced in Drosophila cells. Here we describe a mechanism of chromatin regulation whereby the variant H3.3 is deposited at particular loci, including active rDNA arrays. While the major H3 is incorporated strictly during DNA replication, amino acid changes toward H3.3 allow replication-independent (RI) deposition. In contrast to replication-coupled (RC) deposition, RI deposition does not require the N-terminal tail. H3.3 is the exclusive substrate for RI deposition, and its counterpart is the only substrate retained in yeast. RI substitution of H3.3 provides a mechanism for the immediate activation of genes that are silenced by histone modification. Inheritance of newly deposited nucleosomes may then mark sites as active loci.     

Human CAF-1-dependent nucleosome assembly in a defined system

Replication-coupled nucleosome assembly is a critical step in packaging newly synthesized DNA into chromatin. Previous studies have defined the importance of the histone chaperones CAF-1 and ASF1A, the replicative clamp PCNA, and the clamp loader RFC for the assembly of nucleosomes during DNA replication. Despite significant progress in the field, replication-coupled nucleosome assembly is not well understood. One of the complications in elucidating the mechanisms of replication-coupled nucleosome assembly is the lack of a defined system that faithfully recapitulates this important biological process in vitro. We describe here a defined system that assembles nucleosomal arrays in a manner dependent on the presence of CAF-1, ASF1A-H3-H4, H2A-H2B, PCNA, RFC, NAP1L1, ATP, and strand breaks. The loss of CAF-1 p48 subunit causes a strong defect in packaging DNA into nucleosomes by this system. We also show that the defined system forms nucleosomes on nascent DNA synthesized by the replicative polymerase δ. Thus, the developed system reproduces several key features of replication-coupled nucleosome assembly.