Interactive Fly, Drosophila

Nucleosome assembly protein 1


Yeast and plant NAPs

Yeast DNA coding for nucleosome assembly protein I (NAP-I), which facilitates nucleosome assembly in vitro at physiological ionic conditions, was cloned and its gene product characterized. A monoclonal antibody against human NAP-I (58 kDa) was used to screen a genomic library of Saccharomyces cerevisiae constructed into lambda gt11. A 60-kDa protein was detected by immunoblotting in the extracts of Escherichia coli lysogenized with a positive clone. The 60-kDa protein purified from the extracts has an activity equivalent to that of NAP-I from mouse and human cells. The amino acid sequence deduced from the gene coding for the yeast NAP-I defines a polypeptide of molecular mass 47,848 Da with three negatively charged regions. While the two regions contain 8 and 10 acidic amino acids out of 13 amino acid residues, the longest stretch has 15 glutamic and 13 aspartic acids out of 38 residues. These regions are probably involved in the interaction with histones. Proteins recognized by the anti-NAP-I antibody are also present in Xenopus oocytes and Drosophila cultured cells. Possible roles of NAP-I are discussed in relation to other nucleosome assembly proteins (Ishimi, 1991).

A nucleosome assembly protein (NAP1) of S. cerevisiae, a member of the NAP/SET family, facilitates the association of histones with DNA to form nucleosomes in vitro when physiological ionic conditions prevail. The internal fragment containing the residues 43-365 is necessary and sufficient for the activity, and a long stretch of a negatively charged region near the carboxyl terminus is dispensable. This minimal size fragment can form the 12 S NAP1-histone complex as well as can the whole protein, whereas deleted fragments on either side can bind with core histones to form only aggregates (Fujii-Nakata, 1992). Nucleosome assembly protein 1

Yeast nucleosome assembly protein 1 (NAP1), a member of the NAP/SET family stimulates transcription factor binding and nucleosome displacement in a manner similar to that of nucleoplasmin. Disruption of the histone octamer is required both for the stimulation of transcription factor binding to nucleosomal DNA and for transcription factor-induced nucleosome displacement mediated by nucleoplasmin or NAP1. While NAP1 and nucleoplasmin stimulate the binding of a fusion protein (GAL4-AH) to control nucleosome cores, this stimulation is lost upon covalent histone-histone cross-linking within the histone octamers. In addition, both NAP1 and nucleoplasmin are able to mediate histone displacement upon the binding of five GAL4-AH dimers to control nucleosome cores; however, this activity is also forfeited when the histone octamers are cross-linked. Thus octamer disruption is required for both stimulation of factor binding and factor-dependent histone displacement by nucleoplasmin and NAP1. By contrast, transcription factor-induced histone transfer onto nonspecific competitor DNA does not require disruption of the histone octamer. Thus, histone displacement in this instance occurs by transfer of complete histone octamers, a mechanism distinct from that mediated by the histone-binding proteins nucleoplasmin and NAP1 (Walter, 1995).

A 60-kD protein called NAP1/SET (not to be confused with Drosophila NAP1) has been purified form Xenopus, S. cerevisiae, and Drosophila. The yeast protein is known to function in nucleosome assembly. Members of the NAP/SET family of proteins interact specifically with B-type cyclins. This interaction is highly conserved during evolution: NAP1/SET interacts with cyclins B1 and B2, but not with cyclin A; the S. cerevisiae homolog interacts with B-type cyclins Clb2 but not Clb3. The yeast NAP1/SET protein is cytoplasmic. Genetic experiments in budding yeast indicate that NAP1/SET plays an important role in the function of Clb2, while biochemical experiments demonstrate that purifed NAP1 can be phosphorylated by cyclin B/p34cdc2 kinase complexes but not cyclin A/p34cdc2 kinase complexes. These results suggest that NAP1/SET is a protein involved in the specific functions of cyclin B/p34cdc2 kinase complexes (Kellogg, 1995a).

NAP1/SET is a 60-kD protein that interacts specifically with mitotic cyclins in budding yeast and frogs. The yeast B-type mitotic cyclin Clb2 is unable to carry out its full range of functions without NAP1, even though Clb2/p34CDC28-associated kinase activity rises to normal levels. In the absence of NAP1/SET, Clb2 is unable to efficiently induce mitotic events, and cells undergo a prolonged delay at the short spindle stage with normal levels of Clb2/p34CDC28 kinase activity. NAP1/SET is also required for the ability of Clb2 to induce the switch from polar to isotropic bud growth. As a result, polar bud growth continues during mitosis, giving rise to highly elongated cells. These experiments also suggest that NAP1/SET is required for the ability of the Clb2/p34CDC28 kinase complex to amplify its own production, and that NAP1/SET plays a role in regulation of microtubule dynamics during mitosis. Together, these results demonstrate that NAP1/SET is required for the normal function of the activated Clb2/p34CDC28 kinase complex, and provide a step towards understanding how cyclin-dependent kinase complexes induce specific events during the cell cycle (Kellogg, 1995b).

NAP-1, a protein first isolated from mammalian cells, can introduce supercoils into relaxed circular DNA in the presence of purified core histones. Based on its in vitro activity, it has been suggested that NAP-1 may be involved in nucleosome assembly in vivo. SNAP-1, a soybean NAP-1 homolog, has been cloned. The SNAP-1 cDNA contains an open reading frame of 358 amino acids residues with a calculated molecular weight of 41 kDa. The deduced amino acid sequence of SNAP-1 shares sequence similarity with yeast NAP-1 (38%) and human hNRP (32%). Notable features of the deduced sequence are two extended acidic regions thought to be involved in histone binding. SNAP-1 expressed in Escherichia coli induces supercoiling in relaxed circular DNA, suggesting that SNAP-1 may possess nucleosome assembly activity. The specific activity of SNAP-1 is comparable to that of HeLa NAP-1 in an in vitro assay. Western analysis reveals that SNAP-1 is expressed in the immature and young tissues that were examined, while mature tissues such as old leaves and roots, show very little or no expression. NAP-1 homologs also appear to be present in other plant species (Yoon, 1995).

Mammalian NAPs

Three genes on chromosome 11p15.5 are known to undergo genomic imprinting. The gene for insulin-like growth factor II (IGF2) is normally expressed from the paternal allele, while H19 and p57KIP2, a cyclin-dependent kinase inhibitor, are expressed from the maternal allele. Five germline balanced chromosomal rearrangement breakpoints from patients with Beckwith-Wiedemann syndrome (BWS) have been mapped to 11p15.5 between p57KIP2 and IGF2, and all are derived from the maternal chromosome. By positional cloning from BWS breakpoints, a gene has been isolated and located 100 kb and 65 kb centromeric to the proximal end of this BWS breakpoint cluster and p57KIP2. This gene is homologous to yeast nucleosome assembly protein (NAP1) and to a human homolog of NAP1, and is designated hNAP2 (human nucleosome assembly protein 2). hNAP2 diverges in its expression pattern from IGF2, H19, and p57KIP2; it shows biallelic expression in all tissues tested. Thus, hNAP2 is functionally insulated from the imprinting domain of 11p15 (Hu, 1996).

A cDNA clone has been isolated from a human thymus cDNA library that encodes a protein with 54% amino acid similarity to yeast nucleosome assembly protein I (NAP-I). The sequence for this newly identified protein, designated hNRP (human NAP-related protein), contains a potential seven-residue nuclear localization motif, three clusters of highly acidic residues and other structural features found in various proteins implicated in chromatin formation. When expressed as a fusion protein in Escherichia coli, hNRP reacts specifically with a monoclonal antibody raised against human NAP-I. The hNRP transcript is detected in all tissues and cell lines studied, but levels are somewhat increased in rapidly proliferating cells. Moreover, levels of both hNRP mRNA and protein increased rapidly in cultured T-lymphocytes induced to proliferate by incubation with phorbol ester and ionomycin. Phorbol/ionomycin-induced cells show increases in both hNRP mRNA and mitogenesis, as measured by thymidine incorporation. These responses are markedly inhibited, however, in cells treated with an hNRP antisense oligonucleotide. Thus there is a correlation between induction of hNRP expression and mitogenesis; taken together with the structural similarities between hNRP and yeast NAP-I they suggest that the hNRP gene product participates in DNA replication and thereby plays an important role in the process of cell proliferation (Simon, 1994).

Histones are thought to play a key role in regulating gene expression at the level of DNA packaging. Recent evidence suggests that transcriptional activation requires competition of transcription factors with histones for binding to regulatory regions and that there may be several mechanisms by which this is achieved. A human nucleosome assembly protein, NAP-2, has been characterized. NAP-2 was identified by positional cloning at 11p15.5, a region implicated in several disease processes including Wilms tumor (WT) etiology. The deduced amino acid sequence of NAP-2 indicates that it encodes a protein with a potential nuclear localization motif and two clusters of highly acidic residues. Functional analysis of recombinant NAP-2 protein purified from Escherichia coli demonstrates that this protein can interact with both core and linker histones. Recombinant NAP-2 can transfer histones onto naked DNA templates. Deletion mutagenesis of NAP-2 demonstrates that both NH3- and COOH-terminal domains are required for histone transfer activity. Subcellular localization studies of NAP-2 indicate that it can shuttle between the cytoplasm and the nucleus, suggesting a role as a histone chaperone. Given the potential role of the human NAP-2 gene (HGMW-approved symbol NAP1L4) in WT etiology, the exon/intron structure of this gene has been elucidated and the mutational status of NAP-2 in sporadic WTs has been analyzed. These results, coupled with tumor suppression assays in G401 WT cells, do not support a role for NAP-2 in the etiology of WT. A putative role for NAP-2 in regulating cellular differentiation is discussed (Rodriguez, 1997).

For the activation of replication and transcription from DNA in a chromatin structure, a variety of factors are thought to be needed that alter the chromatin structure. Template activating factor-I (TAF-I) has been identified as such a host factor required for replication of the adenovirus (Ad) genome complexed with viral basic core proteins (Ad core). TAF-I also stimulates transcription from the Ad core DNA. Using mutant TAF-I proteins, it has been shown that the acidic stretch present in the carboxyl terminal region is essential for the stimulation of transcription from the Ad core. A genomic footprinting experiment with restriction endonuclease reveals that TAF-I causes a structural change in the Ad core. TAF-I has been shown to have significant amino acid similarity to nucleosome assembly protein-I (NAP-I), which is involved in the formation of the chromatin structure. TAF-I can be substituted for by NAP-I in the activation of the cell-free Ad core transcription system. Two of the tripartite acidic regions and the region homologous to TAF-I in NAP-I are required for the maximal TAF-I activity of NAP-I. TAF-I has been shown to have NAP-I activity; the acidic region of TAF-I is required for this activity. Since TAF-I causes the structural change of the Ad core and thereby activates transcription, TAF-I is thought to be one of the proteins that is involved in chromatin remodeling. NAP-I is structurally related to TAF-I and functionally substitutes for TAF-I. Furthermore, TAF-I exhibits NAP-I activity. These observations suggest that this type of molecule has dual functions, possibly by participating in facilitating the assembly of the chromatin structure as well as perturbing the chromatin structure to allow transcription to proceed (Kawase, 1997).

The CENP-B dimer may play a critical role in the assembly of higher order structures of the human centromere by juxtaposing CENP-B boxes in long alpha-satellite arrays. Therefore, studies have been carried out of the nucleosome structure formed from alpha-satellite DNA bound with CENP-B and core histones. The dimeric structure of CENP-B is sufficiently stable to bundle together two 3.5 kbp DNA fragments when each DNA fragment contains a CENP-B box. When the same length of DNA includes two CENP-B boxes, the intra-molecular interaction with the CENP-B dimer predominates, resulting in the formation of loop structures. The in vitro assembly of CENP-B/alpha-satellite DNA/core histone complexes with the aid of nucleosome assembly protein-1 (NAP-1) permitted an investigation into the nucleosome arrangement in alpha-satellite DNA with CENP-B bound to CENP-B boxes. Footprint analyses with micrococcal nuclease (MNase) reveal that CENP-B causes nucleosome positioning between pairs of CENP-B boxes with unique hypersensitive sites created on both sides. It has been proposed that CENP-B functions as a structural factor in the centromere region in order to establish a unique, centromere specific pattern of nucleosome positioning (Yoda, 1999).

NAPs and sperm chromatin sperm decondensation

Previous studies have shown that the nuclear phosphoprotein nucleoplasmin performs the first stage of chromatin decondensation of Xenopus sperm at fertilization. It binds and removes sperm basic proteins replacing them with histones. This activity depends upon the massive hyperphosphorylation of nucleoplasmin that occurs when oocytes mature into eggs. Egg extracts or purified hyperphosphorylated egg nucleoplasmin decondense sperm chromatin and remove sperm basic proteins much faster than oocyte extracts or hypophosphorylated oocyte nucleoplasmin. Furthermore, dephosphorylation of egg nucleoplasmin slows sperm decondensation and prevents basic protein removal from sperm chromatin. It is concluded that hyperphosphorylation of nucleoplasmin is used to modulate the rapid changes in chromatin structure that accompany early development in Xenopus (Leno, 1996).

Nucleoplasmin, an acidic thermostable protein abundant in the nucleus of Xenopus laevis oocytes, has been found to dissociate complexes of pUC19 DNA and protein phi 1, an intermediate protamine present in ripe sperm from the mollusc Mytilus edulis. Cruder preparations of nucleoplasmin, such as the amphibian oocyte S150 extract and its thermostable fraction, also dissociate the heterologous DNA-phi 1 complexes and, in addition, promote the assembly of plasmid DNA into a minichromosome displaying regular nucleosomal periodicity, as revealed by micrococcal nuclease digestion. In contrast, purified nucleoplasmin complemented with rat hepatocyte core histone octamers in the presence of DNA topoisomerase I, although capable of inducing nucleoprotein formation onto the complexed DNA, fails to position nucleosomes at the native spacings seen in chromatin in vivo. These data favour the existence of a general mechanism to bring about, in a concerted manner, removal of sperm-specific nuclear proteins and reconstitution of somatic chromatin following fertilization (Ruiz-Lara, 1996).

NAPs, nucleosome chaperones, and chromatin remodeling

An examination of the organization and acetylation of nascent histones prior to their stable incorporation into chromatin detected two somatic non-nucleosomal histone complexes: one containing nascent H3 and H4, and a second containing H2A (and probably H2B) in association with the nonhistone protein NAP-1. The H3/H4 complex has a sedimentation coefficient of 5-6S, consistent with the presence of one or more escort proteins. H4 in the cytosolic H3/H4 complex is diacetylated, fully in accord with the acetylation state of newly synthesized H4 in chromatin. The diacetylation of nascent human H4 is therefore completed prior to nucleosome assembly. HeLa histone acetyltransferase B (HAT B) acetylates H4 but not H3 in vitro, and maximally diacetylates H4 even in the presence of sodium butyrate. Human HAT B acetylates H4 exclusively on the lysine residues at positions 5 and 12, in complete agreement with the highly conserved acetylation pattern of nascent nucleosomal H4 and has a native molecular weight of approximately 100 kDa. Based on these findings a model is presented for the involvement of histone acetylation and NAP-1 in H2A/H2B deposition and exchange, during nucleosome assembly and chromatin remodeling in vivo (Chang, 1997).

To investigate mechanisms of chromatin remodeling, the fate of a single nucleosome core was examined within a spaced nucleosome array upon the binding of transcription factors. Tandem repeats of the Sea Urchin 5S rRNA gene were employed. GAL4 binding to the single nucleosome within an array results in the establishment of DNase I hypersensitivity adjacent to the bound factors mimicking in vivo hypersensitive sites. The positions of adjacent nucleosomes are unchanged upon GAL4 binding, suggesting that histone octamer sliding does not occur. Novel assays were used to determine whether the histones remained present during factor binding. GAL4 binding alone does not independently dislodge or move the underlying histones, which remain in a ternary complex with the bound GAL4. GAL4 binding does, however, specifically predispose the histones contained in this nucleosome to displacement in trans. By trans-displacement is meant the removal of the histone octamer from the DNA bound nucleosomal array. Trans-displacement is distinguished from cis-displacement, the transfer of nucleosomes from the trnasription factor-bound sequences onto other segments of chromosomal DNA through direct transfer. Addition of the histone binding protein, Xenopus nucleoplasmin, mediates the displacement of the core histones in the GAL4-bound nucleosome, resulting in the formation of a nucleosome-free region. These data illustrate trans-displacement of histones as one mechanism for transcription factor-targeted generation of a nucleosome-free region in chromatin. They also illustrate the limitations of nuclease digestions in analyzing changes in chromatin structure and provide important mechanistic details beyond the basic phenomenon of DNase I hypersensitivity (Owen-Hughes, 1996).

A purified recombinant chromatin assembly system, including ACF (Acf-1 + ISWI) and NAP-1, has been used to examine the role of histone acetylation in ATP-dependent chromatin remodeling. The binding of a transcriptional activator (Gal4-VP16) to chromatin assembled using this recombinant assembly system dramatically enhances the acetylation of nucleosomal core histones by the histone acetyltransferase p300. This effect requires both the presence of Gal4-binding sites in the template and the VP16-activation domain. Order-of-addition experiments indicate that prior activator-meditated, ATP-dependent chromatin remodeling by ACF is required for the acetylation of nucleosomal histones by p300. Thus, chromatin remodeling, which requires a transcriptional activator, ACF and ATP, is an early step in the transcriptional process that regulates subsequent core histone acetylation. Glycerol gradient sedimentation and immunoprecipitation assays demonstrate that the acetylation of histones by p300 facilitates the transfer of H2A-H2B from nucleosomes to NAP-1. The results from these biochemical experiments suggest that (1) transcriptional activators (e.g., Gal4-VP16) and chromatin remodeling complexes (e.g., ACF) induce chromatin remodeling in the absence of histone acetylation; (2) transcriptional activators recruit histone acetyltransferases (e.g., p300) to promoters after chromatin remodeling has occurred; and (3) histone acetylation is important for a step subsequent to chromatin remodeling and results in the transfer of histone H2A-H2B dimers from nucleosomes to a histone chaperone such as NAP-1. These results indicate a precise role for histone acetylation, namely to alter the structure of nucleosomes (e.g., facilitate the loss of H2A-H2B dimers) that have been remodeled previously by the action of ATP-dependent chromatin remodeling complexes. Thus, transcription from chromatin templates is ordered and sequential, with precise timing and roles for ATP-dependent chromatin remodeling, subsequent histone acetylation, and alterations in nucleosome structure. The presence of altered (i.e., H2A-H2B-depleted) nucleosomes at a transcriptionally active, chromatin-remodeled promoter may help to maintain an open chromatin structure conducive to multiple rounds of activated transcription (Ito, 2000).

Post-translational modification of NAPs

Nucleoplasmin is a phosphorylated nuclear-accumulating protein. The kinetics of its cytoplasm to nucleus transport are affected by its degree of phosphorylation. Nucleoplasmin co-isolates by two independent methods in a complex including a kinase which phosphorylates nucleoplasmin. The co-purifying kinase is casein kinase II-like because: (1) it phosphorylates casein; (2) its phospho-transferase activity can be competed out by GTP; (3) it is stimulated by polylysine and (4) it is inhibited by heparin. A polyclonal antibody to the alpha (38 kDa) and alpha' (36 kDa) catalytic subunits of casein kinase II specifically recognizes 38 and 36 kDa polypeptides in the nucleoplasmin-complex, and a specific inhibitor of casein kinase II inhibits nucleoplasmin's nuclear transport. Phosphorylation of nucleoplasmin by its associated casein kinase II is strongly inhibited by histones; in addition to nucleoplasmin, another protein (p100) in the nucleoplasmin-complex is phosphorylated by casein kinase II (Vancurova, 1995).

NAP and development

Nucleosome assembly proteins have been identified in all eukaryotic species investigated to date and their suggested roles include histone shuttle, histone acceptor during transcriptional chromatin remodelling and cell cycle regulator. To examine the role of these proteins during early development, the cDNA encoding Xenopus NAP1L was isolated, an antibody against recombinant xNAP1L was raised and the expression pattern of this mRNA and protein was examined. Expression in adults is predominantly in ovaries. This maternal protein remains a major component of xNAP1L within the embryo until swimming tadpole stages. xNAP1L mRNA is initially throughout the embryo but by gastrula stages it is predominantly in the presumptive ectoderm. Later, mRNA is detected in the neural crest, neural tube, eyes, tailbud and ventral blood islands. In order to test whether xNAP1L has a potential role in gene regulation this protein was overexpressed in animal pole explants, and the effect on expression of a series of potential target genes was tested. The mRNA encoding the transcription factor GATA-2 was markedly up-regulated by this overexpression. These data support a role for xNAP1L in tissue-restricted gene regulation (Steer, 2003).

Nucleosome assembly protein 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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