Histone Chaperone Nap1 Research Articles

Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A.

Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. We found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesin subunit stromalin (SA). NAP1 binding to cohesin is cell-cycle regulated and increases during G2/M phase. This causes the dissociation of protein phosphatase 2A (PP2A) from cohesin, increased phosphorylation of SA and cohesin removal in early mitosis. PP2A depletion led to a loss of centromeric cohesion. The distinct mitotic phenotypes caused by the loss of either PP2A or NAP1, were both rescued by their concomitant depletion. We conclude that the balanced antagonism between NAP1 and PP2A controls cohesin dissociation during mitosis.

Proteomics identifies ubiquitin–proteasome targets and new roles for chromatin-remodeling in the Arabidopsis response to phosphate starvation

In order to identify new regulators of the phosphate (Pi) starvation signaling pathway in plants, we analyzed variation in the abundance of nuclear-enriched proteins isolated from Arabidopsis roots that depends on Pi supply. We used 2-D fluorescence difference gel electrophoresis and MALDI-TOF/TOF techniques for proteome separation, visualization and relative protein abundance quantification and identification. Pi-controlled proteins identified in our analysis included components of the chromatin remodeling, DNA replication, and mRNA splicing machineries. In addition, by combining Pi starvation conditions with proteasome inhibitor treatments, we characterized the role of the ubiquitin-proteasome system, a major mechanism for targeted protein degradation in eukaryotes, in the control of the stability of Pi-responsive proteins. Among Pi-responsive proteins, the histone chaperone NAP1;2 was selected for further characterization, and was shown to display differential nucleo-cytoplasmic accumulation in response to Pi deprivation. We also found that mutants affecting three members of the NAP1 family accumulate lower Pi levels and display reduced expression of Pi starvation-inducible genes, reflecting a potential regulatory role for these chromatin-remodeling proteins in Pi homeostasis. In this study, we explore the feasibility of nuclear proteomics to identify regulatory proteins and ubiquitin-proteasome targets within a specific stress signaling pathway in plants, in our case phosphate starvation signaling in Arabidopsis. It will be of interest for researchers involved in the dissection of any signaling pathway in plants, in particular those with an interest in the ubiquitin-proteasome functions, and for the plant nutrition community.

Histone Chaperones Nap1 and Vps75 Regulate Histone Acetylation during Transcription Elongation

Histone chaperones function in chromatin assembly and disassembly, suggesting they have important regulatory roles in transcription elongation. The Saccharomyces cerevisiae proteins Nap1 and Vps75 are structurally related, evolutionarily conserved histone chaperones. We showed that Nap1 genetically interacts with several transcription elongation factors and that both Nap1 and Vps75 interact with the RNA polymerase II kinase, CTK1. Loss of NAP1 or VPS75 suppressed cryptic transcription within the open reading frame (ORF) observed when strains are deleted for the kinase CTK1. Loss of the histone acetyltransferase Rtt109 also suppressed ctk1-dependent cryptic transcription. Vps75 regulates Rtt109 function, suggesting that they function together in this process. Histone H3 K9 was found to be the important lysine that is acetylated by Rtt109 during ctk1-dependent cryptic transcription. We showed that both Vps75 and Nap1 regulate the relative level of H3 K9 acetylation in the STE11 ORF. This supports a model in which Nap1, like Vps75, directly regulates Rtt109 activity or regulates the assembly of acetylated chromatin. Although Nap1 and Vps75 share many similarities, due to their distinct interactions with SET2, Nap1 and Vps75 may also play separate roles during transcription elongation. This work sheds further light on the importance of histone chaperones as general regulators of transcription elongation.

Fluorescence strategies for high-throughput quantification of protein interactions

Advances in high-throughput characterization of protein networks in vivo have resulted in large databases of unexplored protein interactions that occur during normal cell function. Their further characterization requires quantitative experimental strategies that are easy to implement in laboratories without specialized equipment. We have overcome many of the previous limitations to thermodynamic quantification of protein interactions, by developing a series of in-solution fluorescence-based strategies. These methods have high sensitivity, a broad dynamic range, and can be performed in a high-throughput manner. In three case studies we demonstrate how fluorescence (de)quenching and fluorescence resonance energy transfer can be used to quantitatively probe various high-affinity protein–DNA and protein–protein interactions. We applied these methods to describe the preference of linker histone H1 for nucleosomes over DNA, the ionic dependence of the DNA repair enzyme PARP1 in DNA binding, and the interaction between the histone chaperone Nap1 and the histone H2A–H2B heterodimer.

Identification of a Rapidly Formed Nonnucleosomal Histone-DNA Intermediate that Is Converted into Chromatin by ACF

Chromatin assembly involves the combined action of histone chaperones and ATP-dependent motor proteins. Here, we investigate the mechanism of nucleosome assembly with a purified chromatin assembly system containing the histone chaperone NAP1 andthe ATP-dependent motor protein ACF. These studies revealed the rapid formation of a stable nonnucleosomal histone-DNA intermediate that is converted into canonical nucleosomes by ACF. The histone-DNA intermediate does not supercoil DNA like a canonical nucleosome, but has a nucleosome-like appearance by atomic force microscopy. This intermediate contains all four core histones, lacks NAP1, and is formed by the initial deposition of histones H3-H4. Conversion of the intermediate into histone H1-containing chromatin results in increased resistance to micrococcal nuclease digestion. These findings suggest that the histone-DNA intermediate corresponds to nascent nucleosome-like structures, such as those observed at DNA replication forks. Related complexes might be formed during other chromatin-directed processes such as transcription, DNA repair, and histone exchange.

The Histone Chaperones Nap1 and Vps75 Bind Histones H3 and H4 in a Tetrameric Conformation

SummaryHistone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly.

Nucleosome eviction and activated transcription require p300 acetylation of histone H3 lysine 14

Histone posttranslational modifications and chromatin dynamics are inextricably linked to eukaryotic gene expression. Among the many modifications that have been characterized, histone tail acetylation is most strongly correlated with transcriptional activation. In Metazoa, promoters of transcriptionally active genes are generally devoid of physically repressive nucleosomes, consistent with the contemporaneous binding of the large RNA polymerase II transcription machinery. The histone acetyltransferase p300 is also detected at active gene promoters, flanked by regions of histone hyperacetylation. Although the correlation between histone tail acetylation and gene activation is firmly established, the mechanisms by which acetylation facilitates this fundamental biological process remain poorly understood. To explore the role of acetylation in nucleosome dynamics, we utilized an immobilized template carrying a natural promoter reconstituted with various combinations of wild-type and mutant histones. We find that the histone H3 N-terminal tail is indispensable for activator, p300, and acetyl-CoA-dependent nucleosome eviction mediated by the histone chaperone Nap1. Significantly, we identify H3 lysine 14 as the essential p300 acetylation substrate required for dissociation of the histone octamer from the promoter DNA. Together, a total of 11 unique mutant octamer sets corroborated these observations and revealed a striking correlation between nucleosome eviction and strong activator and acetyl-CoA-dependent transcriptional activation. These novel findings uncover an exclusive role for H3 lysine 14 acetylation in facilitating the ATP-independent and transcription-independent disassembly of promoter nucleosomes by Nap1. Furthermore, these studies directly couple nucleosome disassembly with strong, activator-dependent transcription.

Nucleosome thermodynamics, histone modifications, and histone chaperone function

Genome accessibility is regulated to a large part by the posttranslational modification of histones (PTM). Surprisingly, and despite the fact that nucleosomes were first characterized over 35 years ago, very little is known about the free energy required to assemble and maintain nucleosomes under physiological conditions, and even less is known about the effect of PTMs on nucleosome structure and stability. We have determined the structure of nucleosome bearing several PTMs, and investigated their effect on higher order structure. We also present a thermodynamic framework to describe nucleosome stability and how it is affected by histone modifications. A picture emerges in which various histone modifications have very differentiated effects on nucleosome and chromatin structure and stability, thereby fine‐tuning chromatin structure. Histone chaperones are acidic proteins that interact directly with histones and are implicated in nucleosome (dis)assembly through unknown mechanisms. We show that nucleosome assembly requires elimination of non‐nucleosomal histone‐DNA interactions through the action of the histone chaperone Nap1. Predictions from this unexpected model concur perfectly with our in vivo findings, where the absence of Nap1 results in an excess of non‐nucleosomal histone‐DNA complexes in cells. The accumulation of these atypical complexes has a dramatic effect on transcription activation and repression in vivo.

The Histone Chaperone Nap1 Promotes Nucleosome Assembly by Eliminating Nonnucleosomal Histone DNA Interactions

The organization of the eukaryotic genome into nucleosomes dramatically affects the regulation of gene expression. The delicate balance between transcription and DNA compaction relies heavily on nucleosome dynamics. Surprisingly, little is known about the free energy required to assemble these large macromolecular complexes and maintain them under physiological conditions. Here, we describe the thermodynamic parameters that drive nucleosome formation in vitro. To demonstrate the versatility of our approach, we test the effect of DNA sequence and H3K56 acetylation on nucleosome thermodynamics. Furthermore, our studies reveal the mechanism of action of the histone chaperone nucleosome assembly protein 1 (Nap1). We present evidence for a paradigm in which nucleosome assembly requires the elimination of competing, nonnucleosomal histone-DNA interactions by Nap1. This observation is confirmed in vivo, wherein deletion of the NAP1 gene in yeast results in a significant increase in atypical histone-DNA complexes, as well as in deregulated transcription activation and repression.

Avoiding a Fatal Attraction: Properties of Nucleosomes and a Histone Chaperone Revealed under Physiological Conditions

Andrews et al. report a comprehensive thermodynamic analysis of nucleosomes in low salt, providing new insights into chromatin assembly and revealing an unexpected mechanism used by the histone chaperone Nap1 to prevent aberrant chromatin formation in vitro and in vivo.

Nap1 and Chz1 have Separate Htz1 Nuclear Import and Assembly Functions

We analyzed the nuclear import and regulation of the yeast histone variant Htz1 (H2A.Z), and the role of histone chaperones Nap1 and Chz1 in this process. Copurification suggested that Htz1 and H2B dimerized in the cytoplasm prior to import. Like H2B, Htz1 contained a nuclear localization signal (NLS) in its N-terminus that is recognized by multiple karyopherins (also called importins), indicating multiple transport pathways into the nucleus. However, Kap114 and Kap123 appeared to play the major role in Htz1 import. We also identified a role for Nap1 in the import of Htz1/H2B heterodimers, and Nap1 formed a RanGTP-insensitive import complex with Htz1/H2B and Kap114. Nap1 was necessary for maintaining a soluble pool of Htz1, indicating that its chaperone function may be important for the dynamic exchange of histones within nucleosomes. In contrast, Chz1 was imported by a distinct import pathway, and Chz1 did not appear to interact with Htz1 in the cytoplasm. Genetic analysis indicated that NAP1 has a function in the absence of HTZ1 that is not shared with CHZ1. This provides further evidence that the histone chaperones Nap1 and Chz1 have separate Htz1-dependent and -independent functions.

Nap1 and Chz1 have Separate Htz1 Nuclear Import and Assembly Functions

We analyzed the nuclear import and regulation of the yeast histone variant Htz1 (H2A.Z), and the role of histone chaperones Nap1 and Chz1 in this process. Copurification suggested that Htz1 and H2B dimerized in the cytoplasm prior to import. Like H2B, Htz1 contained a nuclear localization signal (NLS) in its N-terminus that is recognized by multiple karyopherins (also called importins), indicating multiple transport pathways into the nucleus. However, Kap114 and Kap123 appeared to play the major role in Htz1 import. We also identified a role for Nap1 in the import of Htz1/H2B heterodimers, and Nap1 formed a RanGTP-insensitive import complex with Htz1/H2B and Kap114. Nap1 was necessary for maintaining a soluble pool of Htz1, indicating that its chaperone function may be important for the dynamic exchange of histones within nucleosomes. In contrast, Chz1 was imported by a distinct import pathway, and Chz1 did not appear to interact with Htz1 in the cytoplasm. Genetic analysis indicated that NAP1 has a function in the absence of HTZ1 that is not shared with CHZ1. This provides further evidence that the histone chaperones Nap1 and Chz1 have separate Htz1-dependent and -independent functions.

Histone Chaperones ASF1 and NAP1 Differentially Modulate Removal of Active Histone Marks by LID-RPD3 Complexes during NOTCH Silencing

Histone chaperones are involved in a variety of chromatin transactions. By a proteomics survey, we identified the interaction networks of histone chaperones ASF1, CAF1, HIRA, and NAP1. Here, we analyzed the cooperation of H3/H4 chaperone ASF1 and H2A/H2B chaperone NAP1 with two closely related silencing complexes: LAF and RLAF. NAP1 binds RPD3 and LID-associated factors (RLAF) comprising histone deacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets the E(spl) NOTCH-regulated genes. ASF1 facilitates gene-selective removal of the H3K4me3 mark by LAF but has no effect on H3 deacetylation. NAP1 directs high nucleosome density near E(spl) control elements and mediates both H3 deacetylation and H3K4me3 demethylation by RLAF. We conclude that histone chaperones ASF1 and NAP1 differentially modulate local chromatin structure during gene-selective silencing.

Chromatin dynamics during epigenetic reprogramming in the mouse germ line

A unique feature of the germ cell lineage is the generation of totipotency. A critical event in this context is DNA demethylation and the erasure of parental imprints in mouse primordial germ cells (PGCs) on embryonic day 11.5 (E11.5) after they enter into the developing gonads. Little is yet known about the mechanism involved, except that it is apparently an active process. We have examined the associated changes in the chromatin to gain further insights into this reprogramming event. Here we show that the chromatin changes occur in two steps. The first changes in nascent PGCs at E8.5 establish a distinctive chromatin signature that is reminiscent of pluripotency. Next, when PGCs are residing in the gonads, major changes occur in nuclear architecture accompanied by an extensive erasure of several histone modifications and exchange of histone variants. Furthermore, the histone chaperones HIRA and NAP-1 (NAP111), which are implicated in histone exchange, accumulate in PGC nuclei undergoing reprogramming. We therefore suggest that the mechanism of histone replacement is critical for these chromatin rearrangements to occur. The marked chromatin changes are intimately linked with genome-wide DNA demethylation. On the basis of the timing of the observed events, we propose that if DNA demethylation entails a DNA repair-based mechanism, the evident histone replacement would represent a repair-induced response event rather than being a prerequisite.

A genome-wide role for CHD remodelling factors and Nap1 in nucleosome disassembly

Chromatin remodelling factors and histone chaperones were previously shown to cooperatively affect nucleosome assembly and disassembly processes in vitro. Here, we show that Schizosaccharomyces pombe CHD remodellers, the Hrp1 and Hrp3 paralogs physically interact with the histone chaperone Nap1. Genome-wide analysis of Hrp1, Hrp3 and Nap1 occupancy, combined with nucleosome density measurements revealed that the CHD factors and Nap1 colocalized in particular to promoter regions where they remove nucleosomes near the transcriptional start site. Hrp1 and Hrp3 also regulate nucleosome density in coding regions, where they have redundant roles to stimulate transcription. Previously, DNA replication-dependent and -independent nucleosome disassembly processes have been described. We found that nucleosome density increased in the hrp1 mutant in the absence of DNA replication. Finally, regions where nucleosome density increased in hrp1, hrp3 and nap1 mutants also showed nucleosome density and histone modification changes in HDAC and HAT mutants. Thus, this study revealed an important in vivo role for CHD remodellers and Nap1 in nucleosome disassembly at promoters and coding regions, which are linked to changes in histone acetylation.

On the Mechanism of Nucleosome Assembly by Histone Chaperone NAP1

The process of mononucleosome assembly mediated by histone chaperone NAP1 was investigated using DNA fragments 146 and 207 bp in length containing the Lytechinus variegatus 5 S rDNA nucleosome positioning sequence. A quantitative description was derived using gel electrophoresis and fluorescent anisotropy data. First, NAP1-bound H3.H4 was released forming a DNA-histone tetramer complex with a time constant of k(1) = (2.5 +/- 0.7) . 10(4) m(-1) s(-1). The tetrasome was converted quickly (k(2) = (4.1 +/- 3.5) . 10(5) m(-1) s(-1)), by the addition of a single H2A.H2B dimer, into a "hexasome," i.e. a nucleosome lacking one H2A.H2B dimer. From this intermediate a nucleosome was formed by the addition of a second H2A.H2B dimer with an average rate constant k(3) = (6.6 +/- 1.4) . 10(3) m(-1) s(-1). For the back-reaction, significant differences were observed between the 146- and 207-bp DNA upon substitution of the canonical H2A histone with H2A.Z. The distinct nucleosome/hexasome ratios were reflected in the corresponding equilibrium dissociation constants and revealed some differences in nucleosome stability. In a fourth reaction, NAP1 mediated the binding of linker histone H1 to the nucleosome, completing the chromatosome structure with k(4) = (7.7 +/- 3.7) . 10(3) m(-1) s(-1). The activity of the chromatin remodeling complex ACF did not increase the kinetics of the mononucleosome assembly process.

Chromatin remodeling by nucleosome disassembly in vitro

The RSC chromatin-remodeling complex completely disassembles a nucleosome in the presence of the histone chaperone Nap1 and ATP. Disassembly occurs in a stepwise manner, with the removal of H2A/H2B dimers, followed by the rest of the histones and the release of naked DNA. RSC and related chromatin-remodeling complexes may be responsible for the removal of promoter nucleosomes during transcriptional activation in vivo.

Compaction Kinetics on Single DNAs: Purified Nucleosome Reconstitution Systems versus Crude Extract

Kinetics of compaction on single DNA molecules are studied by fluorescence videomicroscopy in the presence of 1), Xenopus egg extracts and 2), purified nucleosome reconstitution systems using a combination of histones with either the histone chaperone Nucleosome Assembly Protein (NAP-1) or negatively charged macromolecules such as polyglutamic acid and RNA. The comparison shows that the compaction rates can differ by a factor of up to 1000 for the same amount of histones, depending on the system used and on the presence of histone tails, which can be subjected to post-translational modifications. Reactions with purified reconstitution systems follow a slow and sequential mechanism, compatible with the deposition of one (H3-H4)2 tetramer followed by two (H2A-H2B) dimers. Addition of the histone chaperone NAP-1 increases both the rate of the reaction and the packing ratio of the final product. These stimulatory effects cannot be obtained with polyglutamic acid or RNA, suggesting that yNAP-1 impact on the reaction cannot simply be explained in terms of charge screening. Faster compaction kinetics and higher packing ratios are reproducibly reached with extracts, indicating a role of additional components present in this system. Data are discussed and models proposed to account for the kinetics obtained in our single-molecule assay.

Forced Unraveling of Nucleosomes Assembled on Heterogeneous DNA Using Core Histones, NAP-1, and ACF

Periodic arrays of nucleosomes were assembled on heterogeneous DNA using core histones, the histone chaperone NAP-1, and ATP-dependent chromatin assembly and remodeling factor (ACF). The mechanical properties of these complexes were interrogated by stretching them with optical tweezers. Abrupt events releasing approximately 55-95 base-pairs of DNA, attributable to the non-equilibrium unraveling of individual nucleosomes, were frequently observed. This finding is comparable with a previous observation of 72-80 bp unraveling events for nucleosomes assembled by salt dialysis on a repeating sea urchin 5 S RNA positioning element, but the unraveling force varied over a wider range ( approximately 5-65 pN, with the majority of events at lower force). Because ACF assembles nucleosomes uniformly on heterogeneous DNA sequences, as in native chromatin, we attribute this variation to a dependence of the unraveling force on the DNA sequence within individual nucleosomes. The mean force increased from 24 pN to 31 pN as NaCl was decreased from 100 mM to 5 mM. Spontaneous DNA re-wrapping events were occasionally observed in real time during force relaxation. The observed wide variations in the dynamic force needed to unravel individual nucleosomes and the occurrences of sudden DNA re-wrapping events may have an important regulatory influence on DNA-directed nuclear processes, such as the binding of transcription factors and the movement of polymerase complexes on chromatin.

Association States of Nucleosome Assembly Protein 1 and Its Complexes with Histones

The histone chaperone NAP1 is a carrier of histones during nuclear import, nucleosome assembly, and chromatin remodeling. Analytical ultracentrifugation was used to determine the association states of NAP1 alone and in complexes with core histones. In addition, the concentration dependence of the association was quantified by determining the equilibrium dissociation constant between different NAP1 species. At physiological protein and salt concentrations the prevalent species were the NAP1 dimer and octamer. These were also the association states found to interact with histones in a stoichiometry of one NAP1 monomer per histone. Based on these results a model for a cell cycle-dependent shift of the NAP1 dimer-octamer equilibrium is proposed that reflects different biological functions of NAP1.