Histone Shuttling Research Articles

Insights on the nuclear shuttling of H2A-H2B histone chaperones

All cellular processes that involve the unwinding of DNA also lead to the systematic shuttling of histones. Histone shuttling across the nuclear membrane is facilitated by a class of proteins known as – histone chaperones. Histone chaperones are classified based on their binding to H3/H4 histones or H2A/H2B histones. During the shuttling process, two types of signals - NLS and NES are recognized by the nuclear transport proteins. However, this is the nuclear transport protein and the mechanism of signal recognition by the protein is still unknown. Thus, in this piece of work, the NLS and NES signals are predicted on important H2A/H2B binding histone chaperones. In addition, cellular localization and potential DNA binding regions of histone chaperones are predicted. Mapping of predicted regions on the histone chaperone’s structure suggested that the critical binding regions mainly lie on the disordered region of the histone chaperones. NLS and NES are present in the N- and C-terminal of the histone chaperones. Most histone chaperones contain bipartiate NLS signals. This article sheds light on the crucial aspect that in addition of being directly engaged in nucleosome synthesis and disassembly in vivo, histone chaperone also performs various specific roles via histone binding activity.

MoNap1, a Nucleosome Assemble Protein 1, Regulates Growth, Development, and Pathogenicity in Magnaporthe oryzae.

Nap1 is an evolutionarily conserved protein from yeast to human and is involved in diverse physiological processes, such as nucleosome assembly, histone shuttling between the nucleus and cytoplasm, transcriptional regulation, and the cell cycle regulation. In this paper, we identified nucleosome assemble protein MoNap1 in Magnaporthe oryzae and investigated its function in pathogenicity. Deletion of MoNAP1 resulted in reduced growth and conidiation, decreased appressorium formation rate, and impaired virulence. MoNap1 affects appressorium turgor and utilization of glycogen and lipid droplets. In addition, MoNap1 is involved in the regulation of cell wall, oxidation, and hyperosmotic stress. The subcellular localization experiments showed that MoNap1 is located in the cytoplasm. MoNap1 interacts with MoNbp2, MoClb3, and MoClb1 in M. oryzae. Moreover, deletion of MoNBP2 and MoCLB3 has no effects on vegetative growth, conidiation, and pathogenicity. Transcriptome analysis reveals that MoNAP1 is involved in regulating pathogenicity, the melanin biosynthetic process. Taken together, our results showed that MoNap1 plays a crucial role in growth, conidiation, and pathogenicity of M. oryzae.

Nucleosome Assembly Protein 1, Nap1, Is Required for the Growth, Development, and Pathogenicity of Magnaporthe oryzae.

Magnaporthe oryzae is the causal agent of rice blast, leading to significant reductions in rice and wheat productivity. Nap1 is a conserved protein in eukaryotes involved in diverse physiological processes, such as nucleosome assembly, histone shuttling between the nucleus and cytoplasm, transcriptional regulation, and the cell cycle. Here, we identified Nap1 and characterized its roles in fungal development and virulence in M. oryzae. MoNap1 is involved in aerial hyphal and conidiophore differentiation, sporulation, appressorium formation, plant penetration, and virulence. ΔMonap1 generated a small, elongated, and malformed appressorium with an abnormally organized septin ring on hydrophobic surfaces. ΔMonap1 was more sensitive to cell wall integrity stresses but more resistant to microtubule stresses. MoNap1 interacted with histones H2A and H2B and the B-type cyclin (Cyc1). Moreover, a nuclear export signal (NES) domain is necessary for Nap1’s roles in the regulation of the growth and pathogenicity of M. oryzae. In summary, NAP1 is essential for the growth, appressorium formation, and pathogenicity of M. oryzae.

Structure-function relationship of H2A-H2B specific plant histone chaperones.

Studies on chromatin structure and function have gained a revived popularity. Histone chaperones are significant players in chromatin organization. They play a significant role in vital nuclear functions like transcription, DNA replication, DNA repair, DNA recombination, and epigenetic regulation, primarily by aiding processes such as histone shuttling and nucleosome assembly/disassembly. Like the other eukaryotes, plants also have a highly orchestrated and dynamic chromatin organization. Plants seem to have more isoforms within the same family of histone chaperones, as compared with other organisms. As some of these are specific to plants, they must have evolved to perform functions unique to plants. However, it appears that only little effort has gone into understanding the structural features of plant histone chaperones and their structure-function relationships. Studies on plant histone chaperones are essential for understanding their role in plant chromatin organization and how plants respond during stress conditions. This review is on the structural and functional aspects of plant histone chaperone families, specifically those which bind to H2A-H2B, viz nucleosome assembly protein (NAP), nucleoplasmin (NPM), and facilitates chromatin transcription (FACT). Here, we also present comparative analyses of these plant histone chaperones with available histone chaperone structures. The review hopes to incite interest among researchers to pursue further research in the area of plant chromatin and the associated histone chaperones.

A β-Hairpin Comprising the Nuclear Localization Sequence Sustains the Self-associated States of Nucleosome Assembly Protein 1

The histone chaperone nucleosome assembly protein 1 (NAP1) is implicated in histone shuttling as well as nucleosome assembly and disassembly. Under physiological conditions, NAP1 dimers exist in a mixture of various high-molecular-weight oligomers whose size may be regulated by the cell cycle-dependent concentration of NAP1. Both the functional and structural significance of the observed oligomers are unknown. We have resolved the molecular mechanism by which yeast NAP1 (yNAP1) dimers oligomerize by applying x-ray crystallographic, hydrodynamic, and functional approaches. We found that an extended β-hairpin that protrudes from the compact core of the yNAP1 dimer forms a stable β-sheet with β-hairpins of neighboring yNAP1 dimers. Disruption of the β-hairpin (whose sequence is conserved among NAP1 proteins in various species) by the replacement of one or more amino acids with proline results in complete loss of yNAP1 dimer oligomerization. The in vitro functions of yNAP1 remain unaffected by the mutations. We have thus identified a conserved structural feature of NAP1 whose function, in addition to presenting the nuclear localization sequence, appears to be the formation of higher-order oligomers.

Vps75, A New Yeast Member of the NAP Histone Chaperone Family

Homologues of nucleosome assembly protein 1 (NAP1) are found throughout eukaryotes. Here we identify and characterize a new NAP family histone chaperone from budding yeast, named Vps75. Purified Vps75 preferentially binds histone H3/H4 tetramers and is capable of assembling nucleosomes in vitro. In vivo, Vps75 is associated with the chromatin of both active and inactive genes and telomeres. Others have previously reported that Vps75 forms a complex with Rtt109, required for acetylation of histone H3 lysine 56 (H3 Lys-56). Cells lacking RTT109 are sensitive to hydroxyurea, pointing to a role in replication. We show that VPS75 is not required for H3 Lys-56 acetylation and that vps75Delta cells are insensitive to hydroxyurea, suggesting that although Rtt109 and Vps75 associate and are likely to be functionally connected, they also have separate roles.

Structure and function of nucleosome assembly proteinsThis paper is one of a selection of papers published in this Special Issue, entitled 27th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

Homologues of nucleosome assembly protein 1 (NAP1) have been identified in all eukaryotes. Although initially identified as histone chaperones and chromatin-assembly factors, additional functions include roles in tissue-specific transcription regulation, apoptosis, histone shuttling, and cell-cycle regulation, and extend beyond those of a simple chaperone and assembly factor. NAP1 family members share a structurally conserved fold, the NAP domain. Here we review current knowledge of the NAP family of proteins within the context of the recently determined crystal structure of the NAP1 family's first representative, NAP1 from yeast.

The structure of nucleosome assembly protein 1.

Nucleosome assembly protein 1 (NAP-1) is an integral component in the establishment, maintenance, and dynamics of eukaryotic chromatin. It shuttles histones into the nucleus, assembles nucleosomes, and promotes chromatin fluidity, thereby affecting the transcription of many genes. The 3.0 A crystal structure of yeast NAP-1 reveals a previously uncharacterized fold with implications for histone binding and shuttling. A long alpha-helix is responsible for homodimerization via a previously uncharacterized antiparallel non-coiled-coil, and an alpha/beta domain is implicated in protein-protein interaction. A nuclear export sequence that is embedded in the dimerization helix is almost completely masked by an accessory domain that contains several target sites for casein kinase II. The four-stranded antiparallel beta-sheet that characterizes the alpha/beta domain is found in all histone chaperones, despite the absence of homology in sequence, structural context, or quaternary structure. To our knowledge, this is the first structure of a member of the large NAP family of proteins and suggests a mechanism by which the shuttling of histones to and from the nucleus is regulated.

NAP1 Modulates Binding of Linker Histone H1 to Chromatin and Induces an Extended Chromatin Fiber Conformation

NAP1 (nucleosome assembly protein 1) is a histone chaperone that has been described to bind predominantly to the histone H2A.H2B dimer in the cell during shuttling of histones into the nucleus, nucleosome assembly/remodeling, and transcription. Here it was examined how NAP1 interacts with chromatin fibers isolated from HeLa cells. NAP1 induced a reversible change toward an extended fiber conformation as demonstrated by sedimentation velocity ultracentrifugation experiments. This transition was due to the removal of the linker histone H1. The H2A.H2B dimer remained stably bound to the native fiber fragments and to fibers devoid of linker histone H1. This was in contrast to mononucleosome substrates, which displayed a NAP1-induced removal of a single H2A.H2B dimer from the core particle. The effect of NAP1 on the chromatin fiber structure was examined by scanning/atomic force microscopy. A quantitative image analysis of approximately 36,000 nucleosomes revealed an increase of the average internucleosomal distance from 22.3 +/- 0.4 to 27.6 +/- 0.6 nm, whereas the overall fiber structure was preserved. This change reflects the disintegration of the chromatosome due to binding of H1 to NAP1 as chromatin fibers stripped from H1 showed an average nucleosome distance of 27.4 +/- 0.8 nm. The findings suggest a possible role of NAP1 in chromatin remodeling processes involved in transcription and replication by modulating the local linker histone content.

Poly(ADP-ribose) molecules formed during DNA repair in vivo

We have found that human cells respond to treatment with an alkylating carcinogen by producing a specific, highly conserved array of poly(ADP-ribose) molecules. Using in situ radiolabeling and boronate affinity chromatography, we have been able to isolate ADP-ribose polymers from living cells and to quantify individual molecular size classes on high resolution polyacrylamide gels. Despite carcinogen-induced changes in poly(ADP-ribose) turnover, the relative frequency of linear polymers was strictly conserved. However, the abundance of branched polymers with high affinity for histones increased 2.6-fold. These polymers were degraded more slowly than linear polymers. Our results indicate that the poly(ADP-ribose) molecules involved in the shuttling of histones on DNA in vitro also play a role in the repair of DNA damage in vivo.

Histone shuttling by poly ADP-ribosylation.

The enzymes poly(ADP-ribose)polymerase and poly(ADP-ribose) glycohydrolase may cooperate to drive a histone shuttle mechanism in chromatin. The mechanism is triggered by binding of the N-terminal zinc-finger domain of the polymerase to DNA strand breaks, which activates the catalytic activities residing in the C-terminal domain. The polymerase converts into a protein carrying multiple ADP-ribose polymers which displace histones from DNA by specifically targeting the histone tails responsible for DNA condensation. As a result, the domains surrounding DNA strand breaks become accessible to other proteins. Poly(ADP-ribose)glycohydrolase attacks ADP-ribose polymers in a specific order and thereby releases histones for reassociation with DNA. Increasing evidence from different model systems suggests that histone shuttling participates in DNA repair in vivo as a catalyst for nucleosomal unfolding.

Histone shuttle driven by the automodification cycle of poly(ADP‐ribose) polymerase

In mammalian cells, the incision step of DNA excision repair triggers a dramatic metabolic response in chromatin. The reaction starts with the binding of a zinc-finger protein, i.e. poly(ADP-ribose)polymerase to DNA nicks, activation of four resident catalytic activities leading to poly(ADP-ribose) synthesis, conversion of the polymerase into a protein modified with up to 28 variably sized ADP-ribose polymers, and rapid degradation of polymerase-bound polymers by poly(ADP-ribose)glycohydrolase. This automodification cycle catalyzes a transient and reversible dissociation of histones from DNA. Shuttling of histones on the DNA allows selected other proteins, such as DNA helicase A and topoisomerase I, to gain access to DNA. Histone shuttling in vitro mimics nucleosomal unfolding/refolding in vivo that accompanies the postincisional steps of DNA excision repair. Suppression of the automodification cycle in mammalian cells prevents nucleosomal unfolding and nucleotide excision repair.

Histone shuttling by poly(ADP-ribosylation).

Histone shuttling by poly(ADP-ribosylation).

Poly ADP-ribosylation: a histone shuttle mechanism in DNA excision repair.

In DNA excision repair of mammalian cells, the processing of ADP-ribose by the poly ADP-ribosylation system of chromatin is stimulated several thousand-fold. Most of this turnover is associated with the automodification reaction of the nuclear enzyme poly(ADP-ribose) polymerase and the degradation of polymerase-bound polymers by the enzyme poly(ADP-ribose) glycohydrolase. The automodification cycle catalyzes a temporary dissociation from and reassociation of histones with DNA. It is proposed that this mechanism, termed "histone shuttle", may guide specific proteins to sites of repair. In addition, histone shuttling driven by the poly ADP-ribosylation system seems to be involved in nucleosomal unfolding of chromatin in DNA excision repair.