PARP Power: A Structural Perspective on PARP1, PARP2, and PARP3 in DNA Damage Repair and Nucleosome Remodelling.

Poly (ADP-ribose) polymerases (PARP) 1-3 are well-known multi-domain enzymes, catalysing the covalent modification of proteins, DNA, and themselves. They attach mono- or poly-ADP-ribose to targets using NAD+ as a substrate. Poly-ADP-ribosylation (PARylation) is central to the important functions of PARP enzymes in the DNA damage response and nucleosome remodelling. Activation of PARP happens through DNA binding via zinc fingers and/or the WGR domain. Modulation of their activity using PARP inhibitors occupying the NAD+ binding site has proven successful in cancer therapies. For decades, studies set out to elucidate their full-length molecular structure and activation mechanism. In the last five years, significant advances have progressed the structural and functional understanding of PARP1-3, such as understanding allosteric activation via inter-domain contacts, how PARP senses damaged DNA in the crowded nucleus, and the complementary role of histone PARylation factor 1 in modulating the active site of PARP. Here, we review these advances together with the versatility of PARP domains involved in DNA binding, the targets and shape of PARylation and the role of PARPs in nucleosome remodelling.

Dynamics of transcription-mediated conversion from euchromatin to facultative heterochromatin at the Xist promoter by Tsix.

The fine-scale dynamics from euchromatin (EC) to facultative heterochromatin (fHC) has remained largely unclear. Here, we focus on Xist and its silencing initiator Tsix as a paradigm of transcription-mediated conversion from EC to fHC. In mouse epiblast stem cells, induction of Tsix recapitulates the conversion at the Xist promoter. Investigating the dynamics reveals that the conversion proceeds in a stepwise manner. Initially, a transient opened chromatin structure is observed. In the second step, gene silencing is initiated and dependent on Tsix, which is reversible and accompanied by simultaneous changes in multiple histone modifications. At the last step, maintenance of silencing becomes independent of Tsix and irreversible, which correlates with occupation of the -1 position of the transcription start site by a nucleosome and initiation of DNA methylation introduction. This study highlights the hierarchy of multiple chromatin events upon stepwise gene silencing establishment.

Complex context relationships between DNA methylation and accessibility, histone marks, and hTERT gene expression in acute promyelocytic leukemia cells: perspectives for all-trans retinoic acid in cancer therapy.

Telomerase (hTERT) reactivation and sustained expression is a key event in the process of cellular transformation. Therefore, the identification of the mechanisms regulating hTERT expression is of great interest for the development of new anticancer therapies. Although the epigenetic state of hTERT gene promoter is important, we still lack a clear understanding of the mechanisms by which epigenetic changes affect hTERT expression. Retinoids are well-known inducers of granulocytic maturation in acute promyelocytic leukemia (APL). We have previously shown that retinoids repressed hTERT expression in the absence of maturation leading to growth arrest and cell death. Exploring the mechanisms of this repression, we showed that transcription factor binding was dependent on the epigenetic status of hTERT promoter. In the present study, we used APL cells lines and publicly available datasets from APL patients to further investigate the integrated epigenetic events that promote hTERT promoter transition from its silent to its active state, and inversely. We showed, in APL patients, that the methylation of the distal domain of hTERT core promoter was altered and correlated with the outcome of the disease. Further studies combining complementary approaches carried out on APL cell lines highlighted the significance of a domain outside the minimal promoter, localized around 5 kb upstream from the transcription start site, in activating hTERT. This domain is characterized by DNA hypomethylation and H3K4Me3 deposition. Our findings suggest a cooperative interplay between hTERT promoter methylation, chromatin accessibility, and histone modifications that force the revisiting of previously proposed concepts regarding hTERT epigenetic regulation. They represent, therefore, a major advance in predicting sensitivity to retinoid-induced hTERT repression and, more generally, in the potential development of therapies targeting hTERT expression in cancers.

Sex-specific phenotypes of histone H4 point mutants establish dosage compensation as the critical function of H4K16 acetylation in Drosophila.

Acetylation of histone H4 at lysine 16 (H4K16) modulates nucleosome-nucleosome interactions and directly affects nucleosome binding by certain proteins. In Drosophila, H4K16 acetylation by the dosage compensation complex subunit Mof is linked to increased transcription of genes on the single X chromosome in males. Here, we analyzed Drosophila containing different H4K16 mutations or lacking Mof protein. An H4K16A mutation causes embryonic lethality in both sexes, whereas an H4K16R mutation permits females to develop into adults but causes lethality in males. The acetyl-mimic mutation H4K16Q permits both females and males to develop into adults. Complementary analyses reveal that males lacking maternally deposited and zygotically expressed Mof protein arrest development during gastrulation, whereas females of the same genotype develop into adults. Together, this demonstrates the causative role of H4K16 acetylation by Mof for dosage compensation in Drosophila and uncovers a previously unrecognized requirement for this process already during the onset of zygotic gene transcription.

High-throughput screening with nucleosome substrate identifies small-molecule inhibitors of the human histone lysine methyltransferase NSD2.

The histone lysine methyltransferase nuclear receptor-binding SET domain protein 2 (NSD2, also known as WHSC1/MMSET) is an epigenetic modifier and is thought to play a driving role in oncogenesis. Both NSD2 overexpression and point mutations that increase its catalytic activity are associated with several human cancers. Although NSD2 is an attractive therapeutic target, no potent, selective, and bioactive small molecule inhibitors of NSD2 have been reported to date, possibly due to the challenges of developing high-throughput assays for NSD2. Here, to establish a platform for the discovery and development of selective NSD2 inhibitors, we optimized and implemented multiple assays. We performed quantitative high-throughput screening with full-length WT NSD2 and a nucleosome substrate against a diverse collection of bioactive small molecules comprising 16,251 compounds. We further interrogated 174 inhibitory compounds identified in the primary screen with orthogonal and counter assays and with activity assays based on the clinically relevant NSD2 variants E1099K and T1150A. We selected five confirmed inhibitors for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. We found that all five NSD2 inhibitors bind the catalytic SET domain and one exhibited apparent activity in cells, validating the workflow and providing a template for identifying selective NSD2 inhibitors. In summary, we have established a robust discovery pipeline for identifying potent NSD2 inhibitors from small-molecule libraries.

Cell Cycle Arrest and Apoptosis Induction via Modulation of Mitochondrial Integrity by Bcl-2 Family Members and Caspase Dependence in Dracaena cinnabari-Treated H400 Human Oral Squamous Cell Carcinoma.

Dracaena cinnabari Balf.f. is a red resin endemic to Socotra Island, Yemen. Although there have been several reports on its therapeutic properties, information on its cytotoxicity and anticancer effects is very limited. This study utilized a bioassay-guided fractionation approach to determine the cytotoxic and apoptosis-inducing effects of D. cinnabari on human oral squamous cell carcinoma (OSCC). The cytotoxic effects of D. cinnabari crude extract were observed in a panel of OSCC cell lines and were most pronounced in H400. Only fractions DCc and DCd were active on H400 cells; subfractions DCc15 and DCd16 exhibited the greatest cytotoxicity against H400 cells and D. cinnabari inhibited cells proliferation in a time-dependent manner. This was achieved primarily via apoptosis where externalization of phospholipid phosphatidylserine was observed using DAPI/Annexin V fluorescence double staining mechanism studied through mitochondrial membrane potential assay cytochrome c enzyme-linked immunosorbent and caspases activities revealed depolarization of mitochondrial membrane potential (MMP) and significant activation of caspases 9 and 3/7, concomitant with S phase arrest. Apoptotic proteins array suggested that MMP was regulated by Bcl-2 proteins family as results demonstrated an upregulation of Bax, Bad, and Bid as well as downregulation of Bcl-2. Hence, D. cinnabari has the potential to be developed as an anticancer agent.

Total triterpenoids from Ganoderma Lucidum suppresses prostate cancer cell growth by inducing growth arrest and apoptosis.

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

Learning protein-DNA interaction landscapes by integrating experimental data through computational models.

MOTIVATION: Transcriptional regulation is directly enacted by the interactions between DNA and many proteins, including transcription factors (TFs), nucleosomes and polymerases. A critical step in deciphering transcriptional regulation is to infer, and eventually predict, the precise locations of these interactions, along with their strength and frequency. While recent datasets yield great insight into these interactions, individual data sources often provide only partial information regarding one aspect of the complete interaction landscape. For example, chromatin immunoprecipitation (ChIP) reveals the binding positions of a protein, but only for one protein at a time. In contrast, nucleases like MNase and DNase can be used to reveal binding positions for many different proteins at once, but cannot easily determine the identities of those proteins. Currently, few statistical frameworks jointly model these different data sources to reveal an accurate, holistic view of the in vivo protein-DNA interaction landscape. RESULTS: Here, we develop a novel statistical framework that integrates different sources of experimental information within a thermodynamic model of competitive binding to jointly learn a holistic view of the in vivo protein-DNA interaction landscape. We show that our framework learns an interaction landscape with increased accuracy, explaining multiple sets of data in accordance with thermodynamic principles of competitive DNA binding. The resulting model of genomic occupancy provides a precise mechanistic vantage point from which to explore the role of protein-DNA interactions in transcriptional regulation. AVAILABILITY AND IMPLEMENTATION: The C source code for compete and Python source code for MCMC-based inference are available at http://www.cs.duke.edu/∼amink. CONTACT: amink@cs.duke.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility.

INTRODUCTION: Neutrophil extracellular traps (NETs) have recently been implicated in a number of autoimmune conditions, including rheumatoid arthritis (RA). We examined the underlying signaling pathways triggering enhanced NETosis in RA and ascertained whether the products of NETosis had diagnostic implications or usefulness. METHODS: Neutrophils were isolated from RA patients with active disease and from controls. Spontaneous NET formation from RA and control neutrophils was assessed in vitro with microscopy and enzyme-linked immunosorbent assay (ELISA) for NETosis-derived products. The analysis of the signal-transduction cascade included reactive oxygen species (ROS) production, myeloperoxidase (MPO), neutrophil elastase (NE), peptidyl arginine deiminase 4 (PAD4), and citrullinated histone 3 (citH3). NET formation was studied in response to serum and synovial fluid and immunoglobulin G (IgG) depleted and reconstituted serum. Serum was analyzed for NETosis-derived products, for which receiver operator characteristic (ROC) curves were calculated. RESULTS: Neutrophils from RA cases exhibited increased spontaneous NET formation in vitro, associated with elevated ROS production, enhanced NE and MPO expression, nuclear translocation of PAD4, PAD4-mediated citrullination of H3, and altered nuclear morphology. NET formation in both anti-citrullinated peptide antibody (ACPA)-positive and -negative RA was abolished by IgG depletion, but restored only with ACPA-positive IgG. NETosis-derived products in RA serum demonstrated diagnostic potential, the ROC area under the curve for cell-free nucleosomes being >97%, with a sensitivity of 91% and a specificity of 92%. No significant difference was observed between ACPA-positive and -negative cases. CONCLUSIONS: Signaling elements associated with the extrusion of NETs are significantly enhanced to promote NETosis in RA compared with healthy controls. NETosis depended on the presence of ACPA in ACPA-positive RA serum. The quantitation of NETosis-derived products, such as cell-free nucleosomes in serum, may be a useful complementary tool to discriminate between healthy controls and RA cases.

Kinetic mechanism of DNA translocation by the RSC molecular motor.

ATP-dependent nucleosome repositioning by chromatin remodeling enzymes requires the translocation of these enzymes along the nucleosomal DNA. Using a fluorescence stopped-flow assay we monitored DNA translocation by a minimal RSC motor and through global analysis of these time courses we have determined that this motor has a macroscopic translocation rate of 2.9 bp/s with a step size of 1.24 bp. From the complementary quantitative analysis of the associated time courses of ATP consumption during DNA translocation we have determined that this motor has an efficiency of 3.0 ATP/bp, which is slightly less that the efficiency observed for several genetically related DNA helicases and which likely results from random pausing by the motor during translocation. Nevertheless, this motor is able to exert enough force during translocation to displace streptavidin from biotinylated DNA. Taken together these results are the necessary first step for quantifying both the role of DNA translocation in nucleosome repositioning by RSC and the efficiency at which RSC couples ATP binding and hydrolysis to nucleosome repositioning.

The yin and yang of yeast transcription: elements of a global feedback system between metabolism and chromatin.

When grown in continuous culture, budding yeast cells tend to synchronize their respiratory activity to form a stable oscillation that percolates throughout cellular physiology and involves the majority of the protein-coding transcriptome. Oscillations in batch culture and at single cell level support the idea that these dynamics constitute a general growth principle. The precise molecular mechanisms and biological functions of the oscillation remain elusive. Fourier analysis of transcriptome time series datasets from two different oscillation periods (0.7 h and 5 h) reveals seven distinct co-expression clusters common to both systems (34% of all yeast ORF), which consolidate into two superclusters when correlated with a compilation of 1,327 unrelated transcriptome datasets. These superclusters encode for cell growth and anabolism during the phase of high, and mitochondrial growth, catabolism and stress response during the phase of low oxygen uptake. The promoters of each cluster are characterized by different nucleotide contents, promoter nucleosome configurations, and dependence on ATP-dependent nucleosome remodeling complexes. We show that the ATP:ADP ratio oscillates, compatible with alternating metabolic activity of the two superclusters and differential feedback on their transcription via activating (RSC) and repressive (Isw2) types of promoter structure remodeling. We propose a novel feedback mechanism, where the energetic state of the cell, reflected in the ATP:ADP ratio, gates the transcription of large, but functionally coherent groups of genes via differential effects of ATP-dependent nucleosome remodeling machineries. Besides providing a mechanistic hypothesis for the delayed negative feedback that results in the oscillatory phenotype, this mechanism may underpin the continuous adaptation of growth to environmental conditions.

Phosphate starvation of maize inhibits lateral root formation and alters gene expression in the lateral root primordium zone.

BACKGROUND: Phosphorus (P) is an essential macronutrient for all living organisms. Maize (Zea mays) is an important human food, animal feed and energy crop throughout the world, and enormous quantities of phosphate fertilizer are required for maize cultivation. Thus, it is important to improve the efficiency of the use of phosphate fertilizer for maize. RESULTS: In this study, we analyzed the maize root response to phosphate starvation and performed a transcriptomic analysis of the 1.0-1.5 cm lateral root primordium zone. In the growth of plants, the root-to-shoot ratio (R/L) was reduced in both low-phosphate (LP) and sufficient-phosphate (SP) solutions, but the ratio (R/L) exhibited by the plants in the LP solution was higher than that of the SP plants. The growth of primary roots was slightly promoted after 6 days of phosphate starvation, whereas the numbers of lateral roots and lateral root primordia were significantly reduced, and these differences were increased when associated with the stress caused by phosphate starvation. Among the results of a transcriptomic analysis of the maize lateral root primordium zone, there were two highlights: 1) auxin signaling participated in the response and the modification of root morphology under low-phosphate conditions, which may occur via local concentration changes due to the biosynthesis and transport of auxin, and LOB domain proteins may be an intermediary between auxin signaling and root morphology; and 2) the observed retardation of lateral root development was the result of co-regulation of DNA replication, transcription, protein synthesis and degradation and cell growth. CONCLUSIONS: These results indicated that maize roots show a different growth pattern than Arabidopsis under low-phosphate conditions, as the latter species has been observed to halt primary root growth when the root tip comes into contact with low-phosphate media. Moreover, our findings enrich our understanding of plant responses to phosphate deficits and of root morphogenesis in maize.

Identification and characterization of a bactericidal and proapoptotic peptide from Cycas revoluta seeds with DNA binding properties.

Nowadays, novel pharmacies have been screened from plants. Among them are the peptides, which show multiple biotechnological activities. In this report, a small peptide (Ala-Trp-Lys-Leu-Phe-Asp-Asp-Gly-Val) with a molecular mass of 1,050 Da was purified from Cycas revoluta seeds by using reversed-phase liquid chromatography. This peptide shows clear deleterious effects against human epidermoid cancer (Hep2) and colon carcinoma cells (HCT15). It caused inhibition of cancer cell proliferation and further disruption of nucleosome structures, inducing apoptosis by direct DNA binding. A remarkable antibacterial activity was also observed in this same peptide. Nevertheless, no significant lysis of normal RBC cells was observed in the presence of peptide. Additionally, an acetylation at the N-termini portion is able to reduce both activities. Bioinformatics tools were also utilized for construction of a three-dimensional model showing a single amphipathic helix. Since in vitro binding studies show that the target of this peptide seems to be DNA, theoretical docking studies were also performed to better understand the interaction between peptide and nucleic acids and also to shed some light on the acetyl group role. Firstly, binding studies showed that affinity contacts basically occur due to electrostatic attraction. The complex peptide-ssDNA was clearly oriented by residues Ala(1), Lys(3), and Asp(6), which form several hydrogen bonds that are able to stabilize the complex. When acetyl was added, hydrogen bonds are broken, reducing the peptide affinity. In summary, it seems that information here provided could be used to design a novel derivative of this peptide which a clear therapeutic potential.

The chaperone-histone partnership: for the greater good of histone traffic and chromatin plasticity.

Histones are highly positively charged proteins that wrap our genome. Their surface properties also make them prone to nonspecific interactions and aggregation. A class of proteins known as histone chaperones is dedicated to safeguard histones by aiding their proper incorporation into nucleosomes. Histone chaperones facilitate ordered nucleosome assembly and disassembly reactions through the formation of semi-stable histone-chaperone intermediates without requiring ATP, but merely providing a complementary protein surface for histones to dynamically interact with. Recurrent 'chaperoning' mechanisms involve the masking of the histone's positive charge and the direct blocking of crucial histone surface sites, including those required for H3-H4 tetramerization or the binding of nucleosomal DNA. This shielding prevents histones from engaging in premature or unwanted interactions with nucleic acids and other cellular components. In this review, we analyze recent structural studies on chaperone-histone interactions and discuss the implications of this vital partnership for nucleosome assembly and disassembly pathways.

Yin and Yang of histone H2B roles in silencing and longevity: a tale of two arginines.

In budding yeast, silent chromatin is defined at the region of telomeres, rDNA loci, and silent mating loci. Although the silent chromatin at different loci shows structural similarity, the underlying mechanism to establish, maintain, and inherit these structures may be fundamentally different. In this study, we found two arginine residues within histone H2B, which are specifically required to maintain either the telomeric or the rDNA silenct chromatin. Arginine 95 (R95) plays a specific role at telomeres, whereas arginine 102 (R102) is required to maintain the silent chromatin at rDNA and to ensure the integrity of rDNA loci by suppressing recombination between rDNA repeats. R95 mutants show enhanced rDNA silencing but a paradoxically low Sir2 protein abundance. Furthermore weakened silencing at telomeres in R95 mutants can be suppressed by a specific SIR3 allele, SIR3-D205N, which increases the affinity of Sir proteins to telomeres, suggesting H2B-R95 may directly mediate telomeric Sir protein-nucleosome interactions. Double mutations of R95 and R102 lead to desilencing of both rDNA and telomeres, indicating both arginines are necessary to ensure integrity of silent chromatin at these loci. Furthermore, mutations of R102 cause accumulation of extrachromosomal rDNA circles and reduce life span, suggesting that histone H2B contributes to longevity.

Fission yeast Iec1-ino80-mediated nucleosome eviction regulates nucleotide and phosphate metabolism.

Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenine-responsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.

A single exposure to hyperbaric oxygen increases levels of circulating nucleosomes but does not induce mononuclear cell apoptosis in divers.

Recent reports that hyperbaric oxygenation (HBO2) induced apoptosis in T-cell lines raised concern about a possible immunosuppressive effect of HBO2. Nucleosomes, DNA fragments wrapped around a histone core, have been observed in the circulation in diseases with increased cell death such as sepsis. Our aim was to investigate, whether HBO2 increases circulating nucleosomes as a marker of cell death and induces apoptosis of peripheral blood mononuclear cells in vivo. After informed consent 29 healthy volunteers were exposed to a 30 minute dive at 2.8 atmospheres absolute in a pressure chamber under resting conditions, while breathing 100% oxygen. Samples were obtained before and 24 hours after exposure. Circulating nucleosomes were measured in serum. Caspase-3 activation, Bcl-2 expression and mRNA of Bcl-2, Bcl-xl and Bax were analyzed in mononuclear cell extracts. Nucleosomes were elevated markedly 24h after exposure (p<0.01), while caspase-3 was not activated significantly. mRNA levels of Bcl-2, Bcl-xl and Bax were not altered. In conclusion, while evidence of elevated levels of circulating nucleosomes was found, mononuclear cell apoptosis was not affected by a single exposure to hyperbaric oxygen.

The linker-protein network: control of nucleosomal DNA accessibility.

Numerous studies have recently addressed the accessibility of nucleosomal DNA to protein factors. Two popular concepts - the histone code and chromatin remodeling - consider the nucleosome as a passive entity that 'waits' to be marked by histone modifications and is 'mobilized' by ATP-dependent remodelers. Here, we propose a holistic view of the nucleosome as an active, dynamic entity, the accessibility of which is controlled by binding of different linker proteins to the DNA entry/exit site. The linker proteins might directly compete for this binding site; alternatively, protein chaperones and/or chromatin remodelers might exchange one linker protein for another. Finally, according to our proposed model, the exchange factors are themselves controlled by post-translational modifications or binding of protein partners, to respond to the ever-changing intra- and extra-cellular environment.

In silico modulation of HMGN-1 binding to histones and gene expression by interplay of phosphorylation and O-GlcNAc modification.

Utilizing different computational methods; phosphorylation, O-GlcNAc modification and Yin Yang sites are predicted in HMGN-1. Prediction results suggest that interplay of phosphorylation and O-GlcNAc modification regulates binding and removal of HMGN-1 with the nucleosome and its translocation from nucleus to cytoplasm and back to nucleus, consequently modulating gene expression.

Up-regulation of stress-inducible genes in tobacco and Arabidopsis cells in response to abiotic stresses and ABA treatment correlates with dynamic changes in histone H3 and H4 modifications.

Animal cells react to mitogenic or stress stimuli by rapid up-regulation of immediate-early (IE) genes and a parallel increase in characteristic modifications of core histones: chromatin changes, collectively termed the nucleosomal response. With regard to plants little is known about the accompanying changes at the chromatin level. We have used tobacco BY-2 and Arabidopsis T87 cell lines to study the nucleosomal response of plant cells to high salinity, cold and exogenous abscisic acid (ABA). When in quiescent stage, both tobacco and Arabidopsis cells show the typical nucleosomal response to high salinity and cold stress, manifested by rapid transient up-regulation of histone H3 Ser-10 phosphorylation, immediately followed by transient up-regulation of H3 phosphoacetylation and histone H4 acetylation. For each of the studied stresses the observed nucleosomal response was strictly correlated with the induction of stress-type specific genes. The dynamics of histone modifications in BY-2 cells in response to exogenous ABA exhibited a more complex pattern than that evoked by the two abiotic stresses, probably due to superposition of the primary and secondary effects of ABA. A rapid increase in H3 Ser-10 phosphorylation was also observed in whole leaves subjected to high salinity; however, the rate of change in this modification was much slower than in cultured cells. Together, these results indicate that the quiescent BY-2 and T87 cell lines show a typical nucleosomal response to abiotic stresses and ABA treatment and may represent suitable models for the study of chromatin-mediated mechanisms of stress tolerance in plants.