Phosphatase and Kinase Substrate Specificity Profiling with Pooled Synthetic Peptides and Mass Spectrometry.

Reversible phosphorylation is a pervasive regulatory event in cellular physiology controlled by reciprocal actions of protein kinases and phosphatases. Determining the inherent substrate specificity of kinases and phosphatases is essential for understanding their cellular roles. Synthetic peptides have long served as substrate proxies for defining intrinsic kinase and phosphatase specificities. Here, we describe a high throughput protocol to simultaneously measure specificity constants (kcat/KM) of many synthetic peptide substrates in a single pool using label-free quantitative mass spectrometry. The generation of specificity constants from a single pooled reaction provides a rigorous and rapid comparison of substrate variants to help define an enzyme's specificity. Equally applicable to kinases and phosphatases, as well as other enzyme classes, the protocol consists of three general steps: (1) reaction of enzyme with pooled peptide substrates, each ideally with a unique mass and at concentrations well below KM, (2) analysis of reaction products using liquid chromatography-coupled mass spectrometry (LC-MS), and (3) automated extraction and integration of elution peaks for each substrate/product pair. We incorporate an ionization correction strategy allowing direct calculation of reaction progress, and subsequently kcat/KM, from substrate and product peak areas in a single sample, obviating the need for stable isotope labeling. Peptide consumption is minimal, and high peptide purity and accurate concentrations are not required. Access to a high-resolution LC-MS system is the only nonstandard equipment need. We present an analysis pipeline consisting entirely of established open-source software tools, and demonstrate proof of principle with the highly selective cell cycle phosphatase Cdc14 from Saccharomyces cerevisiae.

Rods Contribute to Visual Behavior in Larval Zebrafish.

Purpose: Although zebrafish rods begin to develop as early as 2 days postfertilization (dpf), they are not deemed anatomically mature and functional until 15 to 21 dpf. A recent study detected a small electroretinogram (ERG) from rods in a cone mutant called no optokinetic response f (nof) at 5 dpf, suggesting that young rods are functional. Whether they can mediate behavioral responses in larvae is unknown. Methods: We first confirmed rod function by measuring nof ERGs under photopic and scotopic illumination at 6 dpf. We evaluated the role of rods in visual behaviors using two different assays: the visual-motor response (VMR) and optokinetic response (OKR). We measured responses from wild-type (WT) larvae and nof mutants under photopic and scotopic illuminations at 6 dpf. Results: Nof mutants lacked a photopic ERG. However, after prolonged dark adaptation, they displayed scotopic ERGs. Compared with WT larvae, the nof mutants displayed reduced VMRs. The VMR difference during light onset gradually diminished with decreased illumination and became nearly identical at lower light intensities. Additionally, light-adapted nof mutants did not display an OKR, whereas dark-adapted nof mutants displayed scotopic OKRs. Conclusions: Because the nof mutants lacked a photopic ERG but displayed scotopic ERGs after dark adaptation, the mutants clearly had functional rods. WT larvae and the nof mutants displayed comparable scotopic light-On VMRs and scotopic OKRs after dark adaptation, suggesting that these responses were driven primarily by rods. Together, these observations indicate that rods contribute to zebrafish visual behaviors as early as 6 dpf.

Glucosinolate and phenylpropanoid biosynthesis are linked by proteasome-dependent degradation of PAL.

Plants produce several hundreds of thousands of secondary metabolites that are important for adaptation to various environmental conditions. Although different groups of secondary metabolites are synthesized through unique biosynthetic pathways, plants must orchestrate their production simultaneously. Phenylpropanoids and glucosinolates are two classes of secondary metabolites that are synthesized through apparently independent biosynthetic pathways. Genetic evidence has revealed that the accumulation of glucosinolate intermediates limits phenylpropanoid production in a Mediator Subunit 5 (MED5)-dependent manner. To elucidate the molecular mechanism underlying this process, we analyzed the transcriptomes of a suite of Arabidopsis thaliana glucosinolate-deficient mutants using RNAseq and identified misregulated genes that are rescued by the disruption of MED5. The expression of a group of Kelch Domain F-Box genes (KFBs) that function in PAL degradation is affected in glucosinolate biosynthesis mutants and the disruption of these KFBs restores phenylpropanoid deficiency in the mutants. Our study suggests that glucosinolate/phenylpropanoid metabolic crosstalk involves the transcriptional regulation of KFB genes that initiate the degradation of the enzyme phenylalanine ammonia-lyase, which catalyzes the first step of the phenylpropanoid biosynthesis pathway. Nevertheless, KFB mutant plants remain partially sensitive to glucosinolate pathway mutations, suggesting that other mechanisms that link the two pathways also exist.

NOTCH signaling is activated in and contributes to resistance in enzalutamide-resistant prostate cancer cells.

Prostate cancer is the second leading cause of cancer death among men in the United States. The androgen receptor (AR) antagonist enzalutamide is a Food and Drug Administration-approved drug for treatment of patients with late-stage prostate cancer and is currently under clinical study for early-stage prostate cancer treatment. After a short positive response period, tumors will develop drug resistance. In this study using RNA-Seq and bioinformatics analyses, we observed that NOTCH signaling is a deregulated pathway in enzalutamide-resistant cells. NOTCH2 and c-MYC gene expression positively correlated with AR expression in samples from patient with hormone refractory disease in which AR expression levels correspond to those typically observed in enzalutamide resistance. Cleaved NOTCH1, HES1 (Hes family BHLH transcription factor 1), and c-MYC protein expression levels are elevated in two enzalutamide-resistant cell lines, MR49F and C4-2R, indicating NOTCH signaling activation. Moreover, inhibition of the overexpressed ADAM metallopeptidase domain 10 (ADAM10) in the resistant cells induces an exclusive reduction in cleaved NOTCH1 expression. Furthermore, exposure of enzalutamide-resistant cells to both PF-03084014 and enzalutamide increased cell death, decreased colony formation ability, and resensitized cells to enzalutamide. Knockdown of NOTCH1 in C4-2R increased enzalutamide sensitivity by decreasing cell proliferation and increasing cleaved PARP expression. In a 22RV1 xenograft model, PF-03084014 and enzalutamide decreased tumor growth through reducing cell proliferation and increasing apoptosis. These results indicate that NOTCH1 signaling may contribute to enzalutamide resistance in prostate cancer, and inhibition of NOTCH signaling can resensitize resistant cells to enzalutamide.

The Chromatin Remodelers PKL and PIE1 Act in an Epigenetic Pathway That Determines H3K27me3 Homeostasis in Arabidopsis.

Selective, tissue-specific gene expression is facilitated by the epigenetic modification H3K27me3 (trimethylation of lysine 27 on histone H3) in plants and animals. Much remains to be learned about how H3K27me3-enriched chromatin states are constructed and maintained. Here, we identify a genetic interaction in Arabidopsis thaliana between the chromodomain helicase DNA binding chromatin remodeler PICKLE (PKL), which promotes H3K27me3 enrichment, and the SWR1-family remodeler PHOTOPERIOD INDEPENDENT EARLY FLOWERING1 (PIE1), which incorporates the histone variant H2A.Z. Chromatin immunoprecipitation-sequencing and RNA-sequencing reveal that PKL, PIE1, and the H3K27 methyltransferase CURLY LEAF act in a common gene expression pathway and are required for H3K27me3 levels genome-wide. Additionally, H3K27me3-enriched genes are largely a subset of H2A.Z-enriched genes, further supporting the functional linkage between these marks. We also found that recombinant PKL acts as a prenucleosome maturation factor, indicating that it promotes retention of H3K27me3. These data support the existence of an epigenetic pathway in which PIE1 promotes H2A.Z, which in turn promotes H3K27me3 deposition. After deposition, PKL promotes retention of H3K27me3 after DNA replication and/or transcription. Our analyses thus reveal roles for H2A.Z and ATP-dependent remodelers in construction and maintenance of H3K27me3-enriched chromatin in plants.

Inhibition of the Wnt/ß-Catenin Pathway Overcomes Resistance to Enzalutamide in Castration-Resistant Prostate Cancer.

Enzalutamide is a second-generation nonsteroidal antiandrogen clinically approved for the treatment of castration-resistant prostate cancer (CRPC), yet resistance to endocrine therapy has limited its success in this setting. Although the androgen receptor (AR) has been associated with therapy failure, the mechanisms underlying this failure have not been elucidated. Bioinformatics analysis predicted that activation of the Wnt/ß-catenin pathway and its interaction with AR play a major role in acquisition of enzalutamide resistance. To validate the finding, we show upregulation of ß-catenin and AR in enzalutamide-resistant cells, partially due to reduction of ß-TrCP-mediated ubiquitination. Although activation of the Wnt/ß-catenin pathway in enzalutamide-sensitive cells led to drug resistance, combination of ß-catenin inhibitor ICG001 with enzalutamide inhibited expression of stem-like markers, cell proliferation, and tumor growth synergistically in various models. Analysis of clinical datasets revealed a molecule pattern shift in different stages of prostate cancer, where we detected a significant correlation between AR and ß-catenin expression. These data identify activation of the Wnt/ß-catenin pathway as a major mechanism contributing to enzalutamide resistance and demonstrate the potential to stratify patients with high risk of said resistance.Significance: Wnt/ß-catenin inhibition resensitizes prostate cancer cells to enzalutamide. Cancer Res; 78(12); 3147-62. ©2018 AACR.

Genome-Wide Analysis of the Arabidopsis Replication Timing Program.

Eukaryotes use a temporally regulated process, known as the replication timing program, to ensure that their genomes are fully and accurately duplicated during S phase. Replication timing programs are predictive of genomic features and activity and are considered to be functional readouts of chromatin organization. Although replication timing programs have been described for yeast and animal systems, much less is known about the temporal regulation of plant DNA replication or its relationship to genome sequence and chromatin structure. We used the thymidine analog, 5-ethynyl-2'-deoxyuridine, in combination with flow sorting and Repli-Seq to describe, at high-resolution, the genome-wide replication timing program for Arabidopsis (Arabidopsis thaliana) Col-0 suspension cells. We identified genomic regions that replicate predominantly during early, mid, and late S phase, and correlated these regions with genomic features and with data for chromatin state, accessibility, and long-distance interaction. Arabidopsis chromosome arms tend to replicate early while pericentromeric regions replicate late. Early and mid-replicating regions are gene-rich and predominantly euchromatic, while late regions are rich in transposable elements and primarily heterochromatic. However, the distribution of chromatin states across the different times is complex, with each replication time corresponding to a mixture of states. Early and mid-replicating sequences interact with each other and not with late sequences, but early regions are more accessible than mid regions. The replication timing program in Arabidopsis reflects a bipartite genomic organization with early/mid-replicating regions and late regions forming separate, noninteracting compartments. The temporal order of DNA replication within the early/mid compartment may be modulated largely by chromatin accessibility.

Metabolic Adaptation to Nutrients Involves Coregulation of Gene Expression by the RNA Helicase Dbp2 and the Cyc8 Corepressor in Saccharomyces cerevisiae.

Cells fine-tune their metabolic programs according to nutrient availability in order to maintain homeostasis. This is achieved largely through integrating signaling pathways and the gene expression program, allowing cells to adapt to nutritional change. Dbp2, a member of the DEAD-box RNA helicase family in Saccharomyces cerevisiae, has been proposed to integrate gene expression with cellular metabolism. Prior work from our laboratory has reported the necessity of DBP2 in proper gene expression, particularly for genes involved in glucose-dependent regulation. Here, by comparing differentially expressed genes in dbp2∆ to those of 700 other deletion strains from other studies, we find that CYC8 and TUP1, which form a complex and inhibit transcription of numerous genes, corepress a common set of genes with DBP2 Gene ontology (GO) annotations reveal that these corepressed genes are related to cellular metabolism, including respiration, gluconeogenesis, and alternative carbon-source utilization genes. Consistent with a direct role in metabolic gene regulation, loss of either DBP2 or CYC8 results in increased cellular respiration rates. Furthermore, we find that corepressed genes have a propensity to be associated with overlapping long noncoding RNAs and that upregulation of these genes in the absence of DBP2 correlates with decreased binding of Cyc8 to these gene promoters. Taken together, this suggests that Dbp2 integrates nutrient availability with energy homeostasis by maintaining repression of glucose-repressed, Cyc8-targeted genes across the genome.

Targeting the Hsp90 C-terminal domain to induce allosteric inhibition and selective client downregulation.

BACKGROUND: Inhibition of Hsp90 is desirable due to potential downregulation of oncogenic clients. Early generation inhibitors bind to the N-terminal domain (NTD) but C-terminal domain (CTD) inhibitors are a promising class because they do not induce a heat shock response. Here we present a new structural class of CTD binding molecules with a unique allosteric inhibition mechanism. METHODS: A hit molecule, NSC145366, and structurally similar probes were assessed for inhibition of Hsp90 activities. A ligand-binding model was proposed indicating a novel Hsp90 CTD binding site. Client protein downregulation was also determined. RESULTS: NSC145366 interacts with the Hsp90 CTD and has anti-proliferative activity in tumor cell lines (GI50=0.2-1.9µM). NSC145366 increases Hsp90 oligomerization resulting in allosteric inhibition of NTD ATPase activity (IC50=119µM) but does not compete with NTD or CTD-ATP binding. Treatment of LNCaP prostate tumor cells resulted in selective client protein downregulation including AR and BRCA1 but without a heat shock response. Analogs had similar potencies in ATPase and chaperone activity assays and variable effects on oligomerization. In silico modeling predicted a binding site at the CTD dimer interface distinct from the nucleotide-binding site. CONCLUSIONS: A set of symmetrical scaffold molecules with bisphenol A cores induced allosteric inhibition of Hsp90. Experimental evidence and molecular modeling suggest that the binding site is independent of the CTD-ATP site and consistent with unique induction of allosteric effects. GENERAL SIGNIFICANCE: Allosteric inhibition of Hsp90 via a mechanism used by the NSC145366-based probes is a promising avenue for selective oncogenic client downregulation.

Repurposing Approach Identifies Auranofin with Broad Spectrum Antifungal Activity That Targets Mia40-Erv1 Pathway.

Current antifungal therapies have limited effectiveness in treating invasive fungal infections. Furthermore, the development of new antifungal is currently unable to keep pace with the urgent demand for safe and effective new drugs. Auranofin, an FDA-approved drug for the treatment of rheumatoid arthritis, inhibits growth of a diverse array of clinical isolates of fungi and represents a new antifungal agent with a previously unexploited mechanism of action. In addition to auranofin's potent antifungal activity against planktonic fungi, this drug significantly reduces the metabolic activity of Candida cells encased in a biofilm. Unbiased chemogenomic profiling, using heterozygous S. cerevisiae deletion strains, combined with growth assays revealed three probable targets for auranofin's antifungal activity-mia40, acn9, and coa4. Mia40 is of particular interest given its essential role in oxidation of cysteine rich proteins imported into the mitochondria. Biochemical analysis confirmed auranofin targets the Mia40-Erv1 pathway as the drug inhibited Mia40 from interacting with its substrate, Cmc1, in a dose-dependent manner similar to the control, MB-7. Furthermore, yeast mitochondria overexpressing Erv1 were shown to exhibit resistance to auranofin as an increase in Cmc1 import was observed compared to wild-type yeast. Further in vivo antifungal activity of auranofin was examined in a Caenorhabditis elegans animal model of Cryptococcus neoformans infection. Auranofin significantly reduced the fungal load in infected C. elegans. Collectively, the present study provides valuable evidence that auranofin has significant promise to be repurposed as a novel antifungal agent and may offer a safe, effective, and quick supplement to current approaches for treating fungal infections.

Ebselen exerts antifungal activity by regulating glutathione (GSH) and reactive oxygen species (ROS) production in fungal cells.

BACKGROUND: Ebselen, an organoselenium compound and a clinically safe molecule has been reported to possess potent antifungal activity, but its antifungal mechanism of action and in vivo antifungal activity remain unclear. METHODS: The antifungal effect of ebselen was tested against Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, Cryptococcus neoformans, and C. gattii clinical isolates. Chemogenomic profiling and biochemical assays were employed to identify the antifungal target of ebselen. Ebselen's antifungal activity in vivo was investigated in a Caenorhabditis elegans animal model. RESULTS: Ebselen exhibits potent antifungal activity against both Candida spp. and Cryptococcus spp., at concentrations ranging from 0.5 to 2µg/ml. Ebselen rapidly eradicates a high fungal inoculum within 2h of treatment. Investigation of the drug's antifungal mechanism of action indicates that ebselen depletes intracellular glutathione (GSH) levels, leading to increased production of reactive oxygen species (ROS), and thereby disturbs the redox homeostasis in fungal cells. Examination of ebselen's in vivo antifungal activity in two Caenorhabditis elegans models of infection demonstrate that ebselen is superior to conventional antifungal drugs (fluconazole, flucytosine and amphotericin) in reducing Candida and Cryptococcus fungal load. CONCLUSION: Ebselen possesses potent antifungal activity against clinically relevant isolates of both Candida and Cryptococcus by regulating GSH and ROS production. The potent in vivo antifungal activity of ebselen supports further investigation for repurposing it for use as an antifungal agent. GENERAL SIGNIFICANCE: The present study shows that ebselen targets glutathione and also support that glutathione as a potential target for antifungal drug development.

Transcriptome Profiling Identifies Multiplexin as a Target of SAGA Deubiquitinase Activity in Glia Required for Precise Axon Guidance During Drosophila Visual Development.

The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex is a transcriptional coactivator with histone acetylase and deubiquitinase activities that plays an important role in visual development and function. In Drosophila melanogaster, four SAGA subunits are required for the deubiquitination of monoubiquitinated histone H2B (ubH2B): Nonstop, Sgf11, E(y)2, and Ataxin 7. Mutations that disrupt SAGA deubiquitinase activity cause defects in neuronal connectivity in the developing Drosophila visual system. In addition, mutations in SAGA result in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7). Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration observed in SCA7 patients. Thus, we sought to identify the gene targets of SAGA deubiquitinase activity in glia in the Drosophila larval central nervous system. To do this, we enriched glia from wild-type, nonstop, and sgf11 larval optic lobes using affinity-purification of KASH-GFP tagged nuclei, and then examined each transcriptome using RNA-seq. Our analysis showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. We further show that the SAGA deubiquitinase-activated gene Multiplexin (Mp) is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia.

CellMiner Companion: an interactive web application to explore CellMiner NCI-60 data.

UNLABELLED: The NCI-60 human tumor cell line panel is an invaluable resource for cancer researchers, providing drug sensitivity, molecular and phenotypic data for a range of cancer types. CellMiner is a web resource that provides tools for the acquisition and analysis of quality-controlled NCI-60 data. CellMiner supports queries of up to 150 drugs or genes, but the output is an Excel file for each drug or gene. This output format makes it difficult for researchers to explore the data from large queries. CellMiner Companion is a web application that facilitates the exploration and visualization of output from CellMiner, further increasing the accessibility of NCI-60 data. AVAILABILITY AND IMPLEMENTATION: The web application is freely accessible at https://pul-bioinformatics.shinyapps.io/CellMinerCompanion The R source code can be downloaded at https://github.com/pepascuzzi/CellMinerCompanion.git CONTACT: ppascuzz@purdue.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

EpCAM-regulated intramembrane proteolysis induces a cancer stem cell-like gene signature in hepatitis B virus-infected hepatocytes.

BACKGROUND & AIMS: Hepatocytes in which the hepatitis B virus (HBV) is replicating exhibit loss of the chromatin modifying polycomb repressive complex 2 (PRC2), resulting in re-expression of specific, cellular PRC2-repressed genes. Epithelial cell adhesion molecule (EpCAM) is a PRC2-repressed gene, normally expressed in hepatic progenitors, but re-expressed in hepatic cancer stem cells (hCSCs). Herein, we investigated the functional significance of EpCAM re-expression in HBV-mediated hepatocarcinogenesis. METHODS: Employing molecular approaches (transfections, fluorescence-activated cell sorting, immunoblotting, qRT-PCR), we investigated the role of EpCAM-regulated intramembrane proteolysis (RIP) in HBV replicating cells in vitro, and in liver tumors from HBV X/c-myc mice and chronically HBV infected patients. RESULTS: EpCAM undergoes RIP in HBV replicating cells, activating canonical Wnt signaling. Transfection of Wnt-responsive plasmid expressing green fluorescent protein (GFP) identified a GFP + population of HBV replicating cells. These GFP+/Wnt+ cells exhibited cisplatin- and sorafenib-resistant growth resembling hCSCs, and increased expression of pluripotency genes NANOG, OCT4, SOX2, and hCSC markers BAMBI, CD44 and CD133. These genes are referred as EpCAM RIP and Wnt-induced hCSC-like gene signature. Interestingly, this gene signature is also overexpressed in liver tumors of X/c-myc bitransgenic mice. Clinically, a group of HBV-associated hepatocellular carcinomas was identified, exhibiting elevated expression of the hCSC-like gene signature and associated with reduced overall survival post-surgical resection. CONCLUSIONS: The hCSC-like gene signature offers promise as prognostic tool for classifying subtypes of HBV-induced HCCs. Since EpCAM RIP and Wnt signaling drive expression of this hCSC-like signature, inhibition of these pathways can be explored as therapeutic strategy for this subtype of HBV-associated HCCs. LAY SUMMARY: In this study, we provide evidence for a molecular mechanism by which chronic infection by the hepatitis B virus results in the development of poor prognosis liver cancer. Based on this mechanism our results suggest possible therapeutic interventions.

Regulated Formation of lncRNA-DNA Hybrids Enables Faster Transcriptional Induction and Environmental Adaptation.

Long non-coding (lnc)RNAs, once thought to merely represent noise from imprecise transcription initiation, have now emerged as major regulatory entities in all eukaryotes. In contrast to the rapidly expanding identification of individual lncRNAs, mechanistic characterization has lagged behind. Here we provide evidence that the GAL lncRNAs in the budding yeast S. cerevisiae promote transcriptional induction in trans by formation of lncRNA-DNA hybrids or R-loops. The evolutionarily conserved RNA helicase Dbp2 regulates formation of these R-loops as genomic deletion or nuclear depletion results in accumulation of these structures across the GAL cluster gene promoters and coding regions. Enhanced transcriptional induction is manifested by lncRNA-dependent displacement of the Cyc8 co-repressor and subsequent gene looping, suggesting that these lncRNAs promote induction by altering chromatin architecture. Moreover, the GAL lncRNAs confer a competitive fitness advantage to yeast cells because expression of these non-coding molecules correlates with faster adaptation in response to an environmental switch.

The antitumorigenic function of EGFR in metastatic breast cancer is regulated by expression of Mig6.

Numerous studies by our lab and others demonstrate that epidermal growth factor receptor (EGFR) plays critical roles in primary breast cancer (BC) initiation, growth and dissemination. However, clinical trials targeting EGFR function in BC have lead to disappointing results. In the current study we sought to identify the mechanisms responsible for this disparity by investigating the function of EGFR across the continuum of the metastatic cascade. We previously established that overexpression of EGFR is sufficient for formation of in situ primary tumors by otherwise nontransformed murine mammary gland cells. Induction of epithelial-mesenchymal transition (EMT) is sufficient to drive the metastasis of these EGFR-transformed tumors. Examining growth factor receptor expression across this and other models revealed a potent downregulation of EGFR through metastatic progression. Consistent with diminution of EGFR following EMT and metastasis EGF stimulation changes from a proliferative to an apoptotic response in in situ versus metastatic tumor cells, respectively. Furthermore, overexpression of EGFR in metastatic MDA-MB-231 BC cells promoted their antitumorigenic response to EGF in three dimensional (3D) metastatic outgrowth assays. In line with the paradoxical function of EGFR through EMT and metastasis we demonstrate that the EGFR inhibitory molecule, Mitogen Induced Gene-6 (Mig6), is tumor suppressive in in situ tumor cells. However, Mig6 expression is absolutely required for prevention of apoptosis and ultimate metastasis of MDA-MB-231 cells. Further understanding of the paradoxical function of EGFR between primary and metastatic tumors will be essential for application of its targeted molecular therapies in BC.

In vivo mapping of arabidopsis scaffold/matrix attachment regions reveals link to nucleosome-disfavoring poly(dA:dT) tracts.

Scaffold or matrix attachment regions (S/MARs) are found in all eukaryotes. The pattern of distribution and genomic context of S/MARs is thought to be important for processes such as chromatin organization and modulation of gene expression. Despite the importance of such processes, much is unknown about the large-scale distribution and sequence content of S/MARs in vivo. Here, we report the use of tiling microarrays to map 1358 S/MARs on Arabidopsis thaliana chromosome 4 (chr4). S/MARs occur throughout chr4, spaced much more closely than in the large plant and animal genomes that have been studied to date. Arabidopsis S/MARs can be divided into five clusters based on their association with other genomic features, suggesting a diversity of functions. While some Arabidopsis S/MARs may define structural domains, most occur near the transcription start sites of genes. Genes associated with these S/MARs have an increased probability of expression, which is particularly pronounced in the case of transcription factor genes. Analysis of sequence motifs and 6-mer enrichment patterns show that S/MARs are preferentially enriched in poly(dA:dT) tracts, sequences that resist nucleosome formation, and the majority of S/MARs contain at least one nucleosome-depleted region. This global view of S/MARs provides a framework to begin evaluating genome-scale models for S/MAR function.

Arabidopsis thaliana chromosome 4 replicates in two phases that correlate with chromatin state.

DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins.

Host-mediated phosphorylation of type III effector AvrPto promotes Pseudomonas virulence and avirulence in tomato.

The AvrPto protein from Pseudomonas syringae pv tomato is delivered into plant cells by the bacterial type III secretion system, where it either promotes host susceptibility or, in tomato plants expressing the Pto kinase, elicits disease resistance. Using two-dimensional gel electrophoresis, we obtained evidence that AvrPto is phosphorylated when expressed in plant leaves. In vitro phosphorylation of AvrPto by plant extracts occurs independently of Pto and is due to a kinase activity that is conserved in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), and Arabidopsis thaliana. Three Ser residues clustered in the C-terminal 18 amino acids of AvrPto were identified in vitro as putative phosphorylation sites, and one site at S149 was directly confirmed as an in vivo phosphorylation site by mass spectrometry. Substitution of Ala for S149 significantly decreased the ability of AvrPto to enhance disease symptoms and promote growth of P. s. tomato in susceptible tomato leaves. In addition, S149A significantly decreased the avirulence activity of AvrPto in resistant tomato plants. Our observations support a model in which AvrPto has evolved to mimic a substrate of a highly conserved plant kinase to enhance its virulence activity. Furthermore, residues of AvrPto that promote virulence are also monitored by plant defenses.