[Show abstract][Hide abstract] ABSTRACT: Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD002411 (http://proteomecentral.proteomexchange.org/dataset/PXD002411).
[Show abstract][Hide abstract] ABSTRACT: Lin28, a well-known RNA-binding protein, regulates diverse cellular properties. All physiological functions of Lin28A characterized so far have been attributed to its repression of let-7 miRNA biogenesis or modulation of mRNA translational efficiency. Here we show that Lin28A directly binds to a consensus DNA sequence in vitro and in mouse embryonic stem cells in vivo. ChIP-seq and RNA-seq reveal enrichment of Lin28A binding around transcription start sites and a positive correlation between its genomic occupancy and expression of many associated genes. Mechanistically, Lin28A recruits 5-methylcytosine-dioxygenase Tet1 to genomic binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. Either Lin28A or Tet1 knockdown leads to dysregulated DNA methylation and expression of common target genes. These results reveal a surprising role for Lin28A in transcriptional regulation via epigenetic DNA modifications and have implications for understanding mechanisms underlying versatile functions of Lin28A in mammalian systems.
[Show abstract][Hide abstract] ABSTRACT: Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process.
[Show abstract][Hide abstract] ABSTRACT: Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule–only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the m
Full-text · Article · Aug 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Age-related macular degeneration (AMD) is a major cause of blindness in the western world. While genetic studies have linked both common and rare variants in genes involved in regulation of the complement system to increased risk of development of AMD, environmental factors, such as smoking and nutrition, can also significantly affect the risk of developing the disease and the rate of disease progression. Since epigenetics has been implicated in mediating, in part, the disease risk associated with some environmental factors, we investigated a possible epigenetic contribution to AMD. We performed genome-wide DNA methylation profiling of blood from AMD patients and controls. No differential methylation site reached genome-wide significance; however, when epigenetic changes in and around known GWAS-defined AMD risk loci were explored, we found small but significant DNA methylation differences in the blood of neovascular AMD patients near age-related maculopathy susceptibility 2 (ARMS2), a top-ranked GWAS locus preferentially associated with neovascular AMD. The methylation level of one of the CpG sites significantly correlated with the genotype of the risk SNP rs10490924, suggesting a possible epigenetic mechanism of risk. Integrating genome-wide DNA methylation analysis of retina samples with and without AMD together with blood samples, we further identified a consistent, replicable change in DNA methylation in the promoter region of protease serine 50 (PRSS50). These methylation changes may identify sites in novel genes that are susceptible to non-genetic factors known to contribute to AMD development and progression.
No preview · Article · Jun 2015 · Epigenetics: official journal of the DNA Methylation Society
[Show abstract][Hide abstract] ABSTRACT: Regulatory regions (e.g. promoters and enhancers) play an essential role in human development and disease. Many computational approaches have been developed to predict the regulatory regions using various genomic features such as sequence motifs and evolutionary conservation. However, these DNA sequence-based approaches do not reflect the tissue-specific nature of the regulatory regions. In this work, we propose to predict regulatory regions using multiple features derived from DNA methylation profile.
We discovered several interesting features of the methylated CpG (mCpG) sites within regulatory regions. First, a hypomethylation status of CpGs within regulatory regions, compared to the genomic background methylation level, extended out >1000 bp from the center of the regulatory regions, demonstrating a high degree of correlation between the methylation statuses of neighboring mCpG sites. Second, when a regulatory region was inactive, as determined by histone mark differences between cell lines, methylation level of the mCpG site increased from a hypomethylated state to a hypermethylated state, the level of which was even higher than the genomic background. Third, a distinct set of sequence motifs was overrepresented surrounding mCpG sites within regulatory regions. Using 5 types of features derived from DNA methylation profiles, we were able to predict promoters and enhancers using machine-learning approach (support vector machine). The performances for prediction of promoters and enhancers are quite well, showing an area under the ROC curve (AUC) of 0.992 and 0.817, respectively, which is better than that simply based on methylation level, especially for prediction of enhancers.
Our study suggests that DNA methylation features of mCpG sites can be used to predict regulatory regions.
[Show abstract][Hide abstract] ABSTRACT: We have previously reported that in the Nuc1 rat, which has a spontaneous mutation in Cryba1 (the gene encoding βA3/A1-crystallin), astrocytes exhibit decreased Notch signaling, leading to reduced promoter activity for glial fibrillary acidic protein (GFAP). Interestingly, in both Nuc1 astrocytes and in wild type astrocytes following knockdown of Cryba1, vascular endothelial growth factor (VEGF) secretion is decreased. This has led us to explore signaling mediators that could be regulated by βA3/A1-crystallin to modulate both GFAP and VEGF. Several studies have shown that the signal transducer and activator of transcription 3 (STAT3) is involved in the co-regulation of GFAP and VEGF. We show that STAT3 and βA3/A1-crystallin may co-regulate each other in astrocytes. Such co-regulation would create a positive feedback circuit; i.e., in the cytosol of astrocytes, βA3/A1-crystallin is necessary for the phosphorylation of STAT3, which then dimerizes and translocates to the nucleus to form DNA-binding complexes, activating transcription of Cryba1. This stoichiometric co-regulation of STAT3 and Cryba1 could potentiate expression of GFAP and secretion of VEGF, both of which are essential for maintaining astrocyte and blood vessel homeostasis in the retina. Consistent with this idea, Cryba1 knockout mice exhibit an abnormal astrocyte pattern and defective remodeling of retinal vessels.
Full-text · Article · Mar 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: BackgroundDNA methylation plays an important role in regulating gene expression during many biological processes. However, the mechanism of DNA-methylation-dependent gene regulation is not fully understood. Here, we explore two possible DNA methylation regulatory mechanisms with opposite modes of gene expression regulation.ResultsBy comparing the genome-wide methylation and expression patterns in different tissues, we find that majority of tissue-specific differentially methylated regions (T-DMRs) are negatively correlated with expression of their associated genes (negative T-DMRs), consistent with the classical dogma that DNA methylation suppresses gene expression; however, a significant portion of T-DMRs are positively correlated with gene expression (positive T-DMRs). We observe that the positive T-DMRs have similar genomic location as negative T-DMRs, except that the positive T-DMRs are more enriched in the promoter regions. Both positive and negative T-DMRs are enriched in DNase I hypersensitivity sites (DHSs), suggesting that both are likely to be functional. The CpG sites of both positive and negative T-DMRs are also more evolutionarily conserved than the genomic background. Interestingly, the putative target genes of the positive T-DMR are enriched for negative regulators such as transcriptional repressors, suggesting a novel mode of indirect DNA methylation inhibition of expression through transcriptional repressors. Likewise, two distinct sets of DNA sequence motifs exist for positive and negative T-DMRs, suggesting that two distinct sets of transcription factors (TFs) are involved in positive and negative regulation mediated by DNA methylation.Conclusions
We find both negative and positive association between T-DMRs and gene expression, which implies the existence of two different mechanisms of DNA methylation-dependent gene regulation.
[Show abstract][Hide abstract] ABSTRACT: The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing 'driver' oncogenic mutations of PIK3CA to dissect the signalling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signalling networks. Here, by integrating phosphoproteomic data with human protein microarray-based AKT1 kinase assays, we discover and validate six novel AKT1 substrates, including cortactin. Through mutagenesis studies, we demonstrate that phosphorylation of cortactin by AKT1 is important for mutant PI3K-enhanced cell migration and invasion. Our study describes a quantitative and global approach for identifying mutation-specific signalling events and for discovering novel signalling molecules as readouts of pathway activation or potential therapeutic targets.
[Show abstract][Hide abstract] ABSTRACT: Vertebrate circadian rhythms are organized by the hypothalamic suprachiasmatic nucleus (SCN). Despite its physiological importance, SCN development is poorly understood. Here, we show that Lim homeodomain transcription factor 1 (Lhx1) is essential for terminal differentiation and function of the SCN. Deletion of Lhx1 in the developing SCN results in loss of SCN-enriched neuropeptides involved in synchronization and coupling to downstream oscillators, among other aspects of circadian function. Intact, albeit damped, clock gene expression rhythms persist in Lhx1-deficient SCN; however, circadian activity rhythms are highly disorganized and susceptible to surprising changes in period, phase, and consolidation following neuropeptide infusion. Our results identify a factor required for SCN terminal differentiation. In addition, our in vivo study of combinatorial SCN neuropeptide disruption uncovered synergies among SCN-enriched neuropeptides in regulating normal circadian function. These animals provide a platform for studying the central oscillator's role in physiology and cognition.
[Show abstract][Hide abstract] ABSTRACT: Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor in vitro and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1-/- cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease.
Full-text · Article · Apr 2014 · ACS Chemical Biology
[Show abstract][Hide abstract] ABSTRACT: The retinal pigment epithelium (RPE) performs specialized functions to support retinal photoreceptors, including regeneration
of the visual chromophore. Enzymes and carrier proteins in the visual cycle function sequentially to regenerate and continuously
supply 11-cis-retinal to retinal photoreceptor cells. However, it is unknown how the expression of the visual cycle genes is coordinated
at the transcriptional level. Here, we show that the proximal upstream regions of six visual cycle genes contain chromatin-accessible
sex-determining region Y box (SOX) binding sites, that SOX9 and LIM homeobox 2 (LHX2) are coexpressed in the nuclei of mature
RPE cells, and that SOX9 acts synergistically with orthodenticle homeobox 2 (OTX2) to activate the RPE65 and retinaldehyde binding protein 1 (RLBP1) promoters and acts synergistically with LHX2 to activate the retinal G protein-coupled receptor (RGR) promoter. ChIP reveals that SOX9 and OTX2 bind to the promoter regions of RPE65, RLBP1, and RGR and that LHX2 binds to those of RPE65 and RGR in bovine RPE. ChIP with human fetal RPE cells shows that SOX9 and OTX2 also bind to the human RPE65, RLBP1, and RGR promoters. Conditional inactivation of Sox9 in mouse RPE results in reduced expression of several visual cycle genes, most dramatically Rpe65 and Rgr. Furthermore, bioinformatic analysis predicts that multiple common microRNAs (miRNAs) regulate visual cycle genes, and cotransfection
of miRNA mimics with luciferase reporter constructs validated some of the predicted miRNAs. These results implicate SOX9 as
a key regulator of visual cycle genes, reveal for the first time the functional role of LHX2 in the RPE, and suggest the possible
regulation of visual cycle genes by common miRNAs.
Preview · Article · Mar 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: How cells degenerate from oxidative stress in aging-related disease is incompletely understood. The study's intent was to identify key cytoprotective pathways activated by oxidative stress, and determine the extent of their protection. Using an unbiased strategy with microarray analysis, retinal pigmented epithelial (RPE) cells treated with cigarette smoke extract (CSE) had over-represented genes involved in the antioxidant and unfolded protein response (UPR). Differentially expressed antioxidant genes were predominantly located in the cytoplasm, with no induction of genes that neutralize superoxide and H2O2 in the mitochondria, resulting in accumulation of superoxide and decreased ATP production. Simultaneously, CSE induced the UPR sensors IRE1α, p-PERK, and ATP6, including CHOP, which was cytoprotective because CHOP knockdown decreased cell viability. In mice given intravitreal CSE, the RPE had increased IRE1α and decreased ATP, and developed epithelial-mesenchymal transition, as suggested by decreased LRAT abundance, altered ZO1 immunolabeling, and dysmorphic cell shape. Mildly degenerated RPE from early AMD samples had prominent IRE1α, but minimal mitochondrial TOM20 immunolabeling. While oxidative stress is thought to induce an antioxidant response with cooperation between the mitochondria and ER, herein, we show that mitochondria become impaired sufficiently to induce epithelial-mesenchymal transition despite a protective UPR. With similar responses in early AMD samples, these results suggest that mitochondria are vulnerable to oxidative stress despite a protective UPR during early phases of aging-related disease.
Full-text · Article · Jan 2014 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: Age-related macular degeneration (AMD) is a leading cause of visual impairment worldwide. Aberrant DNA methylation within the promoter of IL17RC in peripheral blood mononuclear cells has recently been reported in AMD. To validate this association, we examined DNA methylation of the IL17RC promoter in peripheral blood. First, we used Illumina Human Methylation450 Bead Arrays, a widely accepted platform for measuring global DNA methylation. Second, methylation status at multiple sites within the IL17RC promoter was determined by bisulfite pyrosequencing in two cohorts. Third, a methylation-sensitive quantitative PCR-based assay was performed on a subset of samples. In contrast to previous findings, we did not find evidence of differential methylation between AMD cases and age-matched controls. We conclude that hypomethylation within the IL17RC gene promoter in peripheral blood is not suitable for use as a clinical biomarker of AMD. This study highlights the need for considerable replication of epigenetic association studies prior to clinical application.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
MicroRNAs (miRNAs) are a class of endogenous noncoding RNA and post transcriptionally modulate gene expression during development and disease. Our study investigated the differential miRNA expression in human Fuchs' endothelial corneal dystrophy (FECD) compared with normal endothelium to identify miRNA sequences that are involved in the pathogenesis of FECD.
Comparative miRNA expression profiles of endothelial samples obtained from FECD patients during lamellar corneal transplant surgery and from normal donor globes were generated using OpenArray plate technology. Differential expression of individual miRNAs was validated in the original and in independent samples using stem-loop RT qPCR assays. Expression of miRNA target genes was assessed using qPCR and tissue microarray (TMA) immunolabeling.
Our results demonstrate downregulation of 87 miRNAs in FECD compared with normal endothelium (>3-fold change; P < 0.01). Correspondingly, DICER1, (encoding an endoribonuclease critical to miRNA biogenesis) showed a moderate but significant decrease in FECD samples (P < 0.05). Significant repression of three miR-29 family members (miR-29a-3p, miR-29b-2-5p, and miR-29c-5p) was paralleled by upregulation of their extracellular matrix associated mRNA targets collagen I and collagen IV. Tissue microarray immunolabeling showed histologically verifiable subendothelial collagen I and collagen IV deposition and increased endothelial laminin protein expression in FECD samples.
The present study provides the first miRNA profile in FECD and normal endothelial cells and demonstrates widespread miRNA downregulation in FECD. Decreased endothelial expression of miR-29 family members may be associated with increased subendothelial extracellular matrix accumulation in FECD.
No preview · Article · Dec 2013 · Investigative ophthalmology & visual science
[Show abstract][Hide abstract] ABSTRACT: Phosphorylation plays a very important role in cellular signal transduction. Current phosphorylation-related databases often focus on the phosphorylation sites, which are mainly determined by mass spectrometry. Here, we present PhosphoNetworks, a phosphorylation database built upon a high-resolution map of phosphorylation networks. This high-resolution map of phosphorylation networks provides not only the kinase-substrate relationships (KSRs), but also the specific phosphorylation sites on which the kinases act on the substrates. The database contains the most comprehensive dataset for KSRs, including the relationships from a recent high throughput project for identification of KSRs using protein microarrays, as well as known KSRs curated from the literature. In addition, the database also includes several analytical tools for dissecting phosphorylation networks. PhosphoNetworks is expected to play a prominent role in proteomics and phosphorylation related disease research.
http://www.phosphonetworks.org CONTACT: email@example.com.