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1.  W-ChIPeaks: a comprehensive web application tool for processing ChIP-chip and ChIP-seq data 
Bioinformatics  2010;27(3):428-430.
Summary: ChIP-based technology is becoming the leading technology to globally profile thousands of transcription factors and elucidate the transcriptional regulation mechanisms in living cells. It has evolved rapidly in recent years, from hybridization with spotted or tiling microarray (ChIP-chip), to pair-end tag sequencing (ChIP-PET), to current massively parallel sequencing (ChIP-seq). Although there are many tools available for identifying binding sites (peaks) for ChIP-chip and ChIP-seq, few of them are available as easy-accessible online web tools for processing both ChIP-chip and ChIP-seq data for the ChIP-based user community. As such, we have developed a comprehensive web application tool for processing ChIP-chip and ChIP-seq data. Our web tool W-ChIPeaks employed a probe-based (or bin-based) enrichment threshold to define peaks and applied statistical methods to control false discovery rate for identified peaks. The web tool includes two different web interfaces: PELT for ChIP-chip, BELT for ChIP-seq, where both were tested on previously published experimental data. The novel features of our tool include a comprehensive output for identified peaks with GFF, BED, bedGraph and .wig formats, annotated genes to which these peaks are related, a graphical interpretation and visualization of the results via a user-friendly web interface.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3031039  PMID: 21138948
2.  High resolution epifluorescence and TOF-SIMS chemical imaging comparisons of single DNA microarray spots 
Analytical chemistry  2012;84(24):10628-10636.
DNA microarray assay performance is commonly compromised by spot-spot probe and signal variations as well as heterogeneity within printed microspots. Accurate metrics for captured DNA target signal rely upon uniform spot distribution of both probe and target DNA to yield reliable hybridized signal. While often presumed, this is neither easily achieved nor often proven experimentally. High resolution imaging techniques were used to determine spot heterogeneity in identical DNA array microspots comprising varied ratios of unlabeled and dye-labeled DNA probes contact printed onto commercial arraying surfaces. Epifluorescence imaging data for individual array microspots were correlated with time-of-flight secondary ion mass spectrometry (TOF-SIMS) chemical state imaging of the same spots. Epifluorescence imaging intensity distinguished varying DNA density distributed both within a given spot and from spot-to-spot. TOF-SIMS chemical analysis confirmed these heterogeneous printed DNA distributions by tracking bound Cy3 dye-, DNA base, and phosphate- specific ion fragments often correlating to fluorescence patterns within identical spots. TOF-SIMS ion fragments originating from probe DNA and Cy3 dye are enriched in microspot centers, correlating with high fluorescence intensity regions. Both TOF-SIMS and epifluorescence supports Marangoni flow effects on spot drying, with high density DNA-Cy3 located in spot centers and non-homogeneous DNA distribution within printed spots. Microspot image dimensional analysis results for DNA droplet spreading show differing DNA densities across printed spots. The study directly supports different DNA probe chemical and spatial microenvironments within spots that yield spot-spot signal variations known to affect DNA target hybridization efficiencies and kinetics. These variations critically affect probe-target duplex formation and DNA array signal generation.
PMCID: PMC3525714  PMID: 23150996
microarray; chemical imaging; fluorescence; mass spectrometry; surface analysis; nucleic acid; hybridization
3.  Protein Kinase PKR Plays a Stimulus- and Virus-Dependent Role in Apoptotic Death and Virus Multiplication in Human Cells▿  
Journal of Virology  2007;81(15):8192-8200.
The protein kinase regulated by double-stranded RNA (dsRNA), PKR, is implicated in a range of biologic processes, including apoptotic death and interferon antiviral responses, based in part on studies with mouse cells genetically deficient in Pkr. To test the role of the PKR protein in human cells, an RNA interference silencing strategy was used to generate stable HeLa cell lines with less than 2% of the PKR protein (PKR deficient) compared to either parental or control knockdown HeLa lines. Phosphorylation of the α subunit of eukaryotic initiation factor 2 on serine 51 was not detectably increased in response to dsRNA in PKR-deficient HeLa cells but was elevated severalfold in PKR-sufficient cells. PKR-deficient cells displayed reduced dsRNA-induced apoptosis compared to PKR-sufficient cell lines, whereas tumor necrosis factor alpha (TNF-α)-induced apoptosis was comparable between the HeLa lines. NF-κB was activated to a comparable extent in PKR-deficient and PKR-sufficient HeLa cells upon treatment with either dsRNA or TNF-α. The antiviral response against vesicular stomatitis virus was reduced in interferon-treated PKR-deficient compared to PKR-sufficient HeLa cells. However, the growth of two human viruses, adenovirus and reovirus, was unaffected by the PKR knockdown. Surprisingly, the yield of mutant adenovirus that fails to encode VAI RNA was not enhanced in PKR-deficient cells, indicating the importance of host factors in addition to PKR in conferring the VAI RNA phenotype.
PMCID: PMC1951329  PMID: 17522227
4.  Specific NFκB subunit activation and kinetics of cytokine induction in adenoviral keratitis 
Molecular Vision  2009;15:2879-2889.
Corneal inflammation associated with ocular adenoviral infection is caused by leukocytic infiltration of the subepithelial stroma in response to expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by infected corneal cells. We have shown that these two chemokines are activated by the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 for IL-8, and Jun-terminal kinase (JNK) for MCP-1. It is also well established that transcription of each of these chemokines is tightly controlled by the nuclear factor kappa B (NFκB) transcription factor family. Therefore, we sought to better understand the differential regulation of chemokine expression by NFκB in adenoviral infection of the cornea.
Primary keratocytes derived from human donor corneas were treated with signaling inhibitors and small interfering RNA specific to MAPKs, and infected with adenovirus for different time periods before analysis. Activation of specific NFκB subunits was analyzed by western blot, confocal microscopy, electromobility shift assay, and chromatin immunoprecipitation, and chemokine expression was quantified by enzyme-linked immunosorbent assay.
Upon adenoviral infection, NFκB p65, p50, and cREL subunits translocate to the nucleus. This translocation is blocked by inhibitors of specific MAPK signaling pathways. Confocal microscopy showed that inhibitors of the p38, JNK, and ERK pathways differentially inhibited NFκB nuclear translocation, while PP2, an inhibitor of Src family kinases, completely inhibited NFκB nuclear translocation. Western blot analysis revealed that activation of specific NFκB subunits was time dependent following infection. Chromatin immunoprecipitation experiments indicated that binding of NFκB p65 and p50 subunits to the IL-8 promoter upon viral infection was differentially reduced by chemical inhibitors of MAPKs. Electromobility shift assay and luciferase assay analysis revealed that transactivation of IL-8 occurred with binding by the NFκB p65 homodimer or NFκB p65/p50 heterodimer as early as 1 h post infection, whereas MCP-1 expression was dependent upon the NFκB cREL but not the p65 subunit, and occurred 4 h after IL-8 induction. Finally, knockdown of NFκB p65 by short interfering RNA abrogated IL-8 but not MCP-1 expression after adenoviral infection.
The kinetics of NFκB subunit activation are partly responsible for the observed pattern of acute inflammation in the adenoviral-infected cornea. MAPKs differentially regulate chemokine expression in adenoviral keratitis by differential and time-dependent activation of specific NFκB subunits.
PMCID: PMC2797044  PMID: 20038977
5.  Statistics of protein-DNA binding and the total number of binding sites for a transcription factor in the mammalian genome 
BMC Genomics  2010;11(Suppl 1):S12.
Transcription factor (TF)-DNA binding loci are explored by analyzing massive datasets generated with application of Chromatin Immuno-Precipitation (ChIP)-based high-throughput sequencing technologies. These datasets suffer from a bias in the information about binding loci availability, sample incompleteness and diverse sources of technical and biological noises. Therefore adequate mathematical models of ChIP-based high-throughput assay(s) and statistical tools are required for a robust identification of specific and reliable TF binding sites (TFBS), a precise characterization of TFBS avidity distribution and a plausible estimation the total number of specific TFBS for a given TF in the genome for a given cell type.
We developed an exploratory mixture probabilistic model for a specific and non-specific transcription factor-DNA (TF-DNA) binding. Within ChiP-seq data sets, the statistics of specific and non-specific DNA-protein binding is defined by a mixture of sample size-dependent skewed functions described by Kolmogorov-Waring (K-W) function (Kuznetsov, 2003) and exponential function, respectively. Using available Chip-seq data for eleven TFs, essential for self-maintenance and differentiation of mouse embryonic stem cells (SC) (Nanog, Oct4, sox2, KLf4, STAT3, E2F1, Tcfcp211, ZFX, n-Myc, c-Myc and Essrb) reported in Chen et al (2008), we estimated (i) the specificity and the sensitivity of the ChiP-seq binding assays and (ii) the number of specific but not identified in the current experiments binding sites (BSs) in the genome of mouse embryonic stem cells. Motif finding analysis applied to the identified c-Myc TFBSs supports our results and allowed us to predict many novel c-Myc target genes.
We provide a novel methodology of estimating the specificity and the sensitivity of TF-DNA binding in massively paralleled ChIP sequencing (ChIP-seq) binding assay. Goodness-of fit analysis of K-W functions suggests that a large fraction of low- and moderate- avidity TFBSs cannot be identified by the ChIP-based methods. Thus the task to identify the binding sensitivity of a TF cannot be technically resolved yet by current ChIP-seq, compared to former experimental techniques. Considering our improvement in measuring the sensitivity and the specificity of the TFs obtained from the ChIP-seq data, the models of transcriptional regulatory networks in embryonic cells and other cell types derived from the given ChIp-seq data should be carefully revised.
PMCID: PMC2822526  PMID: 20158869
6.  Using ChIP-chip and ChIP-seq to study the regulation of gene expression: genome-wide localization studies reveal widespread regulation of transcription elongation 
Methods (San Diego, Calif.)  2009;48(4):398-408.
Transcription is a sophisticated multi-step process in which RNA polymerase II (Pol II) transcribes a DNA template into RNA in concert with a broad array of transcription initiation, elongation, capping, termination, and histone modifying factors. Recent global analyses of Pol II distribution have indicated that many genes are regulated during the elongation phase, shedding light on a previously underappreciated mechanism for controlling gene expression. Understanding how various factors regulate transcription elongation in living cells has been greatly aided by chromatin immunoprecipitation (ChIP) studies, which can provide spatial and temporal resolution of protein-DNA binding events. The coupling of ChIP with DNA microarray and high-throughput sequencing technologies (ChIP-chip and ChIP-seq) has significantly increased the scope of ChIP studies and genome-wide maps of Pol II or elongation factor binding sites can now be readily produced. However, while ChIP-chip/ChIP-seq data allow for high-resolution localization of protein-DNA binding sites, they are not sufficient to dissect protein function. Here we describe techniques for coupling ChIP-chip/ChIP-seq with genetic, chemical, and experimental manipulation to obtain mechanistic insight from genome-wide protein-DNA binding studies. We have employed these techniques to discern immature promoter-proximal Pol II from productively elongating Pol II, and infer a critical role for the transition between initiation and full elongation competence in regulating development and gene induction in response to environmental signals.
PMCID: PMC3431615  PMID: 19275938
transcription elongation; gene expression; ChIP-chip; ChIP-seq
7.  Development of a High-Throughput Candida albicans Biofilm Chip 
PLoS ONE  2011;6(4):e19036.
We have developed a high-density microarray platform consisting of nano-biofilms of Candida albicans. A robotic microarrayer was used to print yeast cells of C. albicans encapsulated in a collagen matrix at a volume as low as 50 nL onto surface-modified microscope slides. Upon incubation, the cells grow into fully formed “nano-biofilms”. The morphological and architectural complexity of these biofilms were evaluated by scanning electron and confocal scanning laser microscopy. The extent of biofilm formation was determined using a microarray scanner from changes in fluorescence intensities due to FUN 1 metabolic processing. This staining technique was also adapted for antifungal susceptibility testing, which demonstrated that, similar to regular biofilms, cells within the on-chip biofilms displayed elevated levels of resistance against antifungal agents (fluconazole and amphotericin B). Thus, results from structural analyses and antifungal susceptibility testing indicated that despite miniaturization, these biofilms display the typical phenotypic properties associated with the biofilm mode of growth. In its final format, the C. albicans biofilm chip (CaBChip) is composed of 768 equivalent and spatially distinct nano-biofilms on a single slide; multiple chips can be printed and processed simultaneously. Compared to current methods for the formation of microbial biofilms, namely the 96-well microtiter plate model, this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize labor intensive steps, and dramatically reduce assay costs. Such a chip should accelerate the antifungal drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously.
PMCID: PMC3081316  PMID: 21544190
8.  Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method 
Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR)-based assays and enzyme-linked immunosorbent assays (ELISA) are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS) method was applied to detect HBV and HCV antibodies rapidly and simultaneously.
Chemically modified glass slides were used as solid supports (named chip), on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens) were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA) was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay.
We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 ± 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05) existed between the results determined by our assay and ELISA respectively.
Results showed that our assay can be applied with serology for the detection of HBV and HCV antibodies rapidly and simultaneously in clinical detection.
PMCID: PMC1182366  PMID: 15998472
9.  Biocompatible Hydrogels for Microarray Cell Printing and Encapsulation 
Biosensors  2015;5(4):647-663.
Conventional drug screening processes are a time-consuming and expensive endeavor, but highly rewarding when they are successful. To identify promising lead compounds, millions of compounds are traditionally screened against therapeutic targets on human cells grown on the surface of 96-wells. These two-dimensional (2D) cell monolayers are physiologically irrelevant, thus, often providing false-positive or false-negative results, when compared to cells grown in three-dimensional (3D) structures such as hydrogel droplets. However, 3D cell culture systems are not easily amenable to high-throughput screening (HTS), thus inherently low throughput, and requiring relatively large volume for cell-based assays. In addition, it is difficult to control cellular microenvironments and hard to obtain reliable cell images due to focus position and transparency issues. To overcome these problems, miniaturized 3D cell cultures in hydrogels were developed via cell printing techniques where cell spots in hydrogels can be arrayed on the surface of glass slides or plastic chips by microarray spotters and cultured in growth media to form cells encapsulated 3D droplets for various cell-based assays. These approaches can dramatically reduce assay volume, provide accurate control over cellular microenvironments, and allow us to obtain clear 3D cell images for high-content imaging (HCI). In this review, several hydrogels that are compatible to microarray printing robots are discussed for miniaturized 3D cell cultures.
PMCID: PMC4697138  PMID: 26516921
microarray; cell encapsulation; hydrogel; bioprinting; miniaturized 3D cell culture
10.  Fabrication and use of MicroEnvironment microArrays (MEArrays) 
The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. For instance, on the single cell level, distinct microenvironments can impose specific differentiation, migration, and proliferation of phenotypes, and on the tissue level the microenvironment may control processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within them, but also does the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and –soluble factor interactions, in addition to physical characteristics, the microenvironment is highly complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the molecular and physical components of the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with the printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).
PMCID: PMC3490308  PMID: 23093325
Microenvironment; microarray; MEArray; functional cell-based assay
11.  Direct Activation of Ribosome-Associated Double-Stranded RNA-Dependent Protein Kinase (PKR) by Deoxynivalenol, Anisomycin and Ricin: A New Model for Ribotoxic Stress Response Induction 
Toxins  2014;6(12):3406-3425.
Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a critical upstream mediator of the ribotoxic stress response (RSR) to the trichothecene deoxynivalenol (DON) and other translational inhibitors. Here, we employed HeLa cell lysates to: (1) characterize PKR’s interactions with the ribosome and ribosomal RNA (rRNA); (2) demonstrate cell-free activation of ribosomal-associated PKR and (3) integrate these findings in a unified model for RSR. Robust PKR-dependent RSR was initially confirmed in intact cells. PKR basally associated with 40S, 60S, 80S and polysome fractions at molar ratios of 7, 2, 23 and 3, respectively. Treatment of ATP-containing HeLa lysates with DON or the ribotoxins anisomycin and ricin concentration-dependently elicited phosphorylation of PKR and its substrate eIF2α. These phosphorylations could be blocked by PKR inhibitors. rRNA immunoprecipitation (RNA-IP) of HeLa lysates with PKR-specific antibody and sequencing revealed that in the presence of DON or not, the kinase associated with numerous discrete sites on both the 18S and 28S rRNA molecules, a number of which contained double-stranded hairpins. These findings are consistent with a sentinel model whereby multiple PKR molecules basally associate with the ribosome positioning them to respond to ribotoxin-induced alterations in rRNA structure by dimerizing, autoactivating and, ultimately, evoking RSR.
PMCID: PMC4280541  PMID: 25521494
PKR; ribotoxic stress; rRNA; deoxynivalenol; anisomycin; ricin; translational inhibitor; quantitiative Western analysis; RNA immunoprecipitation
12.  Estrogen Modulates NFκB Signaling by Enhancing IκBα Levels and Blocking p65 Binding at the Promoters of Inflammatory Genes via Estrogen Receptor-β 
PLoS ONE  2012;7(6):e36890.
NFκB signaling is critical for expression of genes involved in the vascular injury response. We have shown that estrogen (17β-estradiol, E2) inhibits expression of these genes in an estrogen receptor (ER)-dependent manner in injured rat carotid arteries and in tumor necrosis factor (TNF)-α treated rat aortic smooth muscle cells (RASMCs). This study tested whether E2 inhibits NFκB signaling in RASMCs and defined the mechanisms.
Methodology/Principal Findings
TNF-α treated RASMCs demonstrated rapid degradation of IκBα (10–30 min), followed by dramatic increases in IκBα mRNA and protein synthesis (40–60 min). E2 enhanced TNF-α induced IκBα synthesis without affecting IκBα degradation. Chromatin immunoprecipitation (ChIP) assays revealed that E2 pretreatment both enhanced TNF-α induced binding of NFκB p65 to the IκBα promoter and suppressed TNF-α induced binding of NFκB p65 to and reduced the levels of acetylated histone 3 at promoters of monocyte chemotactic protein (MCP)-1 and cytokine-induced neutrophil chemoattractant (CINC)-2β genes. ChIP analyses also demonstrated that ERβ can be recruited to the promoters of MCP-1 and CINC-2β during co-treatment with TNF-α and E2.
These data demonstrate that E2 inhibits inflammation in RASMCs by two distinct mechanisms: promoting new synthesis of IκBα, thus accelerating a negative feedback loop in NFκB signaling, and directly inhibiting binding of NFκB to the promoters of inflammatory genes. This first demonstration of multifaceted modulation of NFκB signaling by E2 may represent a novel mechanism by which E2 protects the vasculature against inflammatory injury.
PMCID: PMC3378567  PMID: 22723832
13.  Viral dsRNA inhibitors prevent self-association and auotphosphorylation of PKR. 
Journal of molecular biology  2007;372(1):103-113.
Host response to viral RNA genomes and replication products represents an effective strategy to combat viral invasion. PKR is a Ser/Thr protein kinase that binds to dsRNA, autophosphorylates its kinase domain, and subsequently phosphorylates eukaryotic initiation factor 2α (eIF2α). This results in attenuation of protein translation, preventing synthesis of necessary viral proteins. In certain DNA viruses, PKR function can be evaded by transcription of highly structured virus-encoded dsRNA inhibitors that bind to and inactivate PKR. We probe here the mechanism of PKR inhibition by two viral inhibitor RNAs, EBERI (from Epstein-Barr) and VAI (from human adenovirus). Native gel shift mobility assays and isothermal titration calorimetry experiments confirmed that the RNA-binding domains of PKR are sufficient and necessary for the interaction with dsRNA inhibitors. Both EBERI and VAI are effective inhibitors of PKR activation by preventing trans-autophosphorylation between two PKR molecules. The RNA inhibitors prevent self-association of PKR molecules, providing a mechanistic basis for kinase inhibition. A variety of approaches indicated that dsRNA inhibitors remain associated with PKR under activating conditions, as opposed to activator dsRNA molecules that dissociate due to reduced affinity for the phosphorylated form of PKR. Finally, we show using a HeLa cell extract system that inhibitors of PKR result in translational recovery by the protein synthesis machinery. These data indicate that inhibitory dsRNAs bind preferentially to the latent, dephosphorylated form of PKR and prevent dimerization that is required for trans-autophosphorylation.
PMCID: PMC3710116  PMID: 17619024
RNA; PKR; kinase; inhibition; virus
14.  Integrated Assessment and Prediction of Transcription Factor Binding 
PLoS Computational Biology  2006;2(6):e70.
Systematic chromatin immunoprecipitation (chIP-chip) experiments have become a central technique for mapping transcriptional interactions in model organisms and humans. However, measurement of chromatin binding does not necessarily imply regulation, and binding may be difficult to detect if it is condition or cofactor dependent. To address these challenges, we present an approach for reliably assigning transcription factors (TFs) to target genes that integrates many lines of direct and indirect evidence into a single probabilistic model. Using this approach, we analyze publicly available chIP-chip binding profiles measured for yeast TFs in standard conditions, showing that our model interprets these data with significantly higher accuracy than previous methods. Pooling the high-confidence interactions reveals a large network containing 363 significant sets of factors (TF modules) that cooperate to regulate common target genes. In addition, the method predicts 980 novel binding interactions with high confidence that are likely to occur in so-far untested conditions. Indeed, using new chIP-chip experiments we show that predicted interactions for the factors Rpn4p and Pdr1p are observed only after treatment of cells with methyl-methanesulfonate, a DNA-damaging agent. We outline the first approach for consistently integrating all available evidences for TF–target interactions and we comprehensively identify the resulting TF module hierarchy. Prioritizing experimental conditions for each factor will be especially important as increasing numbers of chIP-chip assays are performed in complex organisms such as humans, for which “standard conditions” are ill defined.
Transcription factors (TFs) bind close to their target genes for regulating transcript levels depending on cellular conditions. Each gene may be regulated differently from others through the binding of specific groups of TFs (TF modules). Recently, a wide variety of large-scale measurements about transcriptional networks has become available. Here the authors present a framework for consistently integrating all of this evidence to systematically determine the precise set of genes directly regulated by each TF (i.e., TF–target interactions). The framework is applied to the yeast Saccharomyces cerevisiae using seven distinct sources of evidences to score all possible TF–target interactions in this organism. Subsequently, the authors employ another newly developed algorithm to reveal TF modules based on the top 5,000 TF–target interactions, yielding more than 300 TF modules. The new scoring scheme for TF–target interactions allows predicting the binding of TFs under so-far untested conditions, which is demonstrated by experimentally verifying interactions for two TFs (Pdr1p, Rpn4p). Importantly, the new methods (scoring of TF–target interactions and TF module identification) are scalable to much larger datasets, making them applicable to future studies in humans, which are thought to have substantially larger numbers of TF–target interactions.
PMCID: PMC1479087  PMID: 16789814
15.  Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFκB survival axis downstream of Death receptor-3 
BMC Cancer  2011;11:285.
Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells.
Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument.
Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT29 and SW620 express higher levels of a splice variant of DR3 that has no trans-membrane domain and no death domain.
Colon cancer cells acquire an increased capacity to survive via the activation of the PI3K/NFκB pathway following the stimulation of DR3 by E-selectin. Generation of a DR3 splice variant devoid of death domain can further contribute to protect against apoptosis.
PMCID: PMC3177907  PMID: 21722370
Death receptor-3; E-selectin; colon cancer; PI3 kinase; splice variant
Virology  2005;345(2):10.1016/j.virol.2005.10.003.
The long terminal repeat (LTR) region of leukemia viruses plays a critical role in tissue tropism and pathogenic potential of the viruses. We have previously reported that U3-LTR from Moloney murine and feline leukemia viruses (Mo-MuLV and FeLV) upregulate specific cellular genes in trans in an integration-independent way. The U3-LTR region necessary for this action does not encode a protein but instead makes a specific RNA transcript. Because several cellular genes transactivated by the U3-LTR can also be activated by NFκB, and because the antiapoptotic and growth promoting activities of NFκB have been implicated in leukemogenesis, we investigated whether FeLV U3-LTR can activate NFκB signaling. Here we demonstrate that FeLV U3-LTR indeed upregulates NFκB signaling pathway via activation of Ras-Raf-IκB kinase (IKK) and degradation of IκB. LTR-mediated transcriptional activation of genes did not require new protein synthesis suggesting an active role of the LTR transcript in the process. Using Toll-like receptor (TLR) deficient HEK293 cells and PKR−/− mouse embryo fibroblasts we further demonstrate that although dsRNA activated protein kinase R (PKR) is not necessary, TLR3 is required for the activation of NFκB by the LTR. Our study thus demonstrates involvement of a TLR3 dependent but PKR independent dsRNA mediated signaling pathway for NFκB activation and thus provides a new mechanistic explanation of LTR-mediated cellular gene transactivation.
PMCID: PMC3808874  PMID: 16289658
Leukemia virus; LTR; transactivation; NFκB; TLR3
17.  From Understanding Cellular Function to Novel Drug Discovery: The Role of Planar Patch-Clamp Array Chip Technology 
All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions – including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells.
PMCID: PMC3184600  PMID: 22007170
patch-clamp whole-cell recordings; planar patch-clamp chip; giga-seal; synaptic transmission; cultured neuron pair; cell placement; chemical patterning
18.  PKR promotes choroidal neovascularization via upregulating the PI3K/Akt signaling pathway in VEGF expression 
Molecular Vision  2016;22:1361-1374.
The aim of this study was to investigate the functions of dsRNA-activated protein kinase (PKR) in choroidal neovascularization (CNV) and related signaling pathways in the production of vascular endothelial growth factor (VEGF).
A chemical hypoxia model of in vitro RF/6A cells, a rhesus choroid-retinal endothelial cell line, was established by adding cobalt chloride (CoCl2) to the culture medium. PKR, phosphophosphatidylinositol 3-kinase (p-PI3K), phosphoprotein kinase B (p-Akt), and VEGF protein levels in RF/6A cells were detected with western blotting. PKR siRNA and the PI3K inhibitor LY294002 were used to evaluate the roles of the PKR and PI3K signaling pathways in VEGF expression with western blotting. In an ARPE-19 (RPE cell line) and RF/6A cell coculture system, proliferation, migration, and tube formation of RF/6A cells under hypoxic conditions were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Transwell, and Matrigel Transwell assays, respectively. In vivo CNV lesions were induced in C57BL/6J mice using laser photocoagulation. The mice were euthanized in a timely manner, and the eyecups were dissected from enucleated eyes. PKR, p-PI3K, p-Akt, and VEGF protein levels in tissues were detected with western blotting. To evaluate the leakage area, fundus fluorescein angiography and choroidal flat mount were performed on day 7 after intravitreal injection of an anti-PKR monoclonal antibody.
The in vitro RF/6A cell chemical hypoxia model showed that PKR expression was upregulated in parallel with p-PI3K, p-Akt, and VEGF expression, peaking at 12 h. PKR siRNA downregulated PKR, p-PI3K, p-Akt, and VEGF expression. In addition, the PI3K inhibitor LY294002 greatly decreased the p-PI3K, p-Akt, and VEGF protein levels, but PKR expression was unaffected, indicating that Akt was a downstream molecule of PKR that upregulated VEGF expression. In the ARPE-19 (RPE cell line) and RF/6A cell coculture system, PKR siRNA reduced the migration and tube formation of the RF/6A cells. In vivo, PKR, p-PI3K, p-Akt, and VEGF expression increased and peaked at 7 days in the mouse CNV model induced by laser photocoagulation. Furthermore, on the RPE and choroid cryosections, PKR colocalized with CD31, suggesting that PKR was expressed by the vascular endothelium. The intravitreal injection of an anti-PKR monoclonal antibody decreased the progression and leakage area of CNV in mice.
PKR promotes CNV formation via the PI3K/Akt signaling pathway in VEGF expression. Additionally, the anti-PKR monoclonal antibody significantly decreased CNV in a mouse model, showing the antibody may have therapeutic potential in human CNV.
PMCID: PMC5135740  PMID: 27994435
19.  Combined targeting of Stat3/NFκB/Cox-2/EP4 for effective management of pancreatic cancer 
Near equal rates of incidence and mortality emphasize the need for novel targeted approaches for better management of pancreatic cancer patients. Inflammatory molecules NFκB and Stat3 are overexpressed in pancreatic tumors. Inhibition of one protein allows cancer cells to survive using the other. The goal of the present study is to determine whether targeting Stat3/NFκB cross talk with a natural product Nexrutine (Nx) can inhibit inflammatory signaling in pancreatic cancer.
Experimental design
HPNE, HPNE-Ras, BxPC3, Capan-2, MIA PaCa-2 and AsPC-1 cells were tested for growth, apoptosis, Cox-2, NFκB and Stat3 level in response to Nx treatment. Transient expression, gel shift, ChIP was used to examine transcriptional regulation of Cox-2. Stat3 knockdown was used to decipher Stat3/NFκB cross talk. Histopathological and immunoblotting evaluation was performed on BK5-Cox2 transgenic mice treated with Nx. In vivo expression of prostaglandin receptor EP4 was analyzed in a retrospective cohort of pancreatic tumors using a TMA.
Nx treatment inhibited growth of pancreatic cancer cells through induction of apoptosis. Reduced levels and activity of Stat3, NFκB and their cross talk led to transcriptional suppression of Cox-2 and subsequent decreased levels of PGE2 and PGF2. Stat3 knockdown studies suggest Stat3 as negative regulator of NFκB activation. Nx intervention reduced the levels of NFκB, Stat3 and fibrosis in vivo. Expression of prostaglandin receptor EP4 that is known to play a role in fibrosis was significantly elevated in human pancreatic tumors.
Dual inhibition of Stat3-NFκB by Nx may overcome problems associated with inhibition of either pathway.
PMCID: PMC3969421  PMID: 24520096
Pancreatic cancer; Nexrutine; Stat3; NFκB; Prostaglandin receptor EP4; Cox-2
20.  Using ChIP-Seq Technology to Generate High-Resolution Profiles of Histone Modifications 
The dynamic modification of DNA and histones plays a key role in transcriptional regulation through altering the packaging of DNA and modifying the nucleosome surface. These chromatin states, also referred to as the epigenome, are distinctive for different tissues, developmental stages, and disease states and can also be altered by environmental influences. New technologies allow the genome-wide visualization of the information encoded in the epigenome. For example, the chromatin immunoprecipitation (ChIP) assay allows investigators to characterize DNA–protein interactions in vivo. ChIP followed by hybridization to microarrays (ChIP-chip) or by high-throughput sequencing (ChIP-seq) are both powerful tools to identify genome-wide profiles of transcription factors, histone modifications, DNA methylation, and nucleosome positioning. ChIP-seq technology, which can now interrogate the entire human genome at high resolution with only one lane of sequencing, has recently surpassed ChIP-chip technology for epigenomic analyses. Importantly, for the study of primary cells and tissues, epigenetic profiles can be generated using as little as 1 μg of chromatin. In this chapter, we describe in detail the steps involved in performing ChIP assays (with a focus on characterizing histone modifications in primary cells) either manually or using the IP-Star ChIP robot, followed by a detailed protocol to prepare successful libraries for Illumina sequencing. Critical quality control checkpoints are discussed. Although not a focus of this chapter, we also point the reader to several methods by which massive ChIP-seq data sets can be analyzed to extract the tremendous information contained within.
PMCID: PMC4151291  PMID: 21913086
Chromatin immunoprecipitation; ChIP-seq; Next generation sequencing; Epigenomics; Histone modifications; IP-Star; ChIP robot
21.  The Receptor Tyrosine Kinase FGFR4 Negatively Regulates NF-kappaB Signaling 
PLoS ONE  2010;5(12):e14412.
NFκB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs), including the Fibroblast Growth Factor Receptors (FGFRs) are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFκB activation has not been previously described, although FGFs have been known to antagonize TNFα-induced apoptosis.
Methodology/Principal Findings
Here, we demonstrate an interaction between FGFR4 and IKKβ (Inhibitor of NFκB Kinase β subunit), an essential component in the NFκB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen [1] and confirmed by coimmunoprecipitation and mass spectrometry analysis. We demonstrate tyrosine phosphorylation of IKKβ in the presence of activated FGFR4, but not kinase-dead FGFR4. Following stimulation by TNFα (Tumor Necrosis Factor α) to activate NFκB pathways, FGFR4 activation results in significant inhibition of NFκB signaling as measured by decreased nuclear NFκB localization, by reduced NFκB transcriptional activation in electophoretic mobility shift assays, and by inhibition of IKKβ kinase activity towards the substrate GST-IκBα in in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFα-treated DU145 prostate cancer cells also leads to a decrease in IKKβ activity, concomitant reduction in NFκB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFα treatment of DU145 cells, in comparison with TNFα alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFκB signaling.
These results identify a compelling link between FGFR4 signaling and the NFκB pathway, and reveal that FGFR4 activation leads to a negative effect on NFκB signaling including an inhibitory effect on proapoptotic signaling. We anticipate that this interaction between an RTK and a component of NFκB signaling will not be limited to FGFR4 alone.
PMCID: PMC3008709  PMID: 21203561
22.  Role of ChIP-seq in the discovery of transcription factor binding sites, differential gene regulation mechanism, epigenetic marks and beyond 
Cell Cycle  2014;13(18):2847-2852.
Many biologically significant processes, such as cell differentiation and cell cycle progression, gene transcription and DNA replication, chromosome stability and epigenetic silencing etc. depend on the crucial interactions between cellular proteins and DNA. Chromatin immunoprecipitation (ChIP) is an important experimental technique for studying interactions between specific proteins and DNA in the cell and determining their localization on a specific genomic locus. In recent years, the combination of ChIP with second generation DNA-sequencing technology (ChIP-seq) allows precise genomic functional assay. This review addresses the important applications of ChIP-seq with an emphasis on its role in genome-wide mapping of transcription factor binding sites, the revelation of underlying molecular mechanisms of differential gene regulation that are governed by specific transcription factors, and the identification of epigenetic marks. Furthermore, we also describe the ChIP-seq data analysis workflow and a perspective for the exciting potential advancement of ChIP-seq technology in the future.
PMCID: PMC4614920  PMID: 25486472
chromatin; high throughput; immunoprecipitation; sequencing
23.  Regulation of heat shock protein 60 and 72 expression in the failing heart 
Heart failure, a progressive, fatal disease of the heart muscle, is a state of chronic inflammation and injury. Heat shock protein (HSP) 72, a ubiquitous protective protein that is well-established as cardioprotective, is not increased in heart failure. In contrast, HSP60 levels are doubled in the failing heart. We hypothesized that HSF-1 is not activated in heart failure and that the increased expression of HSP60 was driven by NFκB activation. To test this hypothesis, we measured levels of heat shock factor (HSF) −1 and −2, the transcription factors controlling HSP expression, which were increased in heart failure. There was no increased phosphorylation of serine 230 or serine 303/307 in HSF-1, which are thought to regulate its activity; EMSA showed no increase in HSF binding activity with heart failure. Nonetheless, mRNA was increased for HSP60, but not HSP72. In contrast to HSF, NFκB activity was increased in heart failure. HSP60, but not HSP72, contained NFκB binding elements. ChIP assay demonstrated increased binding of NFκB to both of the NFκB binding elements in the heart failure HSP60 gene. TNFα treatment was used to test the role of NFκB activation in HSP60 expression in a cardiac cell line. TNFα increased HSP60 expression, and this could be prevented by pretreatment with siRNA inhibiting p65 expression. In conclusion, HSP72 is not increased in heart failure because HSF activity is not changed; increased expression of HSP60 may be driven by NFκB activation.
PMCID: PMC2814075  PMID: 19945465
HSP60; HSP72; Heart failure; NFκB; ERK 1/2; GSK
24.  Role of PKR and Type I IFNs in Viral Control during Primary and Secondary Infection 
PLoS Pathogens  2010;6(6):e1000966.
Type I interferons (IFNs) are known to mediate viral control, and also promote survival and expansion of virus-specific CD8+ T cells. However, it is unclear whether signaling cascades involved in eliciting these diverse cellular effects are also distinct. One of the best-characterized anti-viral signaling mechanisms of Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. Here, we have investigated the role of PKR and Type I IFNs in regulating viral clearance and CD8+ T cell response during primary and secondary viral infections. Our studies demonstrate differential requirement for PKR, in viral control versus elicitation of CD8+ T cell responses during primary infection of mice with lymphocytic choriomeningitis virus (LCMV). PKR-deficient mice mounted potent CD8+ T cell responses, but failed to effectively control LCMV. The compromised LCMV control in the absence of PKR was multifactorial, and linked to less effective CD8+ T cell-mediated viral suppression, enhanced viral replication in cells, and lower steady state expression levels of IFN-responsive genes. Moreover, we show that despite normal expansion of memory CD8+ T cells and differentiation into effectors during a secondary response, effective clearance of LCMV but not vaccinia virus required PKR activity in infected cells. In the absence of Type I IFN signaling, secondary effector CD8+ T cells were ineffective in controlling both LCMV and vaccinia virus replication in vivo. These findings provide insight into cellular pathways of Type I IFN actions, and highlight the under-appreciated importance of innate immune mechanisms of viral control during secondary infections, despite the accelerated responses of memory CD8+ T cells. Additionally, the results presented here have furthered our understanding of the immune correlates of anti-viral protective immunity, which have implications in the rational design of vaccines.
Author Summary
Type I interferons (IFNs) constitute the first line of defense against viral infections, promote antigen presentation by dendritic cells, and play a crucial role in directly stimulating anti-viral T cell responses. However, the mechanisms underlying the diverse cellular effects of Type I IFNs are not well defined. One of the best-characterized anti-viral signaling mechanisms induced by Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. We show that requirement for cellular PKR activity could be a distinguishing feature between Type I IFN actions that mediate viral control or stimulate CD8+ T cell expansion during an acute infection with lymphocytic choriomeningitis virus (LCMV). Typically, innate immune mechanisms including Type I IFNs are considered important for viral control during a primary infection. However, we find that presence of vaccine-induced CD8+ T cell memory and accelerated generation of secondary effectors are necessary but not sufficient to provide effective protective immunity to re-infection, without the aid of innate effectors PKR and Type I IFNs. These findings have improved our understanding of virus-immune system interactions and immune correlates of anti-viral protective immunity, which might have implications in the development of effective anti-viral vaccines and immunotherapies.
PMCID: PMC2891951  PMID: 20585572
25.  Site-specific chromatin immunoprecipitation: a selective method to individually analyze neighboring transcription factor binding sites in vivo 
BMC Research Notes  2012;5:109.
Transcription factors (TFs) and their binding sites (TFBSs) play a central role in the regulation of gene expression. It is therefore vital to know how the allocation pattern of TFBSs affects the functioning of any particular gene in vivo. A widely used method to analyze TFBSs in vivo is the chromatin immunoprecipitation (ChIP). However, this method in its present state does not enable the individual investigation of densely arranged TFBSs due to the underlying unspecific DNA fragmentation technique. This study describes a site-specific ChIP which aggregates the benefits of both EMSA and in vivo footprinting in only one assay, thereby allowing the individual detection and analysis of single binding motifs.
The standard ChIP protocol was modified by replacing the conventional DNA fragmentation, i. e. via sonication or undirected enzymatic digestion (by MNase), through a sequence specific enzymatic digestion step. This alteration enables the specific immunoprecipitation and individual examination of occupied sites, even in a complex system of adjacent binding motifs in vivo. Immunoprecipitated chromatin was analyzed by PCR using two primer sets - one for the specific detection of precipitated TFBSs and one for the validation of completeness of the enzyme digestion step. The method was established exemplary for Sp1 TFBSs within the egfr promoter region. Using this site-specific ChIP, we were able to confirm four previously described Sp1 binding sites within egfr promoter region to be occupied by Sp1 in vivo. Despite the dense arrangement of the Sp1 TFBSs the improved ChIP method was able to individually examine the allocation of all adjacent Sp1 TFBS at once. The broad applicability of this site-specific ChIP could be demonstrated by analyzing these SP1 motifs in both osteosarcoma cells and kidney carcinoma tissue.
The ChIP technology is a powerful tool for investigating transcription factors in vivo, especially in cancer biology. The established site-specific enzyme digestion enables a reliable and individual detection option for densely arranged binding motifs in vivo not provided by e.g. EMSA or in vivo footprinting. Given the important function of transcription factors in neoplastic mechanism, our method enables a broad diversity of application options for clinical studies.
PMCID: PMC3312844  PMID: 22348285

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