Supershift Electrophoretic Mobility Shift Assay Research Articles

An electrophoretic mobility shift assay using the protein isolated from host plants

BackgroundThe electrophoretic mobility shift assay (EMSA) is a common technology to detect DNA-protein interactions. However, in most cases, the protein used in EMSA is obtained via prokaryotic expression, and rarely from plants. At the same time, the proteins expressed from prokaryotic systems usually cannot fold naturally and have no post translationally modification, which may affect the binding of proteins to DNA.ResultsHere, we develop a technique to quickly isolate proteins of interest from host plants and then analyze them using fluorescent EMSA. This technology system is called: protein from plants fluorescent EMSA method (PPF-EMSA). In PPF-EMSA, a special transient transformation method is employed to transiently deliver genes into the plant, enabling efficient synthesis the encoded proteins. Then, the target protein is isolated using immunoprecipitation, and the DNA probes were labeled with cyanine 3 (Cy3). Both fluorescent EMSA and super-shift fluorescent EMSA can be performed using the proteins from plants. Three kinds of plants, Betula platyphylla, Populus. davidiana×P. bolleana and Arabidopsis thaliana, are used in this study. The proteins isolated from plants are in a natural state, can fold naturally and are posttranslationally modified, enabling true binding to their cognate DNAs.ConclusionAs transient transformation can be performed quickly and not depended on whether stable transformation is available or not, we believe this method will have a wide application, enabling isolation of proteins from host plant conveniently.

Transcriptional Regulation of Vih by Oct4 and Sox9 in Scylla paramamosain.

Mud crab (Scylla paramamosain) is one of the most economically-important marine crabs in China. However, research on mechanisms of reproductive regulation is not sufficient. Vitellogenesis-inhibiting hormone (VIH) is a member of the crustacean hyperglycemia hormones (CHH) family, which plays an essential role in the regulation of gonadal development and maturation in crustaceans, and current studies on the regulation of Vih transcription in crabs are relatively rare. Our previous studies on the transcriptional regulation of mud crab Vih (SpVih) have proved that the binding site of Oct4/Sox9 transcription factor may be the key region for positively regulating the expression of SpVih. In this study, the electrophoretic mobility shift assay (EMSA) experiment confirmed that the nuclear protein extracted from the eyestalk could bind to the key region of SpVih promoter, and these specific bindings were dependent on the presence of Oct4/Sox9 binding sites. Two specific binding complex bands were detected in the supershift group of EMSA supershift experiments by Oct4 and Sox9 antibodies, further confirming the specific recognition of these two transcription factors on the key regulatory region of SpVih. In vitro, Oct4 and Sox9 gene overexpression vectors and SpVih core promoter fragment vector were constructed and co-transfected into HEK293T cells. As a result, SpVih activity increased with the concentration of transcription factors. In vivo, when Oct4 and Sox9 dsRNA were injected into the eyestalks of mud crab, respectively, the expression level of SpVih decreased significantly after interference with Oct4 or Sox9, and the expression level of SpVtg in the ovary and hepatopancreatic increased. Both in vitro and in vivo experiments showed that Oct4 and Sox9 had a positive regulatory effect on SpVih. The GST pull-down experiment was carried out by purified Oct4 and Sox9 proteins, and the results showed that there was an interaction between them. It was speculated that they regulated the expression of SpVih through the interaction.

Lead Neurotoxicity on Human Neuroblastoma Cell Line SH-SY5Y is Mediated via Transcription Factor EGR1/Zif268 Induced Disrupted in Scherophernia-1 Activation.

Lead (Pb2+) is a well-known type of neurotoxin and chronic exposure to Pb2+ induces cognition dysfunction. In this work, the potential role of early growth response gene 1 (EGR1) in the linkage of Pb2+ exposure and disrupted in scherophernia-1 (DISC1) activity was investigated. Human neuroblastoma cell line SH-SY5Y was subjected to different concentrations of lead acetate (PbAc) to determine the effect of Pb2+ exposure on the cell viability, apoptosis, and activity of EGR1 and DISC1. Then the expression of EGR1 in SH-SY5Y cells was knocked down with specific siRNA to assess the function of EGR1 in Pb2+ induced activation of DISC1. The interaction between EGR1 and DISC1 was further validated with dual luciferase assay, Supershift electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP)-PCR. Administration of PbAc decreased cell viability and induced apoptosis in SH-SY5Y cells in a dose-dependent manner. Additionally, exposure to PbAc also up-regulated expression of EGR1 and DISC1 at all concentrations. Knockdown of EGR1 blocked the effect of PbAc on SH-SY5Y cells, indicating the central role of EGR1 in the function of Pb2+ on activity of DISC1. Based on the results of dual luciferase assay, Supershift EMSA, and ChIP-PCR, EGR1 mediated the effect of Pb2+ on DISC1 by directly bound to the promoter region of DISC1 gene. The current study elaborated the mechanism involved in the effect of Pb2+ exposure on expression of DISC1 for the first time: EGR1 activated by Pb2+ substitution of zinc triggered the transcription of DISC1 gene by directly binding to its promoter.

Regulation of Antimicrobial Peptides in Aedes aegypti Aag2 Cells.

Antimicrobial peptides (AMPs) are an important group of immune effectors that play a role in combating microbial infections in invertebrates. Most of the current information on the regulation of insect AMPs in microbial infection have been gained from Drosophila, and their regulation in other insects are still not completely understood. Here, we generated an AMP induction profile in response to infections with some Gram-negative, -positive bacteria, and fungi in Aedes aegypti embryonic Aag2 cells. Most of the AMP inductions caused by the gram-negative bacteria was controlled by the Immune deficiency (Imd) pathway; nonetheless, Gambicin, an AMP gene discovered only in mosquitoes, was combinatorially regulated by the Imd, Toll and JAK-STAT pathways in the Aag2 cells. Gambicin promoter analyses including specific sequence motif deletions implicated these three pathways in Gambicin activity, as shown by a luciferase assay. Moreover, the recognition between Rel1 (refer to Dif/Dorsal in Drosophila) and STAT and their regulatory sites at the Gambicin promoter site was validated by a super-shift electrophoretic mobility shift assay (EMSA). Our study provides information that increases our understanding of the regulation of AMPs in response to microbial infections in mosquitoes. And it is a new finding that the A. aegypti AMPs are mainly regulated Imd pathway only, which is quite different from the previous understanding obtained from Drosophila.

A novel kindred with inherited STAT2 deficiency and severe viral illness

A novel kindred with inherited STAT2 deficiency and severe viral illness

Novel Synthetic Promoters from the Cestrum Yellow Leaf Curling Virus.

Constitutive promoters direct gene expression uniformly in most tissues and cells at all stages of plant growth and development; they confer steady levels of transgene expression in plant cells and hence their demand is high in plant biology. The gene silencing due to promoter homology can be avoided by either using diverse promoters isolated from different plant and viral genomes or by designing synthetic promoters. The aim of this chapter was to describe the basic protocols needed to develop and analyze novel, synthetic, nearly constitutive promoters from Cestrum yellow leaf curling virus (CmYLCV) through promoter/leader deletion and activating cis-sequence analysis. We also describe the methods to evaluate the strength of the promoters efficiently in various transient expression systems like agroinfiltration assay, gene-gun method, and assay in tobacco protoplasts. Besides, the detailed methods for developing transgenic plants (tobacco and Arabidopsis) for evaluation of the promoter using the GUS reporter gene are also described. The detailed procedure for electrophoretic mobility shift assay (EMSA) coupled with super-shift EMSA analysis are also described for showing the binding of tobacco transcription factor, TGA1a to cis-elements in the CmYLCV distal promoter region.

Human Obesity Associated with an Intronic SNP in the Brain-Derived Neurotrophic Factor Locus.

Brain-derived neurotrophic factor (BDNF) plays a key role in energy balance. In population studies, SNPs of the BDNF locus have been linked to obesity, but the mechanism by which these variants cause weight gain is unknown. Here, we examined human hypothalamic BDNF expression in association with 44 BDNF SNPs. We observed that the minor C allele of rs12291063 is associated with lower human ventromedial hypothalamic BDNF expression (p < 0.001) and greater adiposity in both adult and pediatric cohorts (p values < 0.05). We further demonstrated that the major T allele for rs12291063 possesses a binding capacity for the transcriptional regulator, heterogeneous nuclear ribonucleoprotein D0B, knockdown of which disrupts transactivation by the T allele. Binding and transactivation functions are both disrupted by substituting C for T. These findings provide a rationale for BDNF augmentation as a targeted treatment for obesity in individuals who have the rs12291063 CC genotype.

A Region Containing an as-1 Element of Dahlia Mosaic Virus (DaMV) Subgenomic Transcript Promoter Plays a Key Role in Green Tissue- and Root-Specific Expression in Plants

A subgenomic transcript (Sgt) promoter was isolated from the genomic clone of dahlia mosaic virus (DaMV), which is a double-stranded DNA virus of the Caulimoviridae family. The DaMVSgt promoter, which is linked to the heterologous β-glucuronidase (GUS) reporter gene, was characterized in transient protoplasts and in transgenic tobacco, as well as in Arabidopsis plants. The 5′- and 3′-deletion analysis of a 591-bp DaMVSgt promoter fragment indicated that a 441-bp promoter fragment (−372 to +69 from the transcription start site; TSS) was sufficient for maximal promoter activity. A 141-bp promoter fragment (−72 to +69 from TSS) was the minimal promoter region that also showed relatively strong activity. The three activation sequence-1 (as-1) elements and the border regions were primarily responsible for the promoter activity, as revealed by a finer internal deletion and mutation analysis of the cis-elements and of the immediate border sequence of the activation domain. Electrophoretic mobility shift assay (EMSA), supershift EMSA, DNase I footprinting, Southwestern blotting, and UV cross-linking studies demonstrated the binding of a tobacco transcription factor, TGA1a, that correlated with 2,4-dichlorophenylacetic acid (2,4D)-induced transcriptional activity of the DaMVSgt promoter. Histological GUS staining and the GUS enzymatic assay demonstrated that the 441-bp DaMVSgt4 promoter and 141-bp minimal DaMVSgt4F are 5.5 and 4.6 times, respectively, stronger than the CaMV 35S promoter. The minimal DaMVSgt4F promoter is more active than CaMV 35S in all types of green tissues and roots, without any detectable expression in reproductive tissues and seeds. The DaMVSgt4F promoter may be useful for transgene containment applications.

HNRNPA1 interacts with a 5′-flanking distal element of interleukin-6 and upregulates its basal transcription

Interleukin-6 (IL-6) is an important pro-inflammatory cytokine involved in many autoimmune and inflammatory diseases. We have shown previously that a region from −5307 to −5202 bp upstream of the IL-6 transcriptional start site is responsible for basal IL-6 gene expression and that there were DNA binding proteins involved from EMSA and transient expression experiments. Here we have combined surface plasmon resonance technology with mass spectrometry analysis and identified nuclear proteins bound to this region. HNRNPA1 and HNRNPA2B1 were found consistently. EMSA supershift and chromatin immunoprecipitation assays confirmed the involvement of HNRNPA1, but not HNRNPA2B1. Knocking down HNRNPA1 expression by siRNA resulted in reduced IL-6 transcriptional activity as assessed from transfection experiments using reporter constructs, mRNA and protein measurements. Overexpression of HNRNPA1 cDNA increased IL-6 mRNA expression. This regulation was dependent on the presence of the sequence from −5307 to −5202 bp of the IL-6 gene. Thus, HNRNPA1 is a novel transcriptional regulator of IL-6 expression, acting via the 5′ flanking sequence of the gene.

The Identification of Trans-acting Factors That Regulate the Expression of GDF5 via the Osteoarthritis Susceptibility SNP rs143383

rs143383 is a C to T transition SNP located in the 5′untranslated region (5′UTR) of the growth differentiation factor 5 gene GDF5. The T allele of the SNP is associated with increased risk of osteoarthritis (OA) in Europeans and in Asians. This susceptibility is mediated by the T allele producing less GDF5 transcript relative to the C allele, a phenomenon known as differential allelic expression (DAE). The aim of this study was to identify trans-acting factors that bind to rs143383 and which regulate this GDF5 DAE. Protein binding to the gene was investigated by two experimental approaches: 1) competition and supershift electrophoretic mobility shift assays (EMSAs) and 2) an oligonucleotide pull down assay followed by quantitative mass spectrometry. Binding was then confirmed in vivo by chromatin immunoprecipitation (ChIP), and the functional effects of candidate proteins investigated by RNA interference (RNAi) and over expression. Using these approaches the trans-acting factors Sp1, Sp3, P15, and DEAF-1 were identified as interacting with the GDF5 5′UTR. Knockdown and over expression of the factors demonstrated that Sp1, Sp3, and DEAF-1 are repressors of GDF5 expression. Depletion of DEAF-1 modulated the DAE of GDF5 and this differential allelic effect was confirmed following over expression, with the rs143383 T allele being repressed to a significantly greater extent than the rs143383 C allele. In combination, Sp1 and DEAF-1 had the greatest repressive activity. In conclusion, we have identified four trans-acting factors that are binding to GDF5, three of which are modulating GDF5 expression via the OA susceptibility locus rs143383.

Identification of non-canonical NF-κB signaling as a critical mediator of Smac mimetic-stimulated migration and invasion of glioblastoma cells

As inhibitor of apoptosis (IAP) proteins can regulate additional signaling pathways beyond apoptosis, we investigated the effect of the second mitochondrial activator of caspases (Smac) mimetic BV6, which antagonizes IAP proteins, on non-apoptotic functions in glioblastoma (GBM). Here, we identify non-canonical nuclear factor-κB (NF-κB) signaling and a tumor necrosis factor-α (TNFα)/TNF receptor 1 (TNFR1) autocrine/paracrine loop as critical mediators of BV6-stimulated migration and invasion of GBM cells. In addition to GBM cell lines, BV6 triggers cell elongation, migration and invasion in primary, patient-derived GBM cells at non-toxic concentrations, which do not affect cell viability or proliferation, and also increases infiltrative tumor growth in vivo underscoring the relevance of these findings. Molecular studies reveal that BV6 causes rapid degradation of cellular IAP proteins, accumulation of NIK, processing of p100 to p52, translocation of p52 into the nucleus, increased NF-κB DNA binding and enhanced NF-κB transcriptional activity. Electrophoretic mobility shift assay supershift shows that the NF-κB DNA-binding subunits consist of p50, p52 and RelB further confirming the activation of the non-canonical NF-κB pathway. BV6-stimulated NF-κB activation leads to elevated mRNA levels of TNFα and additional NF-κB target genes involved in migration (i.e., interleukin 8, monocyte chemoattractant protein 1, CXC chemokine receptor 4) and invasion (i.e., matrix metalloproteinase-9). Importantly, inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor prevents the BV6-stimulated cell elongation, migration and invasion. Similarly, specific inhibition of non-canonical NF-κB signaling by RNA interference-mediated silencing of NIK suppresses the BV6-induced cell elongation, migration and invasion as well as upregulation of NF-κB target genes. Intriguingly, pharmacological or genetic inhibition of the BV6-stimulated TNFα autocrine/paracrine loop by the TNFα-blocking antibody Enbrel or by knockdown of TNFR1 abrogates BV6-induced cell elongation, migration and invasion. By demonstrating that the Smac mimetic BV6 at non-toxic concentrations promotes migration and invasion of GBM cells via non-canonical NF-κB signaling, our findings have important implications for the use of Smac mimetics as cancer therapeutics.

Elevated hepatic multidrug resistance-associated protein 3/ATP-binding cassette subfamily C 3 expression in human obstructive cholestasis is mediated through tumor necrosis factor alpha and c-Jun NH2-terminal kinase/stress-activated protein kinase-signali

Multidrug resistance-associated protein 3 (MRP3, ABC subfamily C [ABCC]3) plays an important role in protecting hepatocytes and other tissues by excreting an array of toxic organic anion conjugates, including bile salts. MRP3/ABCC3 expression is increased in the liver of some cholestatic patients, but the molecular mechanism of this up-regulation remains elusive. In this report, we assessed liver MRP3/ABCC3 expression in patients (n = 22) with obstructive cholestasis caused by gallstone blockage of bile ducts and noncholestatic patient controls (n = 22). MRP3/ABCC3 messenger RNA (mRNA) and protein expression were significantly increased by 3.4- and 4.6-fold, respectively, in these cholestatic patients where elevated plasma tumor necrosis factor alpha (TNFα) (4.7-fold; P < 0.01) and hepatic specificity protein 1 transcription factor (SP1) and liver receptor homolog 1 expression (3.1- and 2.1-fold at mRNA level, 3.5- and 2.5-fold at protein level, respectively) were also observed. The induction of hepatic MRP3/ABCC3 mRNA expression is significantly positively correlated with the level of plasma TNFα in these patients. In HepG2 cells, TNFα treatment induced SP1 and MRP3/ABCC3 expression in a dose- and time-dependent manner, where increased phosphorylation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) was also detected. These inductions were significantly reduced in the presence of the JNK inhibitor, SP600125. TNFα treatment enhanced HepG2 cell nuclear extract-binding activity to the MRP3/ABCC3 promoter, but was abolished by SP600125, as demonstrated by electrophoretic mobility shift assay (EMSA). An increase in nuclear protein-binding activity to the MRP3/ABCC3 promoter, consisting primarily of SP1, was also observed in liver samples from cholestatic patients, as assessed by supershift EMSA assays. Our findings indicate that up-regulation of hepatic MRP3/ABCC3 expression in human obstructive cholestasis is likely triggered by TNFα, mediated by activation of JNK/SAPK and SP1.

Identification of Transcription Factors Regulating CTNNAL1 Expression in Human Bronchial Epithelial Cells

Adhesion molecules play important roles in airway hyperresponsiveness or airway inflammation. Our previous study indicated catenin alpha-like 1 (CTNNAL1), an alpha-catenin-related protein, was downregulated in asthma patients and animal model. In this study, we observed that the expression of CTNNAL1 was increased in lung tissue of the ozone-stressed Balb/c mice model and in acute ozone stressed human bronchial epithelial cells (HBEC). In order to identify the possible DNA-binding proteins regulating the transcription of CTNNAL1 gene in HBEC, we designed 8 oligo- nucleotide probes corresponding to various regions of the CTNNAL1 promoter in electrophoretic mobility shift assays (EMSA). We detected 5 putative transcription factors binding sites within CTNNAL1 promoter region that can recruit LEF-1, AP-2α and CREB respectively by EMSA and antibody supershift assay. Chromatin immunoprecipitation (ChIP) assay verified that AP-2 α and LEF-1 could be recruited to the CTNNAL1 promoter. Therefore we further analyzed the functions of putative AP-2 and LEF-1 sites within CTNNAL1 promoter by site-directed mutagenesis of those sites within pGL3/FR/luc. We observed a reduction in human CTNNAL1 promoter activity of mutants of both AP-2α and LEF-1 sites. Pre-treatment with ASOs targeting LEF-1and AP-2α yielded significant reduction of ozone-stress-induced CTNNAL1 expression. The activation of AP-2α and LEF-1, followed by CTNNAL1 expression, showed a correlation during a 16-hour time course. Our data suggest that a robust transcriptional CTNNAL1 up-regulation occurs during acute ozone-induced stress and is mediated at least in part by ozone-induced recruitments of LEF-1 and AP-2α to the human CTNNAL1 promoter.

Promoter cloning and characterization of the human programmed cell death protein 4 (pdcd4) gene: evidence for ZBP-89 and Sp-binding motifs as essential Pdcd4 regulators

Pdcd4 (programmed cell death protein 4) is an important novel tumour suppressor inhibiting transformation, translation, invasion and intravasation, and its expression is down-regulated in several cancers. However, little is known about the transcriptional regulation and the promoter of this important tumour suppressor. So far the following is the first comprehensive study to describe the regulation of Pdcd4 transcription by ZBP-89 (zinc-finger-binding protein 89), besides characterizing the gene promoter. We identified the transcriptional start sites of the human pdcd4 promoter, a functional CCAAT-box, and the basal promoter region. Within this basal region, computer-based analysis revealed several potential binding sites for ZBPs, especially for Sp (specificity protein) family members and ZBP-89. We identified four Sp1/Sp3/Sp4-binding elements to be indispensable for basal promoter activity. However, overexpression of Sp1 and Sp3 was not sufficient to enhance Pdcd4 protein expression. Analysis in different solid cancer cell lines showed a significant correlation between pdcd4 and zbp-89 mRNA amounts. In contrast with Sp transcription factors, overexpression of ZBP-89 led to an enhanced expression of Pdcd4 mRNA and protein. Additionally, specific knockdown of ZBP-89 resulted in a decreased pdcd4 gene expression. Reporter gene analysis showed a significant up-regulation of basal promoter activity by co-transfection with ZBP-89, which could be abolished by mithramycin treatment. Predicted binding of ZBP-89 to the basal promoter was confirmed by EMSA (electrophoretic mobility-shift assay) data and supershift analysis for ZBP-89. Taken together, data for the first time implicate ZBP-89 as a regulator of Pdcd4 by binding to the basal promoter either alone or by interacting with Sp family members.

The role of oct-1 in the regulation of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) expression in bovine mammary epithelial cells

Lingual antimicrobial peptide (LAP) and tracheal antimicrobial peptide (TAP) are two important β-defensins of antimicrobial peptide family, which are evolutionarily conserved effector molecules of the innate immune response. Although known to be sensitive to pathogenic challenge, the control of their expression remains unclear. Both LAP and TAP genes showed constitutive and inducible expression in bovine mammary epithelial tissues, and the aim of this study was to investigate the mechanisms underlying their expression and regulation. Reporter plasmids fused with 5' regions of the two gene promoter regions were constructed and transiently transfected into a bovine mammary epithelial (BME) cell line. Initial serial deletion of the promoter regions from both genes identified two positive regulatory elements within the 1 kb regions upstream the transcription start sites, which co-operatively contribute to LAP and TAP gene expression. Further luciferase reporter assays revealed that an enhancer and a 61-bp region proximal to both genes are important for basal expression and regulation of transcription. Electrophoretic mobility shift assays (EMSA) indicated the involvement of the Oct-1 protein-DNA complex in regulating the promoter activity, which was confirmed by super shift EMSA with Oct-1 antibody and by knockdown of Oct-1 with small interfering RNA. The Oct-1 binding motif was also shown to be responsive to phorbol 12-myristate 13-acetate but not LPS stimulation. The results from this study clearly demonstrate the involvement of the Oct-1 transcription factor in the regulation of LAP and TAP expression.

NF-κB Is Constitutively Activated in EBV-Infected T or NK Cells and Can Be a Molecular Target for EBV-Positive T/NK-Cell Lymphoproliferative Disease Treatment

AbstractAbstract 1998Epstein-Barr virus (EBV) can infect not only B cells but also rarely T or NK cells and causes EBV-positive T/NK-cell lymphoproliferative disease (EBV-T/NK-LPD), such as extranodal NK/T-cell lymphoma (ENKL), aggressive NK-cell leukemia, and chronic active EBV infection (CAEBV). However, why and how EBV infects T or NK cells and the mechanism of action responsible for the development of these EBV-induced malignancies have not been elucidated to date. Furthermore, optimal chemotherapy for these, especially for CAEBV, has not been established and the prognosis of EBV-T/NK-LPD remains very poor. NF-kB is a transcription factor that mediates anti-apoptotic molecular signaling and promotes proliferation of cancer cells. NF-kB is known to be constitutively activated in some hematological malignancies; the proteasome inhibitor bortezomib suppresses its activity and is used clinically as an anticancer reagent. To clarify the molecular mechanism underlying the development of EBV-T/NK-LPD, we focused on NF-kB in four EBV-positive T- and NK-cell lines, SNT8, SNT15, SNT16, and SNK6, which had been established from primary lesions of ENKL patients (SNT8 and SNK6) and the peripheral blood (PB) of a CAEBV patient (SNT15, 16), respectively. In these cells, it was demonstrated that p50, p52, RelA, and RelB—the components of NF-kB—existed constitutively in the nucleus. On the other hand, the EBV-negative T-cell lines, Jurkat and Molt4, and an NK-cell line, KHYG1, were negative for nuclear localization of these molecules. Elevated nuclear NF- kB–DNA binding activity was demonstrated by electrophoretic mobility shift assay (EMSA), using oligonucleotides encoding an NF-kB-binding sequence as a probe. Supershift EMSA revealed that NF- kB–DNA binding complexes in these cells involved p50, p52, and RelA. In addition, reporter assay using NF-kB-dependent luciferase reporter gene consisting of an NF-kB-binding site as a promoter demonstrated constitutive activation of NF-kB in SNT8 cells, whereas not in Jurkat cells. From these results, we concluded that NF-kB was constitutively activated through the canonical and noncanonical pathways in these EBV-infected T- or NK-cell (EBV-T/NK cell) lines. Next, we investigated NF-kB activation in EBV-T/NK cells derived from CAEBV patients. CAEBV was diagnosed according to the following criteria: presence of persistent infectious mononucleosis-like symptoms, elevation of EBV-DNA titer in PB, and detection of EBV-infected T or NK cells with clonal proliferation. To detect infected cells, we isolated peripheral mononuclear cells and divided them into CD19-, CD4-, CD8-, or CD56-positive fractions using antibody-conjugated magnetic beads. The EBV DNA of each fraction was quantified using a real-time quantitative polymerase chain reaction assay. Ten patients (aged 8–72 years; 4 male, 6 female; infected cell types CD4:3, CD8:3, γδ:1, and CD56:3) were diagnosed as CAEBV. Then, we examined NF-kB activation in these cells. Similar to the cell lines, immunoblotting and EMSA demonstrated evidence of constitutive activation of NF-kB such as nuclear localization and DNA binding of p50, p52, and RelA in EBV-T/NK cells from CAEBV patients. To investigate the molecular mechanism of NF-kB activation in EBV-T/NK cells, we examined the influence of viral proteins expressed in EBV-T/NK cells, including LMP1, LMP2A, LMP2B, and EBNA1 (latency type 2). We performed luciferase reporter assay using expression vectors for these proteins in Jurkat cells. LMP1, most significantly, and LMP2A, to a lesser extent, upregulated NF-kB-dependent reporter gene expression in Jurkat cells, whereas the other viral proteins did not, suggesting that LMP1 and LMP2A mediated NF-kB activation in T/NK cells. Finally we investigated the contribution of NF-kB to the development of EBV-T/NK-LPD. Bortezomib, in a dose-dependent manner, inhibited translocation of p50, p52, and RelA to the nucleus and reduced the expression of Bcl-XL, which was a transcriptional target of NF-kB in SNT8, SNK6, and CAEBV patients' cells. In addition, it was also confirmed that bortezomib (0.5nM -5nM) suppressed the survival of and induced apoptosis of EBV-T/NK cells derived from 5 CAEBV patients. These results suggest that constitutive activation of NF-kB contributes to the development of EBV-T/NK-LPD and that the NF-kB signaling pathway can be an attractive molecular target for treatment. Disclosures:No relevant conflicts of interest to declare.

Application of statistical and functional methodologies for the investigation of genetic determinants of coronary heart disease biomarkers: lipoprotein lipase genotype and plasma triglycerides as an exemplar

Genome-wide association studies have proved very successful in identifying novel single-nucleotide polymorphisms (SNPs) associated with disease or traits, but the related, functional SNP is usually unknown. In this paper, we describe a methodology to locate and validate candidate functional SNPs using lipoprotein lipase (LPL), a gene previously associated with triglyceride levels, as an exemplar. Two thousand seven hundred and eighty-six healthy middle-aged men from the NPHSII UK prospective study (with up to six measures of plasma lipid levels) were genotyped for 20 LPL tagging (t)SNPs using Illumina Bead technology. Using model-selection procedures and haplotypes, we identified eight SNPs that consistently maximized the fit of the model to the phenotype. Fifteen SNPs in high linkage disequilibrium with these were identified, and functional assays were carried out on all 23 SNPs. Electrophoretic mobility shift assay (EMSA) was used to identify SNPs that had the potential to alter DNA–protein interactions, reducing the number to eight possible candidate SNPs. These were examined for ability to alter expression using a luciferase reporter assay, and two regulatory SNPs, showing genotype differences, rs327 and rs3289, were identified. Finally, multiplexed-competitor-EMSA (MC-EMSA) and supershift EMSA identified FOXA2 to rs327T, and CREB-binding protein (CBP) and CCAAT displacement protein (CDP) to rs3289C as the factors responsible for transcription binding. We have identified two novel candidate functional SNPs in LPL and presented a procedure aimed to efficiently detect SNPs potentially causal to genetic association. We believe that this methodology could be successfully applied to future re-sequencing data.

Principles and problems of the electrophoretic mobility shift assay

Introduction The electrophoretic mobility shift assay (EMSA) is classically used to detect DNA binding proteins, the tenet of the EMSA is that DNA with protein bound, migrates through a polyacrylamide gel more slowly than the corresponding free unbound DNA. Methods The classical EMSA protocol has 4 major steps: 1) The isolation of proteins from cells. Since the vast majority of active DNA binding proteins are present within the nucleus, a sequential membrane lysis protocol is used which yields purified nuclear protein. 2) Manufacture and radiolabelling of the DNA probe. Phosphorous 32 ( 32P) is attached to the 5′ ends of the DNA probe through use of 32P-γATP as a substrate for T4 polynucleotide kinase. DNA probes can both be purchased or custom made. 3) Purified proteins and radiolabelled DNA probes are co-incubated with an EMSA binding buffer to promote binding of the proteins with the DNA probe. If a supershift EMSA is being carried out, the reaction also contains a selective antibody which when bound to the protein–DNA complexes, causes further retardation within the gel. 4) The DNA–protein complexes are loaded and run on a non-denaturing polyacrylamide gel causing separation of the DNA–protein complexes from the free DNA probes. The polyacrylamide gels are then dried down and analysed via autoradiography. Results As a demonstration of the effectiveness of this protocol, we show that tumour necrosis factor (TNF)α and phorbol 12-myristate 13-acetate (PMA) stimulation of A549 cells, results in a number of DNA–protein complexes being induced when compared to untreated cells. We also demonstrate that these complexes contain the p50 and p65 subunits of NF-κB through utilisation of the EMSA supershift protocol. Discussion We provide detailed troubleshooting hints and tips for this technique and discuss the limitations of the EMSA, as well as a number of EMSA variants and alternative techniques.

Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation.

Using libraries of replication origins generated previously, we identified three clones that supported the autonomous replication of their respective plasmids in transformed, but not in normal cells. Assessment of their in vivo replication activity by in situ chromosomal DNA replication assays revealed that the chromosomal loci corresponding to these clones coincided with chromosomal replication origins in all cell lines, which were more active by 2–3-fold in the transformed by comparison to the normal cells. Evaluation of pre-replication complex (pre-RC) protein abundance at these origins in transformed and normal cells by chromatin immunoprecipitation assays, using anti-ORC2, -cdc6 and -cdt1 antibodies, showed that they were bound by these pre-RC proteins in all cell lines, but a 2–3-fold higher abundance was observed in the transformed by comparison to the normal cells. Electrophoretic mobility shift assays (EMSAs) performed on the most efficiently replicating clone, using nuclear extracts from the transformed and normal cells, revealed the presence of a DNA replication complex in transformed cells, which was barely detectable in normal cells. Subsequent supershift EMSAs suggested the presence of transformation-specific complexes. Mass spectrometric analysis of these complexes revealed potential new protein players involved in DNA replication that appear to correlate with cellular transformation.

Regulation of RASSF1A in nasopharyngeal cells and its response to UV irradiation

RASSF1A, which is frequently found inactivated in human cancers, is revealed as a tumor suppressor gene in nasopharyngeal carcinoma (NPC). Using RASSF1A-expressing (NP69 and HK-1) and non- RASSF1A-expressing (C666-1) cell models, the transcriptional regulation of RASSF1A was studied. By deletion analysis of 3.1 kb of 5′ flanking region, the core promoter of RASSF1A was identified in the region between −431 and −1 upstream of the translation start site. Sequence analysis of this core promoter revealed several putative transcription factor binding sties. Using NP69 cells and by block replacement mutagenesis, the presence of three functional GC-boxes were identified, to which by competitive and supershift electrophoretic mobility shift assays (EMSA), the in vitro bindings of Sp1 and Sp3 were suggested. The in vivo functions of Sp-proteins in regulating RASSF1A gene were then investigated by overexpression studies; among the tested Sp-proteins, Sp1 or Sp3, but not Sp4, was able to augment promoter activities. More interestingly, co-expression of Sp1 and Sp3 could synergistically enhance RASSF1A promoter function. UV irradiation induces oxidation stresses and hence is routinely used to investigate expressions of oncogenes and tumor suppressors. In this report, upon UV irradiation, the RASSF1A promoter activity and endogenous transcript levels were found to be reduced. By chromatin immunoprecipitation (ChIP) and EMSA, we demonstrated that the binding of Sp1 and Sp3 onto −431 to −202 were significantly reduced after UV irradiation. This UV-mediated effect on RASSF1A promoter, as shown by specific inhibitors that interrupt cellular pathways, is MEK1-, but not JNK-dependent. In summary, our data provided a simple model to explain the potential development of NPC, via silencing of the tumor suppressor RASSF1A by reduced bindings of activators Sp1 and Sp3 onto the GC-boxes in the core promoter of the gene.