Journal: BMC Gastroenterology

Article Title: Mapping of HNF4α target genes in intestinal epithelial cells

doi: 10.1186/1471-230X-9-68

Figure Lengend Snippet: Analysis of the CDX2 promoter . A) The promoter region cloned in pGL4.10. The coordinates are relative to +1, the transcriptional start site. The arrows indicate primers used in the real-time qPCR. The possible HNF4α binding site and the mutated sequence is shown below. B) HNF4α ChIP-chip and AcHis3 ChIP-chip results for probes spanning the CDX2 promoter. Enrichments are shown as Log2 ratios between immunoprecipitated and input DNA. N = 3. C) Real-time qPCR with CDX2 and IgG intron primers using HNF4α ChIP, HA ChIP and input DNA. Enrichments are presented as percent of total input. N = 4. The CDX2 promoter enrichment is statistical significant (p-values < 0.05, Student T test). D) Gel shift analysis of the HNF4α site in the CDX2 promoter using Caco-2 nuclear extract (lane 1). Competition by unlabelled wt (lane 2), mut-HNF4α oligonucleotides (lane 3) and an unrelated oligonucleotide (lane 4) demonstrating a specific binding. One of the two protein/DNA bands can be supershifted with HNF4α antibody (lane 5). E) Promoter analysis of CDX2 promoter in Caco-2 (grey bars) and COS7 (white bars) cells with and without co-transfection of HNF4α expression plasmid. The binding site for HNF4α was mutated in order to analyze the functional importance of the HNF4α site (pGL4-CDX2 mutHNF4). The luciferase activity was corrected for transfection efficiency and normalized to the expression of pGL4-CDX2, N = 4.

Article Snippet: The amplified ChIP DNA was sent to NimbleGen Systems Inc. for labeling and hybridization to a 1.5 kb promoter array containing probes covering the region from -1350 to +150 of 24,275 human genes. (See http://www.nimblegen.com/products/chip/index.html for a description of the promoter array).

Techniques: Clone Assay, Binding Assay, Sequencing, ChIP-chip, Immunoprecipitation, Gel Shift, Cotransfection, Expressing, Plasmid Preparation, Functional Assay, Luciferase, Activity Assay, Transfection

Journal: BMC Gastroenterology

Article Title: Mapping of HNF4α target genes in intestinal epithelial cells

doi: 10.1186/1471-230X-9-68

Figure Lengend Snippet: Analysis of the trehalase ( TREH ) promoter . A) Map of the TREH promoter region. PCR primers, the HNF4α binding site, and the mutations are shown. B) HNF4α ChIP-chip and AcHis3 ChIP-chip results for probes spanning the TREH promoter. N = 3. C) Real-time qPCR analysis of HNF4α ChIP, HA ChIP and input DNA using TREH and IgG intron primers. N = 3. The TREH promoter enrichment is statistical significant (p-values < 0.05, Student T test). D) Gel shift analysis of the HNF4α site in the TREH promoter. E) Promoter analysis of the TREH promoter in Caco-2 (grey bars) and COS7 (white bars) cells with and without co-transfection of HNF4α expression plasmid and a mutation in the HNF4α site (pGL4-TREH mutHNF4). The luciferase activity was corrected for transfection efficiency and normalized to the expression of pGL4-TREH, N = 4.

Article Snippet: The amplified ChIP DNA was sent to NimbleGen Systems Inc. for labeling and hybridization to a 1.5 kb promoter array containing probes covering the region from -1350 to +150 of 24,275 human genes. (See http://www.nimblegen.com/products/chip/index.html for a description of the promoter array).

Techniques: Binding Assay, ChIP-chip, Gel Shift, Cotransfection, Expressing, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Transfection

Journal: BMC Gastroenterology

Article Title: Mapping of HNF4α target genes in intestinal epithelial cells

doi: 10.1186/1471-230X-9-68

Figure Lengend Snippet: Analysis of the cingulin ( CGN ) promoter . A) Map of the CGN promoter region. PCR primers, the HNF4α binding site, and the mutations are shown. B) HNF4α ChIP-chip and AcHis3 ChIP-chip results for probes spanning the CGN promoter. N = 3. C) Real-time qPCR analysis of HNF4α ChIP, HA ChIP and input DNA using CGN and IgG intron primers. N = 3. The CGN promoter enrichment is statistical significant (p-values < 0.05, Student T test). D) Gel shift analysis of the HNF4α site in the CGN promoter. E) Promoter analysis of the CGN promoter in Caco-2 (grey bars) and COS7 (white bars) cells with and without co-transfection of HNF4α expression plasmid and a mutation in the HNF4α site (pGL4-CGN mutHNF4). The luciferase activity was corrected for transfection efficiency and normalized to the expression of pGL4-CGN, N = 4.

Article Snippet: The amplified ChIP DNA was sent to NimbleGen Systems Inc. for labeling and hybridization to a 1.5 kb promoter array containing probes covering the region from -1350 to +150 of 24,275 human genes. (See http://www.nimblegen.com/products/chip/index.html for a description of the promoter array).

Techniques: Binding Assay, ChIP-chip, Gel Shift, Cotransfection, Expressing, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Transfection

Journal: BMC Gastroenterology

Article Title: Mapping of HNF4α target genes in intestinal epithelial cells

doi: 10.1186/1471-230X-9-68

Figure Lengend Snippet: HNF4α ChIP on mouse small intestinal epithelium . Chromatin immunoprecipitation analysis of HNF4α binding to HNF1α ( Tcf1 ), phosphoenolpyruvate carboxykinase 1 ( Pck1 ), apolipoprotein C3 ( Apoc3 ), Cdx-2 ( Cdx2 ), trehalase ( Treh ), and cingulin ( Cgn ) promoters in mouse small intestinal epithelium. An intron in the IgG gene served as a negative control (IgG). The analyzed promoter regions are all conserved between human and mouse. Enrichments are represented as percent of the total amount of genomic input DNA in ChIP. Significant enrichments are indicated (p-values < 0.001 is shown by ***, p-values < 0.05 is shown by *).

Article Snippet: The amplified ChIP DNA was sent to NimbleGen Systems Inc. for labeling and hybridization to a 1.5 kb promoter array containing probes covering the region from -1350 to +150 of 24,275 human genes. (See http://www.nimblegen.com/products/chip/index.html for a description of the promoter array).

Techniques: Chromatin Immunoprecipitation, Binding Assay, Negative Control

Journal: Molecular Systems Biology

Article Title: Environment-responsive transcription factors bind subtelomeric elements and regulate gene silencing

doi: 10.1038/msb.2010.110

Figure Lengend Snippet: Network cluster negatively regulated by Oaf1p is enriched 5–7 kb from telomeres. ( A ) Histogram of distance to closest telomere for targets of Oaf1p (in a network cluster enriched for those downregulated by Oaf1p) . More than 12% of these targets were found within 10 kb of telomeres, whereas only about 2% of all intergenic regions on the microarray were in this position. In contrast, network clusters upregulated by Oaf1p were not found within 10 kb of telomeres (data not shown). ( B ) High-resolution histogram of DNA segments within 10 kb of telomeres shows that the same targets are enriched 5–7 kb from telomeres (blue bars). The expected binding frequency if targets had no positional preference is shown (green vector). The region within 1 kb of telomeres had no detectable interaction with the factors, but <10% of this region was present on the microarrays (data not shown).

Article Snippet: For each replicate, linkers were annealed to DNA ends in whole-cell extract and IP fractions, and fragments were amplified by ligation-mediated PCR with high-fidelity Taq polymerase, and shipped to NimbleGen Systems of Iceland for labeling, hybridization, scanning and preliminary analysis as described below: Equal amounts of DNA in whole-cell extracts and IP fraction were labeled with Cy3 and Cy5, respectively, combined and co-hybridized at 42°C to microarrays of 50-mer DNA probes that span both strands of the entire genome positioned every 64 bp (resulting in 14 bases of DNA between probes).

Techniques: Microarray, Binding Assay, Plasmid Preparation

Journal: Molecular Systems Biology

Article Title: Environment-responsive transcription factors bind subtelomeric elements and regulate gene silencing

doi: 10.1038/msb.2010.110

Figure Lengend Snippet: Binding profiles of transcription factors (TFs) correlate with X-element positions. Subtelomeric-binding profiles are shown for factors found to enriched at X elements in . The densities of TF binding and X elements present on the microarrays are represented as heat maps. Binding conditions other than rich medium are marked with colored dots as in . With the exception of Phd1p, all factors had maximal binding 5–7 kb from telomeres, matching the position of X element enrichment. Pearson's correlation coefficients ( ρ ) comparing each binding profile to the positions of X elements are shown at the right.

Article Snippet: For each replicate, linkers were annealed to DNA ends in whole-cell extract and IP fractions, and fragments were amplified by ligation-mediated PCR with high-fidelity Taq polymerase, and shipped to NimbleGen Systems of Iceland for labeling, hybridization, scanning and preliminary analysis as described below: Equal amounts of DNA in whole-cell extracts and IP fraction were labeled with Cy3 and Cy5, respectively, combined and co-hybridized at 42°C to microarrays of 50-mer DNA probes that span both strands of the entire genome positioned every 64 bp (resulting in 14 bases of DNA between probes).

Techniques: Binding Assay

Journal: The Open AIDS Journal

Article Title: Cervical HPV Infection in Female Sex Workers: A Global Perspective

doi: 10.2174/1874613601307010058

Figure Lengend Snippet: Background Information on 35 Studies of Female Sex Workers that Include HPV Genotype Testing

Article Snippet: Western Pacific-2011 [39) , Japan , Kyoto , 196 , Samples were obtained. HPV DNA was detected with PCR and genotyped with the Kurabo GeneSquare Microarray system. , 6, 11, 16, 18, 30, 31, 33, 34, 35, 39, 40, 42, 45, 51, 53 54, 56, 58, 59, 61, 66 and 68 , 52.6%.

Techniques: Sampling, Real-time Polymerase Chain Reaction, Hybridization, Software, Surround Optical-fiber Immunoassay, Enzyme-linked Immunosorbent Assay, Western Blot, Clone Assay, Microarray

Journal: PLoS ONE

Article Title: DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

doi: 10.1371/journal.pone.0148887

Figure Lengend Snippet: (A) SEM was used to compare OMV formation. The IVp-II-regulated gene cluster was entirely (Δ3397–3403) or partially (only BF3403, the last gene of the cluster) deleted in the ON/ON genetic background of B . fragilis YCH46. Bar: 1 μm. (B) Comparison of OMV production. OMV production was quantified by measuring the protein content of culture supernatants. The graph indicates OMV production relative to that of the ON/ON (Δ3397–3403) mutant. The data are expressed as means ± standard deviation. The bars labeled with different letters indicate significant differences at p < 0.05.

Article Snippet: The B . fragilis YCH46 DNA microarray from NimbleGen Systems, which includes 4,527 target genes with at least 8 unique 60-mer synthetic oligonucleotide probes for each gene, was used for the comparative transcriptomic analysis of the mutant strains.

Techniques: Comparison, Mutagenesis, Standard Deviation, Labeling

Journal: PLoS ONE

Article Title: DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

doi: 10.1371/journal.pone.0148887

Figure Lengend Snippet: (A) Growth of B . fragilis YCH46 in GAM supplemented with the indicated bile concentrations. (B) qPCR was used to determine the relative abundance of the IVp-I-ON or IVp-II-ON genotypes after exposure to the indicated bile concentration. (C) Effect of bile exposure on the expression of an IVp-I-regulated EPS production gene (BF2767) and IVp-II-regulated membrane protein genes (BF3397 and BF3403). (D) OMV production of B . fragilis increased 7-fold after exposure to 5% bile. (E) Schematic of the 3 x FLAG-tag fusion to the C-terminus of the BF3397-encoded protein. (F) Enhancement of the BF3397-encoded membrane protein in response to 5% bile. A B . fragilis YCH46 derivative with a 3 x FLAG-tag sequence fused to BF3397, as shown in panel E, was exposed to 5% bile: the production of BF3397 was assessed by Western blotting with an anti-FLAG antibody. Upper, middle, and lower bands indicate the anti-FLAG tag antibody used for immunoprecipitation, BF3397 (unprocessed form), and N-terminally processed BF3397 (indicated by arrowheads), respectively.

Article Snippet: The B . fragilis YCH46 DNA microarray from NimbleGen Systems, which includes 4,527 target genes with at least 8 unique 60-mer synthetic oligonucleotide probes for each gene, was used for the comparative transcriptomic analysis of the mutant strains.

Techniques: Concentration Assay, Expressing, Membrane, FLAG-tag, Sequencing, Western Blot, Immunoprecipitation

Journal: PLoS ONE

Article Title: DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

doi: 10.1371/journal.pone.0148887

Figure Lengend Snippet: (A) Competitive growth assay of ON/ON B . fragilis mutant and its parental YCH46 stain in vitro . Equivalent cell mixture of wild and mutant B . fragilis cells was inoculated into GAM broth. Closed circles indicate the periodical change of total viable cell number in GAM broth. Columns indicate the relative abundance of the wild and mutant B . fragilis cell in GAM broth. (B) IVp-I/IVp-II ON/OFF prevalence during in vivo gut colonization IVp-I/IVp-II under in vivo gut colonization by B . fragilis . A cell suspension (7.0 x10 7 CFU in total) of wild-type B . fragilis was inoculated into three 8-week-old male BALB/cA germ-free mice via gavage. At 3, 7, 10, and 14 days after inoculation, feces samples were collected. DNA was extracted from the initial inoculum and the collected feces samples, and the ON/OFF ratio of IVp-I/IVp-II in each sample was determined by qPCR. The data are expressed as means ± standard deviation. The bars labeled with different letters indicate significant differences at p < 0.05. (C) Competitive growth assay of IVp-I/Vp-II-locked B . fragilis mutant and its parental YCH46 stain. Equivalent cell mixture of wild and the OFF/ON mutant cells was inoculated into BALB/c germ-free mice. Colony PCR was performed on at least 96 colonies per sample with primer pair encompassing the deletion site in BF2766 to compare population levels of mutants (closed triangles) with wild type (closed circles) cells in the mouse intestine. In this experiment, mice were kept in a vinyl isolator to maintain their gnotobiotic conditions.

Article Snippet: The B . fragilis YCH46 DNA microarray from NimbleGen Systems, which includes 4,527 target genes with at least 8 unique 60-mer synthetic oligonucleotide probes for each gene, was used for the comparative transcriptomic analysis of the mutant strains.

Techniques: Growth Assay, Mutagenesis, Staining, In Vitro, In Vivo, Suspension, Standard Deviation, Labeling