<i>FasL</i> promoter SNP haplotypes and location of TCF/LEF-1 binding elements.

<p><b>A</b>). Four SNPs (-844C>T, -756A>G, -478A>T, and -205C>G) were identified in the FasL promoter region. <b>B</b>). PHASE program for haplotype reconstruction was used for haplotype analysis from 150 African American donors. The <i>FasL</i> promoter SNP haplotypes were verified from homozygous donors and further confirmed by sequencing genomic DNA clones from heterozygous donors. The <i>FasL</i> haplotype frequencies were calculated according to the established haplotypes. <b>C</b>). Identification of TCF/LEF-1 binding elements (TBEs) in the <i>FasL</i> promoter region. The core sequence of putative distal TCF/LEF-1 element (TBE1) is between nucleotide position -838 to -833 while the core sequence of the putative proximal TCF/LEF-1 element (TBE2) is between nucleotide position -205 and -200. SNP -205C>G is located within the TBE2 core sequence. The nucleotide changes (underlined) were the introduced mutations in the EMSA probes or in the promoter reporter constructs.</p

β-catenin knockdown decreases FasL expression in SW480 cells.

<p>SW480 cells were transfected with either the scrambled control siRNA (open bars) or the β-catenin siRNA (black bars) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026143#s4" target="_blank">Materials and Methods</a>). β-catenin and FasL mRNA level were calculated by relative quantification using GAPDH as the control in real-time RT-PCR assay. Introduction of β-catenin siRNA decreased 88% β-catenin and 42% FasL mRNA expression in SW480 cells (*<i>P</i><0.01). Data were the average of three experiments.</p

Binding of TCF-4 and β-catenin to the distal <i>FasL</i> TCF/LEF-1 binding element (TBE1).

<p><b>A</b>). Radio-labeled wild-type TBE1 probe (lane 1-5) and mutant TBE1 probe (lane 6) were incubated with 8 µg of SW480 cell nuclear extracts for 30 min. Competition experiments were performed by preincubating with 200 fold molar excess of the unlabeled TBE1 probes (Cold SP, lane 3), or non-specific probe (Cold NP, lane 5). Antibody binding experiments were carried out following the vendor's instruction with anti-TCF-4 antibody (lane 4) and rabbit IgG as control (lane 1). The arrow indicates the position of specific transcription factor complexes. Results shown were representative of four experiments. <b>B</b>). Radio-labeled TBE1 (lane 1-4) and mutant TBE1 probes (lane 5) were incubated with 8 µg of Jurkat cell nuclear extracts for 30 min. Antibody binding experiments were carried out with anti-β-catenin antibody (lane 3). The arrows indicate the position of specific transcription factor complexes. Results shown are representative of four experiments. <b>C</b>). TCF-4 bound to the TBE1 of <i>FasL</i> promoter in a Chromatin Immunoprecipitation Assay (ChIP). ChIP assay was performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026143#s4" target="_blank">Materials and Methods</a>”. Rabbit anti-human TCF-4 antibody was used to precipitate Jurkat T cell chromatin complexes containing <i>FasL</i> promoter DNA fragment (lane 5). The appropriate positive (lane 2) and negative controls (lane 3 and 4) were included. Lane 1 contained DNA molecular weight marker (100 bp DNA ladders). <b>D</b>). β-catenin bound to the <i>FasL</i> promoter TBE1 in a ChIP assay. Mouse monoclonal antibody against human β-catenin was used to precipitate Jurkat T cell chromatin complexes containing <i>FasL</i> promoter DNA fragment (lane 5). The appropriate positive (lane 2) and negative controls (lane 3 and 4) were included. Lane 1 contained DNA molecular weight marker (100 bp DNA ladders). The positive PCR products were shown as 163 bp DNA bands (pointed by arrow) in ChIP assays. The identity of DNA band was further confirmed with DNA sequencing.</p

Role of TBEs in <i>FasL</i> promoter.

<p><b>A</b>). Human LEF-1 increases <i>FasL</i> promoter activities in SW480 cells. <i>FasL</i> promoter reporter construct (0.5 µg) was co-transfected with human LEF-1 expression construct or vector control plasmid DNA (0.5 µg) into SW480 cells. LEF-1 significantly increased FasL promoter activities compared to the vector DNA control in SW480 cells. Data represent means ± SEM from three independent experiments (*<i>P</i><0.01).<b> B</b>). Functional TBEs are required for the enhancement of <i>FasL</i> promoter activity by LEF-1 and β-catenin. <i>FasL</i> promoter reporter plasmid DNA (0.5 µg) was co-transfected with LEF-1 (0.5 µg) and β-catenin (1.0 µg) into COS-7 cells. Co-transfection of LEF-1 and β-catenin significantly increased activities of <i>FasL</i> promoter containing functional TBEs (TBE1W/2W) in COS-7 cells (*<i>P</i><0.01). Co-transfection of LEF-1 and β-catenin failed to significantly increase activities of <i>FasL</i> promoter containing mutant TBEs (TBE1M/2M). Data represent means ± SEM from three independent experiments. <b>C</b>). Promoter reporter plasmid DNA (0.5 µg) containing either triplicate wild-type TBE2 (3×TBE2W, -205C allele) or triplicate mutant TBE2 (3×TBE2M, -205G allele) was transfected into SW480 and SW620 cells as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026143#s4" target="_blank">Materials and Methods</a>”. The triplicate wild-type TBE2 (3×TBE2W) significantly increased the promoter activities compared to the triplicate mutant TBE2 (3×TBE2M) in SW620 (4.3 folds for wild-type TBE2 and 1.3 folds for mutant TBE2 over the vector control respectively) and in SW480 (12.7 folds for wild-type TBE2 and 2.9 for mutant TBE2 over the vector control respectively). Data represent means ± SEM from six independent experiments. The star symbol indicates that there are significant differences between 3×TBE2W and 3×TBE2M (*<i>P</i><0.001). <b>D</b>). Role of <i>FasL</i> promoter TBEs in SW480 cells. Mutation of either TBE1 (TBE1M/2W) or TBE2 (TBE1W/2M) failed to affect the <i>FasL</i> promoter activities compared to the wild-type reporter construct (<i>P</i> = 0.50 for TBE1W2M and <i>P</i> = 0.30 for TBE1M/2M). Simultaneous mutations of TBE1 and TBE2 (TBE1M/2M) significantly reduced <i>FasL</i> promoter activities compared to the construct with wild-type TBE1 and TBE2 (TBE1W/2W) in SW480 cells (*<i>P</i><0.01). Data represent means ± SEM from four independent experiments. Relative luciferase light units (RLU) were standardized to Renilla luciferase activities (A and B) or standardized to β-galactosidase activities (C and D).</p

Genomic Copy Number Variants: Evidence for Association with Antibody Response to Anthrax Vaccine Adsorbed

<div><p>Background</p><p>Anthrax and its etiologic agent remain a biological threat. Anthrax vaccine is highly effective, but vaccine-induced IgG antibody responses vary widely following required doses of vaccinations. Such variation can be related to genetic factors, especially genomic copy number variants (CNVs) that are known to be enriched among genes with immunologic function. We have tested this hypothesis in two study populations from a clinical trial of anthrax vaccination.</p><p>Methods</p><p>We performed CNV-based genome-wide association analyses separately on 794 European Americans and 200 African-Americans. Antibodies to protective antigen were measured at week 8 (early response) and week 30 (peak response) using an enzyme-linked immunosorbent assay. We used DNA microarray data (Affymetrix 6.0) and two CNV detection algorithms, hidden markov model (PennCNV) and circular binary segmentation (GeneSpring) to determine CNVs in all individuals. Multivariable regression analyses were used to identify CNV-specific associations after adjusting for relevant non-genetic covariates.</p><p>Results</p><p>Within the 22 autosomal chromosomes, 2,943 non-overlapping CNV regions were detected by both algorithms. Genomic insertions containing <i>HLA-DRB5, DRB1</i> and <i>DQA1/DRA</i> genes in the major histocompatibility complex (MHC) region (chromosome 6p21.3) were moderately associated with elevated early antibody response (β = 0.14, p = 1.78×10⁻³) among European Americans, and the strongest association was observed between peak antibody response and a segmental insertion on chromosome 1, containing <i>NBPF4, NBPF5, STXMP3, CLCC1</i>, and <i>GPSM2</i> genes (β = 1.66, p = 6.06×10⁻⁵). For African-Americans, segmental deletions spanning <i>PRR20, PCDH17</i> and <i>PCH68</i> genes on chromosome 13 were associated with elevated early antibody production (β = 0.18, p = 4.47×10⁻⁵). Population-specific findings aside, one genomic insertion on chromosome 17 (containing <i>NSF, ARL17</i> and <i>LRRC37A</i> genes) was associated with elevated peak antibody response in both populations.</p><p>Conclusion</p><p>Multiple CNV regions, including the one consisting of MHC genes that is consistent with earlier research, can be important to humoral immune responses to anthrax vaccine adsorbed.</p></div

Odds ratios for the protective effect of the rs231778 G allele in African Americans with RA.

<p>Analysis compares number of patients and controls for genotype AA vs those with either AG or GG in a 2×2 Fisher's exact test. OR – odds ratio; CI – confidence interval.</p

This plot, generated from HaploView v3.31 (Broad Institute), shows the r² values between SNPs genotyped in <i>CTLA4</i>.

<p>Darker boxes represent stronger r² values. 5251* is not yet registered in dbSNP as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000424#s2" target="_blank">methods</a>.</p

Mean percentage of European ancestry segregated by genotype of rs231778.

<p>This table provides data for the 347 samples with complete admixture and rs231778 genotyping data. Standard error is provided in parenthesis. Brackets indicate frequency counts used in determining association of the rs231778 G allele with rheumatoid arthritis among 366 samples, as described in the text.</p

Minor allele frequency (MAF) of rs231778 segregated by HLA-DRB1 shared epitope status.

<p>This table combines samples from both CLEAR I and CLEAR II and only incorporates data where HLA-DRB1 shared epitope (SE) status has been determined. The ‘N’ listed below each MAF represents the number of ‘G’ alleles over the total number of alleles for each number of SE alleles present. SE – <i>HLA-DRB1</i> shared epitope.</p

Only SNPs genotyped in this study are listed by name.

<p>SNPs in linkage disequilibrium with SNPs genotyped in this study are shown in brown. SNPs in gray were not genotyped or haplotype tagged due to low minor allele frequency (MAF). SNP - Single Nucleotide Polymorphism. 5251* is not yet registered in dbSNP as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000424#s2" target="_blank">methods</a>.</p