The regulatory element containing the TT>A variants demonstrates enhancer activity.

<p>Sequences carrying risk (-A) and non-risk (TT) polymorphisms were cloned upstream of a minimal thymidine kinase promoter luciferase construct to measure luciferase activation following transient transfection and stimulation. a. HEK293T cells stimulated with P/I for 48 hours. b. THP1 cells stimulated with LPS for 48 hrs. c. THP1 cells stimulated with P/I for 48 hours. Statistical comparisons were performed using a Student's <i>t</i>-test of three biological independent experiments, * indicates p<0.05.</p

SATB1 is required for the TT>A enhancer-promoter interaction and <i>TNFAIP3</i> transcription.

<p>a. Shown is a representative 3C assay from 3 independent experiments in HEK293T cells. Relative crosslinking frequencies (RCF) were normalized to both <i>GAPDH</i> loading control and a <i>TNFAIP3</i> BAC clone and plotted according to its location to <i>TNFAIP3</i> gene and the TT>A enhancer. High local interaction frequencies near the TT>A enhancer serve as a positive control (fragments 30–34). b. The top panel shows the protein expression levels of SATB1 and A20 were detected using Western blots with antibodies against SATB1 and A20, respectively. β-actin was used as loading control. The bottom two panels show densitometer quantification of the relative expression of SATB1 (middle panel) and A20 (lower panel) normalized to β-actin. Error bars represent standard error of the mean from 3 independent experiments. Differential protein levels between SATB1 knockdown and non-silencing shRNA control were calculated using Student's <i>t</i>-test.</p

The TT>A risk variant exhibits reduced enhancer-promoter interaction frequencies and lower expression of <i>TNFAIP3</i>.

<p>a. 3C-qPCR assays were performed in EBV-transformed B cells homozygous for either risk or non-risk alleles in the TT>A enhancer; crosslinking frequencies between the enhancer and the promoter were normalized to <i>GAPDH</i> control. b, c. Protein expression of A20 and phospho-IκBα in homozygous cell lines were determined using Western blot and were normalized to loading control β-actin. Plots represent the relative expression of densitometric measurements for each cell line. d. Allele-specific 3C sequencing results of EBV cell lines heterozygous for the TT>A alleles. The read counts for each paired allele from 3C samples were normalized to the read counts for each allele from parallel analysis of input genomic DNA not subjected to 3C. Comparisons were made between non-risk and risk alleles for each individual. Statistical differences were calculated using paired <i>t</i>-test.</p

3C analysis demonstrates long-distance interactions between the TT>A enhancer and the <i>TNFAIP3</i> gene region.

<p>a. The track on the top is the location of the <i>TNFAIP3</i> primers used to identify potential amplified interaction fragments tested by 3C. Primers 6–10, 16 and 24 produced signals and are marked with asterisks. The middle and bottom track shows the genomic region of <i>TNFAIP3</i> with the location of the promoter CpG island and ENCODE defined transcription factor binding sites. b. Increased relative interaction frequencies (RCF) were detected in three regions of <i>TNFAIP3</i>, the promoter, intron 2 and the 3′ untranslated region. c. Stimulation of THP1 cells with LPS results in increased 3C interactions between the TT>A enhancer and the <i>TNFAIP3</i> promoter along with a concomitant increase in A20 expression and IκBα phosphorylation. Shown is a representative blot from 3 independent experiments. Statistical differences were determined using Student's <i>t</i>-test.</p

The Effect of Inversion at 8p23 on <i>BLK</i> Association with Lupus in Caucasian Population

<div><p>To explore the potential influence of the polymorphic 8p23.1 inversion on known autoimmune susceptibility risk at or near <i>BLK</i> locus, we validated a new bioinformatics method that utilizes SNP data to enable accurate, high-throughput genotyping of the 8p23.1 inversion in a Caucasian population. Methods: Principal components analysis (PCA) was performed using markers inside the inversion territory followed by k-means cluster analyses on 7416 European derived and 267 HapMaP CEU and TSI samples. A logistic regression conditional analysis was performed. Results: Three subgroups have been identified; inversion homozygous, heterozygous and non-inversion homozygous. The status of inversion was further validated using HapMap samples that had previously undergone Fluorescence in situ hybridization (FISH) assays with a concordance rate of above 98%. Conditional analyses based on the status of inversion were performed. We found that overall association signals in the <i>BLK</i> region remain significant after controlling for inversion status. The proportion of lupus cases and controls (cases/controls) in each subgroup was determined to be 0.97 for the inverted homozygous group (1067 cases and 1095 controls), 1.12 for the inverted heterozygous group (1935 cases 1717 controls) and 1.36 for non-inverted subgroups (924 cases and 678 controls). After calculating the linkage disequilibrium between inversion status and lupus risk haplotype we found that the lupus risk haplotype tends to reside on non-inversion background. As a result, a new association effect between non-inversion status and lupus phenotype has been identified ((p = 8.18×10⁻⁷, OR = 1.18, 95%CI = 1.10–1.26). Conclusion: Our results demonstrate that both known lupus risk haplotype and inversion status act additively in the pathogenesis of lupus. Since inversion regulates expression of many genes in its territory, altered expression of other genes might also be involved in the development of lupus.</p></div

Association and comparisons of minor allele frequency of known SNP rs13277113 for lupus according to three identified Caucasian subgroups.

<p>Association and comparisons of minor allele frequency of known SNP rs13277113 for lupus according to three identified Caucasian subgroups.</p

Forward Genetic Screening Identifies a Small Molecule That Blocks <i>Toxoplasma gondii</i> Growth by Inhibiting Both Host- and Parasite-Encoded Kinases

<div><p>The simultaneous targeting of host and pathogen processes represents an untapped approach for the treatment of intracellular infections. Hypoxia-inducible factor-1 (HIF-1) is a host cell transcription factor that is activated by and required for the growth of the intracellular protozoan parasite <i>Toxoplasma gondii</i> at physiological oxygen levels. Parasite activation of HIF-1 is blocked by inhibiting the family of closely related Activin-Like Kinase (ALK) host cell receptors ALK4, ALK5, and ALK7, which was determined in part by use of an ALK4,5,7 inhibitor named SB505124. Besides inhibiting HIF-1 activation, SB505124 also potently blocks parasite replication under normoxic conditions. To determine whether SB505124 inhibition of parasite growth was exclusively due to inhibition of ALK4,5,7 or because the drug inhibited a second kinase, SB505124-resistant parasites were isolated by chemical mutagenesis. Whole-genome sequencing of these mutants revealed mutations in the <i>Toxoplasma</i> MAP kinase, TgMAPK1. Allelic replacement of mutant TgMAPK1 alleles into wild-type parasites was sufficient to confer SB505124 resistance. SB505124 independently impacts TgMAPK1 and ALK4,5,7 signaling since drug resistant parasites could not activate HIF-1 in the presence of SB505124 or grow in HIF-1 deficient cells. In addition, TgMAPK1 kinase activity is inhibited by SB505124. Finally, mice treated with SB505124 had significantly lower tissue burdens following <i>Toxoplasma</i> infection. These data therefore identify SB505124 as a novel small molecule inhibitor that acts by inhibiting two distinct targets, host HIF-1 and TgMAPK1.</p></div

Demographic distribution of the individuals in the study^*.

<p>* Values are the number of patients/number of controls.</p><p>Demographic distribution of the individuals in the study^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115614#nt101" target="_blank">*</a>}.</p

Comparisons of minor allele frequency differences of a known published SNP associated with lupus (rs13277113) in 3 different Caucasian subgroups.

<p>Comparisons of minor allele frequency differences of a known published SNP associated with lupus (rs13277113) in 3 different Caucasian subgroups.</p

A schematic representation of the association map for Lupus at 8p23 before and after conditional analyses.

<p>Red dots =  original association, Blue dots = Association effects after conditioning on inversion. R2 =  correlation coefficient as a measure of linkage disequilibrium between inversion status and all markers in Caucasian population. The highest region in LD with inversion is shown with vertical line at LINC00208.</p