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

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 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

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

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

Comparative genomic analysis of Ctg-3301 (Chromosome A12) and Ctg-465 (Chromosome D12) of Upland cotton with <i>Arabidopsis</i> genome.

<p>Comparative genomic analysis of Ctg-3301 (Chromosome A12) and Ctg-465 (Chromosome D12) of Upland cotton with <i>Arabidopsis</i> genome.</p

Diagrammatic representation of BAC-pools and their homology across the pools and respective BAC clone ABI assemblies in Ctg-3301 and Ctg-465.

<p>Diagrammatic representation of BAC-pools and their homology across the pools and respective BAC clone ABI assemblies in Ctg-3301 and Ctg-465.</p

A schematic diagram representing MTP based BAC-pool sequencing for complex genomes.

<p>454 and Sanger hybrid sequencing approach was used to derive high quality sequence assemblies in allotetraploid cotton.</p

Generation of SBR mutants.

<p>A. Relative plaque formation in HFFs was determined for each parasite strain in the presence of increasing concentrations of SB505124. B–D. Parasite replication was measured by infecting HFFs on glass coverslips in the presence or absence of 3 µM SB505124 and then fixing the cells 24 hours later. Parasites and nuclei were detected with anti-SAG1 antibody and DAPI, respectively. B. Representative images. C. For each replicate, 100 vacuoles were monitored for parasites per vacuole and nuclei per parasite. Vacuoles were designated as being irregular if they contained an irregular number of parasites/vacuole (non 2ⁿ). Shown are averaged percentages and standard deviations of 2 independent experiments with two replicates each. D. Averaged percentages and standard deviations of irregular vacuoles (show in C) by nuclei per parasite.</p

SB505124 reduces parasite growth in <i>Toxoplasma</i>-infected mice.

<p>RH-GFP infected mice were intraperitoneally injected daily with 10 mg/kg SB505124 or DMSO alone. After 5 days post-infection, mice were sacrificed and flow cytometric analysis was performed on peritoneal exudate cells (3–4 mice per treatment group per experiment, 2 independent experiments). A. FACS plots (upper) and histograms (lower) showing percentages of infected (GFP⁺) cells of two representative mice per treatment group. B. Mean percentages of infected cells between treatment groups with standard deviations. C. Relative MFI of infected (GFP⁺) cells with standard deviations. D. ELISA determination of serum IFNγ levels of mock- and drug-treated, intraperitoneally infected mice 5 days post-infection. Shown are average and standard deviations.</p