Additional file 2: Figure S1. of A deletion in the intergenic region upstream of Ednrb causes head spot in the rat strain KFRS4/Kyo

PCR experiments to confirm the deletion found in KFRS4/Kyo. (PDF 68 kb

Additional file 3: Figure S2. of A deletion in the intergenic region upstream of Ednrb causes head spot in the rat strain KFRS4/Kyo

A KFRS4/Kyo-specific deletion of approximately 50 kb in length located approximately 50 kb upstream of Ednrb. (PDF 116 kb

Additional file 1: of Design and application of a target capture sequencing of exons and conserved non-coding sequences for the rat

Table S1. Summary statistics for SNV and INDEL in various depths. Figure S1. Sequence coverage of the target regions for each rat strain. Figure S2. Number of homozygous SNVs identified in WTC/Kyo and PVG/Seac strains for each genomic region. Figure S3. Proportion of the number of SNVs in terms of the each class of regions in the target, i.e., CDS, UTR, CNS, and other regions, for each rat strain. Figure S4. The relationship between the phastCons conservation score and SNV density for each rat strain. (PDF 215 kb

Additional file 4: Figure S3. of A deletion in the intergenic region upstream of Ednrb causes head spot in the rat strain KFRS4/Kyo

Virtual 4C output for the region around Ednrb gene in mouse genome (mm9 assembly). (PDF 308 kb

Identification of Protein Targets of 12/15-Lipoxygenase-Derived Lipid Electrophiles in Mouse Peritoneal Macrophages Using Omega-Alkynyl Fatty Acid

The 12/15-lipoxygenase (12/15-LOX) enzyme introduces peroxyl groups, in a position-specific manner, into polyunsaturated fatty acids to form various kinds of bioactive lipid metabolites, including lipid-derived electrophiles (LDE). The resident peritoneal macrophage is the site of highest 12/15-LOX expression in the mouse. However, the role of the enzyme in the regulation of resident macrophages is not fully understood. Here, we describe a chemoproteomic method to identify the targets of enzymatically generated LDE. By treating mouse peritoneal macrophages with omega-alkynyl arachidonic acid (aAA), we identified a series of proteins adducted by LDE generated through a 12/15-LOX catalyzed reaction. Pathway analysis revealed a dramatic enrichment of proteins involved in energy metabolism and found that glycolytic flux and mitochondrial respiration were significantly affected by the expression of 12/15-LOX. Our findings thus highlight the utility of chemoproteomics using aAA for identifying intracellular targets of enzymatically generated LDE

A Novel Small Compound SH-2251 Suppresses Th2 Cell-Dependent Airway Inflammation through Selective Modulation of Chromatin Status at the <i>Il5</i> Gene Locus

<div><p>IL-5 is a key cytokine that plays an important role in the development of pathological conditions in allergic inflammation. Identifying strategies to inhibit IL-5 production is important in order to establish new therapies for treating allergic inflammation. We found that SH-2251, a novel thioamide-related small compound, selectively inhibits the differentiation of IL-5-producing Th2 cells. SH-2251 inhibited the induction of active histone marks at the <i>Il5</i> gene locus during Th2 cell differentiation. The recruitment of RNA polymerase II, and following expression of the Th2 cell-specific intergenic transcripts around the <i>Il5</i> gene locus was also inhibited. Furthermore, Th2 cell-dependent airway inflammation in mice was suppressed by the oral administration of SH-2251. Gfi1, a transcriptional repressor, was identified as a downstream target molecule of SH-2251 using a DNA microarray analysis. The Gfi1 expression dramatically decreased in SH-2251-treated Th2 cells, and the SH-2251-mediated inhibition of IL-5-producing Th2 cell differentiation was restored by transduction of <i>Gfi1</i>. Therefore, our study unearthed SH-2251 as a novel therapeutic candidate for allergic inflammation that selectively inhibits active histone marks at the <i>Il5</i> gene locus.</p></div

OVA-induced airway inflammation is attenuated by oral administration of SH-2251.

<p><b>(A)</b>, Decreased infiltration of eosinophils in the BAL fluid of asthmatic SH-2251-administered mice. The absolute numbers of eosinophils (Eos.), neutrophils (Neu.), lymphocytes (Lym.) and macrophages (Mac.) in the BAL fluid are shown with standard deviations (n = 5 per group). *<i>P</i><0.01 and **<i>P</i><0.001 by ANOVA and the Bonferroni-test. (B), Quantitative RT-PCR of <i>Il4</i>, <i>Il5</i> and <i>Il13</i> mRNA in the BAL fluid cells of vehicle and SH-2251-administered mice. (C), Quantitative RT-PCR of <i>Il4</i>, <i>Il5</i>, <i>Il13</i> and <i>Ifnγ</i> mRNA in the lung CD4 T cells of vehicle and SH-2251-administered mice. (n = 5 per group). (D), Cytokine production from lung CD4 T cells of vehicle and SH-2251-administered mice stimulated <i>in vitro</i>. The lung CD4 T cells were stimulated with immobilized anti-TCR-β mAb for 48 hours and the concentrations of cytokines in the culture supernatants were determined using ELISA. The lungs were fixed and stained with hematoxylin and eosin (E, left) or periodic acid-Schiff reagent (F). The scale bars represent 500 µm. The numbers of infiltrated leukocytes in the peribronchiolar regions are shown (mean cell numbers/mm²) (E, right). Three independent experiments were performed with similar results. Student's <i>t</i>-test was used for the statistical analyses. *<i>P</i><0.05 and **<i>P</i><0.01 (B, C, D and E)</p

The induction of active histone marks at the <i>Il5</i> gene locus is inhibited by SH-2251.

<p><b>(A)</b>, Naïve CD4 T cells were cultured under Th2-conditions for five days in the presence of the indicated concentration of SH-2251, and a ChIP assay was performed with the indicated antibodies. The relative intensity (/Input) is shown with the standard deviation. <b>(B)</b>, The global patterns of histones H3K4me3 and H3K27ac at the Th2 cytokine gene loci were determined using ChIP-sequencing. <b>(C)</b>, The indicated histone modification status around the <i>Il5</i> gene locus in the SH-2251-treated Th2 cells was determined using a manual ChIP assay. The relative intensity (/Input) is shown with the standard deviation. <b>(D)</b>, Recruitment of RNA polymerase II around the <i>Il5</i> gene locus (upper panel) was determined using a manual ChIP assay. The relative intensity (/Input) is shown with the standard deviation. The transcripts around the <i>Il5</i> gene in the SH-2251-treated Th2 cells (lower panel) were determined using quantitative RT-PCR. The relative intensity (/<i>Hprt</i>) is shown with the standard deviation. Four independent experiments (A, C and D) were performed with similar results.</p

Transduction of <i>Gfi1</i> into SH-2251-treated Th2 cells restores the differentiation of IL-5-producing Th2 cells.

<p><b>(A)</b>, CD4 T cells were cultured under Th2-conditions in the presence or absence of SH-2251 (100 nM) for two days, then the cells were transduced with Mock- or <i>Gfi1</i>-IRES-hNGFR-containing retrovirus vectors. Three days after transduction, the IL-5/IFN-γ staining profiles of the transduced cells (hNGFR-positive cells) were determined with intracellular staining. The percentages of cells in each quadrant are indicated. <b>(B)</b>, The cytokine production from SH-2251-treated Th2 cells transduced with <i>Gfi1</i> was determined. <b>(C)</b>, Histones H3K4me3, H3K9ac and H3K27ac at the <i>Il5</i> gene locus in hNGFR-positive <i>Gfi1</i>-transduced SH-2251-treated Th2 cells. The relative intensity (/Input) is shown with the standard deviation. Three (A, B and C) independent experiments were performed with similar results. <b>(D)</b>, The global pattern of Gfi1 binding around the <i>Il5</i> gene locus was determined using ChIP-sequencing with an anti-Gfi1 mAb. The locations of the PCR primer pairs (triangle) used in a manual ChIP assay are also listed. <b>(E)</b>, The binding of Gfi1 around the <i>Il5</i> gene locus in SH-2251-treated Th2 cells was determined using a manual ChIP assay. The relative intensity (/Input) is shown with the standard deviation. Two independent experiments were performed with similar results.</p

The expression and functions of Gata3 are not impaired by treatment with SH-2251.

<p><b>(A)</b>, The mRNA expression of Gata3 in the SH-2251-trearted Th2 cells was determined using quantitative RT-PCR. The relative intensity (/<i>Hprt</i>) is shown with the standard deviation. <b>(B)</b>, The protein expression level of Gata3 was determined with immunoblotting. The nuclear (Gata3) and cytoplasmic (α-Tubulin) lysates with a three fold serial dilution were used. Three independent experiments (A and B) were performed with similar results. <b>(C)</b>, The global patterns of Gata3 binding at the Th2 cytokine gene loci (upper panel) and the <i>Il5</i> gene locus (lower panel) were determined using ChIP-sequencing with an anti-Gata3 pAb. The locations of PCR primer pairs (triangle) used in a manual ChIP assay are also listed. <b>(D)</b>, The binding of Gata3 around the <i>Il5</i> gene locus (left) panel and the V_A enhancer (V_A E) and intronic enhancer (IE) regions of the <i>Il4</i> gene locus (right panel) in the SH-2251-treated Th2 cells was determined using a manual ChIP assay. The relative intensity (/Input) is shown with the standard deviation. Three independent experiments were performed with similar results. <b>(E)</b>, The effects of SH-2251 on the Gata3-dependent transcriptional activation of the <i>Il5</i> promoter were determined using a Dual luciferase assay. The mean and standard deviation of the relative luciferase activity of three different experiments are shown. Stim: PMA (30 ng/ml)+dbcAMP (100 µM). Four independent experiments (A, B, D and E) were performed with similar results.</p