Cross-enhancement of ANGPTL4 transcription by HIF1 alpha and PPAR beta/delta is the result of the conformational proximity of two response elements.

BACKGROUND: Synergistic transcriptional activation by different stimuli has been reported along with a diverse array of mechanisms, but the full scope of these mechanisms has yet to be elucidated. RESULTS: We present a detailed investigation of hypoxia-inducible factor (HIF) 1 dependent gene expression in endothelial cells which suggests the importance of crosstalk between the peroxisome proliferator-activated receptor (PPAR) ß/δ and HIF signaling axes. A migration assay shows a synergistic interaction between these two stimuli, and we identify angiopoietin-like 4 (ANGPTL4) as a common target gene by using a combination of microarray and ChIP-seq analysis. We profile changes of histone marks at enhancers under hypoxia, PPARß/δ agonist and dual stimulations and these suggest that the spatial proximity of two response elements is the principal cause of the synergistic transcription induction. A newly developed quantitative chromosome conformation capture assay shows the quantitative change of the frequency of proximity of the two response elements. CONCLUSIONS: To the best of our knowledge, this is the first report that two different transcription factors cooperate in transcriptional regulation in a synergistic fashion through conformational change of their common target genes.

Direct evidence for pitavastatin induced chromatin structure change in the KLF4 gene in endothelial cells.

Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.

Expression of the proapoptotic protein Bax is reduced in bronchial mucous cells of asthmatic subjects.

The present studies were designed to determine whether our findings in mice showing that the Bcl-2-associated protein X (Bax), which plays a role in the resolution of allergen-induced mucous cell metaplasia, can be applied to asthma in humans. Immunostaining of autopsy tissues from mild and severe asthmatic subjects showed a significant reduction in the percentage of Bax-positive mucous cells compared with those from nonasthmatic controls. To exclude the possibility that postmortem changes may have affected Bax expression, Bax mRNA levels in airway epithelial cells obtained from nonsmoking asthmatic subjects were compared with those from nonasthmatic controls. Because the number of cells obtained by bronchial brushings is limited, we developed a robust preamplification procedure of cDNA before quantitative real-time PCR to allow detection of 100 gene targets from limited sample size, even when it was prepared from partially degraded RNA. cDNA was prepared by reverse transcription from RNA isolated from bronchial epithelial cells obtained by bronchial brushings from well-characterized subjects without lung disease and from subjects with mild asthma. Quantitative analysis showed that Bax mRNA levels were significantly reduced in samples obtained from asthma patients compared with nonasthma controls. Furthermore, Bax mRNA levels were reduced when primary airway epithelial cells from 10 individuals were treated in culture with the T helper 2 cytokine IL-13. These studies show that Bax expression is reduced in airway epithelial cells of even mild asthmatic subjects and suggest that restoring Bax expression may provide a clinical approach for restoring the normal numbers of epithelial cells and reduced mucous hypersecretion in asthma.

The linkage disequilibrium maps of three human chromosomes across four populations reflect their demographic history and a common underlying recombination pattern.

The extent and patterns of linkage disequilibrium (LD) determine the feasibility of association studies to map genes that underlie complex traits. Here we present a comparison of the patterns of LD across four major human populations (African-American, Caucasian, Chinese, and Japanese) with a high-resolution single-nucleotide polymorphism (SNP) map covering almost the entire length of chromosomes 6, 21, and 22. We constructed metric LD maps formulated such that the units measure the extent of useful LD for association mapping. LD reaches almost twice as far in chromosome 6 as in chromosomes 21 or 22, in agreement with their differences in recombination rates. By all measures used, out-of-Africa populations showed over a third more LD than African-Americans, highlighting the role of the population's demography in shaping the patterns of LD. Despite those differences, the long-range contour of the LD maps is remarkably similar across the four populations, presumably reflecting common localization of recombination hot spots. Our results have practical implications for the rational design and selection of SNPs for disease association studies.