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

Azuma Taguchi ,Jun Ichi Suehiro ,Yijun Ruan,Masaomi Nangaku,Yasuharu Kanki,Junko Stevens ,Juro Sakai,Takao Hamakubo,Shozo Yamamoto ,Naohito Nozaki,Akira Endo,Imari Mimura,Tsuyoshi Inoue,Seiichiro Tsutsumi ,Huay Mei Poh,Guoliang Li,Shoulian Dong,Takeshi Kawamura,Tatsuhiko Kodama,Hideyuki Tanabe,Haruki Shibata,Takahide Kohro,Yoshihiro Ohta,Toshiya Tanaka,Takashi Minami,Hiroko Iwanari,Kiyomi Kaneki,Yasunobu Yoshinaka,Youichiro Wada,Seiichi Tanabe ,Mika Kobayashi,Akira Sugiyama,Hiroshi Kimurâ ,Sigeo Ihara,Hiroyuki Aburatani,Takeshi Doi,Takashi Maejima,Hirofumi Aruga,Kazuki Yamamoto,Xiaoan Ruan,Akimune Asanuma

doi:10.1371/journal.pone.0096005

Abstract

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.

Highlights

IntroductionMultiple lines of evidence, including randomized clinical trials, have suggested that statins directly affect vascular cells, and exert atheroprotective effects through a modification of gene expression [4]
Since the discovery of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors [1], commonly known as statins, they have come to be widely-used cholesterol lowering drugs [2,3].Multiple lines of evidence, including randomized clinical trials, have suggested that statins directly affect vascular cells, and exert atheroprotective effects through a modification of gene expression [4]
To evaluate the gene expression profiles of the Kruppel-like factor (KLF) family members, we performed microarray analysis of Human umbilical vein endothelial cells (HUVECs) treated with pitavastatin for 4 hours

Summary

Introduction

Multiple lines of evidence, including randomized clinical trials, have suggested that statins directly affect vascular cells, and exert atheroprotective effects through a modification of gene expression [4]. In endothelial cells (ECs), statins induce the mRNA level of nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), and this effect is reported to be mediated by the induction of the transcription factor kruppel-like factor 2 (KLF2) acting through the myocyte enhancer factor 2 (MEF2) binding site [5,6]. KLF2 is of particular interest in atherogenesis, because cholesterol accumulation and low shear stress in the vascular wall are two major aspects of atherosclerotic plaque formation, suggesting that the KLF family exerts important biological effects on the cellular phenotype. We focused on KLFs to investigate the molecular mechanism of gene regulation in statin-treated vascular cells

Methods

Results

Conclusion