Direct Evidence for Pitavastatin Induced Chromatin Structure Change in the KLF4 Gene in Endothelial Cells

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.

MEF2 transcription factors are key regulators of sprouting angiogenesis

“…However, this phenotype must be considered in light of other roles for MEF2 factors in the vasculature. MEF2 has been implicated in the regulation of vascular integrity downstream from MMP10 activation and in the activation of the transcription factors KLF2 and KLF4 (Parmar et al 2006;Maejima et al 2014), important regulators of inflammation, vascular tone, and stabilization (Atkins and Jain 2007). Additionally, our work here demonstrates a direct link between MEF2 factor and many other genes involved in sprouting angiogenesis.…”

“…These results indicate that the loss of MEF2A/C results in an overall reduction of angiogenic sprouting and point to a potential role for MEF2 in regulating vascular growth more generally. To establish whether MEF2 factors directly regulate other angiogenesis-related genes and consequently the angiogenic process more generally, we analyzed genome-wide MEF2C-binding peaks associated with enhancer-specific histone marks in HUVECs from publicly available ChIPseq (ChIP combined with high-throughput sequencing) data (Maejima et al 2014). Supporting our earlier analysis, the Dll4in3 enhancer region contained a robust MEF2-binding peak, whereas no similar peak was found around the Dll4-12 nonangiogenic enhancer or elsewhere in the Dll4 locus (Supplemental Fig.…”

“…MEF2C and corresponding H3K27ac ChIP-seq data were obtained using publicly available ChIP-seq data (Maejima et al 2014) accessible at NCBI Gene Expression Omnibus database (Edgar et al 2002), accession numbers GSE32547, GSE32644, GSE32693, and GSE41553. Raw reads were trimmed with Sickle, and duplicate PCR reads were removed with rmdup.…”

HIV vasculopathy

“…Prior studies have suggested that KLF2 and KLF4 may be important transcriptional regulators of this anti-inflammatory effect of statins[36, 39, 66, 67]. Surprisingly in the present study, there was no effect of 24 weeks of statin therapy on KLF2/4 levels in PBMCs.…”

Endothelial Krüppel-Like Factor 4 Mediates the Protective Effect of Statins against Ischemic AKI

“…These results are consistent with those from previous studies showing simvastatin induced KLF4 expression and a shift to vasoprotective phenotype via the activation of the MEK5/ Erk5 cascade in human primary ECs. 31 Moreover, Maejima et al 32 also showed that pitavastatin induced KLF4 expression, accompanied by the chromatin structure changes in the promoterenhancer region of the KLF4 gene in HUVECs. Further studies on the mechanism by which statins induce KLF4 expression may shed light on novel therapeutic targets for treating and preventing ischemic AKI and other vascular diseases.…”