Figure 1.
Genes up- or down-regulated by pitavastatin treatment through KLF4 in HUVECs.
Transcriptome data were derived from the average of an array performed 5 times with 1 µM pitavastatin-treated HUVECs and the average of duplicate arrays using HUVECs transfected with KLF4 siRNA or control (Ctl) siRNA, and treated with 1 µM pitavastatin for 4 hours. Fold induction is the representation of a log2 fold change to standardize the induction rate. Whole clustering analysis (A) using 384 selected genes that had significant changes in expression compared to control treatment were selected (See the details in Methods). The cluster shown in (B) contains the genes induced by pitavastatin and suppressed with siKLF4. Note that NOS3 and THBD are included in addition to KLF4. These genes are indicated with red arrows. KLF2 is shown by black arrow. The cluster shown in (C) includes the genes reduced pitavastatin treatment and induced with siKLF4. The sequences of the applied siRNA are shown in Table S2A in File S1.
Figure 2.
Binding of MEF2C at kb −148 from the TSS of the KLF4 gene is essential to pitavastatin-mediated KLF4 induction.
(A) HUVECs were incubated with 1 µM pitavastatin for 4 hours. As described in Methods, Chromatin immunoprecipitation was performed followed by deep sequencing. The localization and magnitude of MEF2C binding in the KLF4 transcription regulation region are illustrated. Two MEF2C binding sites in the KLF4 locus (−98 and −148 kb, relative to the TSS) were detected by ChIP-seq analysis. The localization of H3K27ac obtained by ChIP-seq is shown in the third lane. (B) Schematic structure of the transcriptional regulation region of the KLF4 gene. The sequences of the primers used are shown in Table S2D in File S1. (C) HUVECs were transiently transfected with a KLF4-luc, (−98 kb)-KLF4-luc and (−148 kb)-KLF4-luc plasmid together with the Renilla luciferase plasmid, and were treated with 1 µM pitavastatin for 12 hours. Luciferase activity was measured as described in the Methods section. Error bars indicate the S.D. (n = 3), *P<0.01 compared with pitavastatin (−), Student's t test. (D) HUVECs were transiently transfected with KLF4-luc, wild-type enhancer (−148 kb)-KLF4-luc and (enhancer −148 kb)-KLF4-luc containing a point mutation in the MEF2 binding element. Pitavastatin-mediated induction of promoter activity was abolished by mutation of the MEF2C binding site. Error bars indicate the S.D. (n = 3), *P<0.01 compared with pitavastatin (−), Student's t test. The Firefly luciferase activity value was normalized by Renilla luciferase activity.
Figure 3.
The frequency of direct interaction between the kb −148 enhancer and promoter in the KLF4 locus was affected by pitavastatin treatment.
HUVECs were harvested and cultivated as described in the Methods section. (A) The localization of active Pol II obtained by ChIP-seq. The black arrow shows the MEF2C binding site identified by ChIP-seq. (B) A ChIA-PET library was constructed and sequenced. From the TSS of KLF4, 15 PETs originated and 13 of them interacted with a locus −148 kb upstream of the TSS, which result is identical with the MEF2C binding site observed by ChIP-seq and validated by luciferase assay in Figure 2. The numbers in the middle indicate the location on chromosome 9 using the hg19 build program. (C) Quantitative 3C assay. HUVECs were incubated with 1 µM pitavastatin for 4 hours. Primers were designed for analyzing the crosslink frequency of the regions connected with the arches. The relative frequencies were compared between DMSO control (black arch) and statin treatment (red arch). The sequences of the primers are shown in Table S2E in File S1. The data (mean ± SD) is representative of three independent experiments with similar results. Note that the interaction between the TSS and kb −148 was increased by statin treatment.
Figure 4.
3D-FISH confirms the proximity of KLF4 and the MEF2C binding region detected by 3C.
HUVECs were incubated with 1 µM pitavastatin for 4 hours. (A) Probe design for the two-color 3D-FISH analysis of the target region on human chromosome 9q31.2. The numbers in the middle indicate the location on chromosome 9 using the hg19 build program. (B) Visualization of two-color 3D-FISH on structurally preserved HUVEC nuclei and an image of the 3D distance. FISH with probes K (red) and M (green) showing the KLF4 gene and MEF2C binding region, respectively. Nuclei were counterstained with TOPRO-3 (blue). 3D reconstruction was carried out on the captured image with Imaris software. The left panel shows the representative image of HUVECs with DMSO and the right panel shows the representative image of HUVECs with statin treatment. Magnified views of each probe sets are shown on top of the whole images. (C) The distance between the KLF4 gene and MEF2C binding region for each condition. The distance was measured using the 3D image processing and analysis software CTMS (Chromosome Territory Measurement Software) (Cybernet Co. Ltd.). 70 chromosomes were analyzed and all of the data are shown in this figure. The average distances between the KLF4 gene and MEF2C binding region are 0.45 µm with DMSO and 0.38 µm with the statin. P<0.05 compared with pitavastatin (−), Wilcoxon rank-sum test.