Table_2_Isolation of ferret astrocytes reveals their morphological, transcriptional, and functional differences from mouse astrocytes.DOCX

Astrocytes play key roles in supporting the central nervous system structure, regulating synaptic functions, and maintaining brain homeostasis. The number of astrocytes in the cerebrum has markedly increased through evolution. However, the manner by which astrocytes change their features during evolution remains unknown. Compared with the rodent brain, the brain of the ferret, a carnivorous animal, has a folded cerebral cortex and higher white to gray matter ratio, which are common features of the human brain. To further clarify the features of ferret astrocytes, we isolated astrocytes from ferret neonatal brains, cultured these cells, and compared their morphology, gene expression, calcium response, and proliferating ability with those of mouse astrocytes. The morphology of cultured ferret astrocytes differed from that of mouse astrocytes. Ferret astrocytes had longer and more branched processes, smaller cell bodies, and different calcium responses to glutamate, as well as had a greater ability to proliferate, compared to mouse astrocytes. RNA sequencing analysis revealed novel ferret astrocyte-specific genes, including several genes that were the same as those in humans. Astrocytes in the ferret brains had larger cell size, longer primary processes in larger numbers, and a higher proliferation rate compared to mouse astrocytes. Our study shows that cultured ferret astrocytes have different features from rodent astrocytes and similar features to human astrocytes, suggesting that they are useful in studying the roles of astrocytes in brain evolution and cognitive functions in higher animals.</p

Table_1_Isolation of ferret astrocytes reveals their morphological, transcriptional, and functional differences from mouse astrocytes.XLSX

Astrocytes play key roles in supporting the central nervous system structure, regulating synaptic functions, and maintaining brain homeostasis. The number of astrocytes in the cerebrum has markedly increased through evolution. However, the manner by which astrocytes change their features during evolution remains unknown. Compared with the rodent brain, the brain of the ferret, a carnivorous animal, has a folded cerebral cortex and higher white to gray matter ratio, which are common features of the human brain. To further clarify the features of ferret astrocytes, we isolated astrocytes from ferret neonatal brains, cultured these cells, and compared their morphology, gene expression, calcium response, and proliferating ability with those of mouse astrocytes. The morphology of cultured ferret astrocytes differed from that of mouse astrocytes. Ferret astrocytes had longer and more branched processes, smaller cell bodies, and different calcium responses to glutamate, as well as had a greater ability to proliferate, compared to mouse astrocytes. RNA sequencing analysis revealed novel ferret astrocyte-specific genes, including several genes that were the same as those in humans. Astrocytes in the ferret brains had larger cell size, longer primary processes in larger numbers, and a higher proliferation rate compared to mouse astrocytes. Our study shows that cultured ferret astrocytes have different features from rodent astrocytes and similar features to human astrocytes, suggesting that they are useful in studying the roles of astrocytes in brain evolution and cognitive functions in higher animals.</p

Direct Evidence for Pitavastatin Induced Chromatin Structure Change in the <i>KLF4</i> Gene in Endothelial Cells

<div><p>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 (<i>KLF</i>) 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 <i>KLF4</i> 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 (<i>NOS3</i>) and thrombomodulin (<i>THBD</i>), were suppressed by <i>KLF4</i> knockdown. Myocyte enhancer factor-2 (<i>MEF2</i>) family activation is reported to be involved in pitavastatin-dependent <i>KLF4</i> induction. We focused on <i>MEF2C</i> among the <i>MEF2</i> family members and identified a novel functional <i>MEF2C</i> binding site 148 kb upstream of the <i>KLF4</i> 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 <i>MEF2C</i>-bound enhancer and transcription start site (TSS) of <i>KLF4</i> 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.</p></div

Binding of <i>MEF2C</i> at kb −148 from the TSS of the <i>KLF4</i> gene is essential to pitavastatin-mediated <i>KLF4</i> induction.

<p>(A) HUVECs were incubated with 1 µM pitavastatin for 4 hours. As described in <i>Methods</i>, Chromatin immunoprecipitation was performed followed by deep sequencing. The localization and magnitude of <i>MEF2C</i> binding in the <i>KLF4</i> transcription regulation region are illustrated. Two <i>MEF2C</i> binding sites in the <i>KLF4</i> 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 <i>KLF4</i> gene. The sequences of the primers used are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096005#pone.0096005.s001" target="_blank">Table S2D in File S1</a>. (C) HUVECs were transiently transfected with a KLF4-luc, (−98 kb)-KLF4-luc and (−148 kb)-KLF4-luc plasmid together with the <i>Renilla</i> luciferase plasmid, and were treated with 1 µM pitavastatin for 12 hours. Luciferase activity was measured as described in the <i>Methods</i> section. Error bars indicate the S.D. (<i>n</i> = 3), *<i>P</i><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 <i>MEF2</i> binding element. Pitavastatin-mediated induction of promoter activity was abolished by mutation of the <i>MEF2C</i> binding site. Error bars indicate the S.D. (<i>n</i> = 3), *<i>P</i><0.01 compared with pitavastatin (−), Student's t test. The <i>Firefly</i> luciferase activity value was normalized by <i>Renilla</i> luciferase activity.</p

3D-FISH confirms the proximity of <i>KLF4</i> and the <i>MEF2C</i> binding region detected by 3C.

<p>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 <i>KLF4</i> gene and <i>MEF2C</i> 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 <i>KLF4</i> gene and <i>MEF2C</i> 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 <i>KLF4</i> gene and <i>MEF2C</i> binding region are 0.45 µm with DMSO and 0.38 µm with the statin. <i>P</i><0.05 compared with pitavastatin (−), Wilcoxon rank-sum test.</p

The frequency of direct interaction between the kb −148 enhancer and promoter in the <i>KLF4</i> locus was affected by pitavastatin treatment.

<p>HUVECs were harvested and cultivated as described in the <i>Methods</i> section. (A) The localization of active Pol II obtained by ChIP-seq. The black arrow shows the <i>MEF2C</i> binding site identified by ChIP-seq. (B) A ChIA-PET library was constructed and sequenced. From the TSS of <i>KLF4</i>, 15 PETs originated and 13 of them interacted with a locus −148 kb upstream of the TSS, which result is identical with the <i>MEF2C</i> binding site observed by ChIP-seq and validated by luciferase assay in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096005#pone-0096005-g002" target="_blank">Figure 2</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096005#pone.0096005.s001" target="_blank">Table S2E in File S1</a>. 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.</p

Genes up- or down-regulated by pitavastatin treatment through <i>KLF4</i> in HUVECs.

<p>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 <i>KLF4</i> 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 <i>Methods</i>). The cluster shown in (B) contains the genes induced by pitavastatin and suppressed with si<i>KLF4</i>. Note that <i>NOS3</i> and <i>THBD</i> are included in addition to <i>KLF4</i>. These genes are indicated with red arrows. <i>KLF2</i> is shown by black arrow. The cluster shown in (C) includes the genes reduced pitavastatin treatment and induced with si<i>KLF4</i>. The sequences of the applied siRNA are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096005#pone.0096005.s001" target="_blank">Table S2A in File S1</a>.</p