9 research outputs found
DNA demethylation of the regulatory region of <i>mil-1</i> resulted in upregulation of its expression in ES cells.
<p>(A) Bisulfite sequencing analysis of the regulatory region of <i>mil-1</i> and (B) quantitative RT-PCR analysis of <i>mil-1</i> expression were performed on embryonic stem (ES) cells with or without siRNA-mediates <i>Dnmt1</i> knockdown (<i>Dnmt1</i> KD/Con KD). (A) The regulatory region became more hypomethylated following <i>Dnmt1</i> knockdown. (B) Histogram represents the relative expression level of <i>mil-1</i> in the <i>Dnmt1</i>-knockdown ES cells. The expression level in the control ES cells (Con KD) was set as 1.0. <i>Gapdh</i> PCR signal was used an internal control to measure relative expression. The data were obtained from four independent experiments. *p<0.05. Error bars represent SEM. (C) Luciferase activity of the reporter vectors with methylated or unmethylated regulatory region of <i>mil-1</i> in ES cells. Luciferase activity was normalized against the activity of a cotransfected <i>Renilla</i> construct. The liciferase activity of the methylated construct (Methylated 3.0k-pCpGL) was set as 1.0. The data were obtained from six independent experiments. *p<0.05. Error bars represent SEM.</p
Flanking regions of the PGC-specific genes also become hypomethylated during PGC development.
<p>(A) Comparison of the <i>Ifitm</i> genes consensus element (ICE) of <i>mil-1</i>/<i>Ifitm3 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046036#pone.0046036-Tanaka3" target="_blank">[25]</a> with the homologous sequences found in the putative regulatory regions flanking <i>Blimp1</i>/<i>Prdm1</i>, <i>Prdm14</i>, <i>Stella</i>/<i>Dppa3</i>, and <i>Nanos3</i>. (B) Bisulfite sequencing analysis of the flanking regions of <i>Blimp1</i>, <i>Stella</i>, and <i>Dazl</i> was performed using epiblasts (E6.0 and E6.75) and PGCs (E7.5 and E10.5). The flanking regions of <i>Blimp1</i> and <i>Stella</i>, like those in <i>mil-1</i>, were also progressively demethylated during PGC development, whereas that of <i>Dazl</i> was maintained hypermethylated in PGCs at E10.5.</p
The regulatory region of <i>mil-1</i> becomes hypomethylated during PGC development.
<p>Bisulfite sequencing analysis of the regulatory region of <i>mil-1</i> was performed using epiblasts, PGCs/somatic cells purified as GFP positive/negative cells from embryos at each embryonic day (E). The rectangle containing <i>mil-1</i> in the top line represents an exon, and the numbers with ‘kb (kilobase)’ under the line indicate distance from the transcription start site (TSS). The box outlined in green represents the regulatory element required for PGC-specific expression and the <i>Ifitm</i> genes consensus element (ICE) is shown in more detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046036#pone-0046036-g004" target="_blank">Figure 4A</a>. Each circle corresponds to a CpG site in the regulatory region, and the degree of gray in each circle corresponds to the level of DNA methylation.</p
Knockdown of <i>Dnmt1</i> causes upregulation of <i>mil-1</i> and <i>Stella</i> expression in PGCLCs.
<p>Quantitative RT-PCR analysis of <i>mil-1</i> and <i>Stella</i> expression in PGCLCs (PGC-like cells) with or without <i>Dnmt1</i> knockdown (<i>Dnmt1</i> KD/Con KD). The expression level in EpiLCs was set as 1.0. Shown is a representative data from two independent experiments.</p
The expression of <i>mil-1</i>, <i>Blimp1</i>, and <i>Stella</i> become upregulated during PGC development.
<p>(A, B, C) Quantitative RT-PCR analysis of the expression of (A) <i>mil-1</i>, (B) <i>Blimp1</i>, and (C) <i>Stella</i> was performed using epiblasts (E6.0) and PGCs (E7.5 and E9.0). Histograms represent relative expression levels of these three genes at each developmental stage. The averages of expression levels in the epiblasts (E6.0) were set as 1.0. <i>Gapdh</i> PCR signal was used as an internal control to measure relative. The data were obtained from three individual embryos. *p<0.05. Error bars represent SEM.</p
Bivalent histone modification on the somatic genes in PGCs.
<p>(A, B) ChIP analysis with the H3K4me3 or H3K27me3 antibodies for the promoter regions of somatic genes (<i>Hoxa1</i>, <i>Hoxb1</i>, and <i>Gfap</i>) and of the PGC-specific genes (<i>mil-1</i>, <i>Blimp1</i>, and <i>Stella</i>) was performed on EpiSCs (A), and male and female PGCs at E13.5 (B), showing the bivalent histone modification. Histogram represents ratios of the immuno-precipitated chromatin to the input chromatin, which was quantified by quantitative PCR analysis. Also shown are results using beads only as a no antibody control (NAC). Shown is a representative data from two independent experiments.</p
Knockdown of <i>Dnmt1</i> causes hypomethylation of <i>Blimp1</i> and <i>Stella</i> flanking regions and upregulation of <i>Blimp1</i> and <i>Stella</i> expression in ES cells.
<p>(A) Bisulfite sequencing analysis of the flanking regions of <i>Blimp1</i> and <i>Stella</i> and (B) quantitative RT-PCR analysis of <i>Blimp1</i> and <i>Stella</i> expression were performed using ES cells with or without <i>Dnmt1</i> knockdown treatment (<i>Dnmt1</i> KD/Con KD).</p
Repression of somatic gene expression does not depend on DNA methylation in PGCs.
<p>(A) Bisulfite sequencing analysis of the flanking regions of <i>Hoxa1</i>, <i>Hoxb1</i>, and <i>Gfap</i> was performed using epiblasts (E6.0), epiblast stem cells (EpiSCs), and PGCs (E13.5), showing hypomethylation in PGCs. (B) Bisulfite sequencing analysis of the regulatory region of <i>Gfap</i> and (C) quantitative RT-PCR analysis of the <i>Gfap</i> expression were performed using ES cells with or without <i>Dnmt1</i> knockdown treatment (<i>Dnmt1</i> KD/Con KD).</p
Transgenic expression of <i>Telomerase reverse transcriptase</i> (Tert) improves cell proliferation of primary cells and enhances reprogramming efficiency into the induced pluripotent stem cell
<p>The enzymatic activity of telomerase is important for the extension of the telomere repeat sequence and overcoming cellular senescence. We generated a conditional transgenic mouse line, carrying the <i>telomerase reverse transcriptase</i> (Tert) expression cassette, controlled by the Cre-<i>loxP</i>-mediated recombination. In our study, Cre recombinase expression efficiently activated Tert expression, resulting in its increased enzymatic activity, which extended the period of cellular proliferation until the keratinocytes entered senescence. This suggests that transgenic Tert expression is effective in enhancing primary cell proliferation. Notably, Tert expression increased colony formation of induced pluripotent stem (iPS) cells after the introduction of four reprogramming factors, Oct-4, klf4, SOX-2, and c-Myc into the transgenic fibroblasts. To the best of our knowledge, this is the first study to show that the transgenic Tert expression enhances reprogramming efficiency of iPS cells, which indicates a critical role for Tert in the reprogramming process.</p> <p>Transgenic expression of telomerase reverse transcriptase improves cell proliferation of primary cells and enhances reprogramming efficiency into the iPS cell.</p