GSE Is a Maternal Factor Involved in Active DNA Demethylation in Zygotes

<div><p>After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified <i>Gse</i> (<u>g</u>onad-<u>s</u>pecific <u>e</u>xpression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5<b> </b>mC) and the accumulation of 5-hydroxymethylcytosine (5<b> </b>hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, <i>Lemd1</i>, <i>Nanog</i> and the upstream regulatory region of the <i>Oct4</i> (also known as <i>Pou5f1</i>) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5<b> </b>mC and 5<b> </b>hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5<b> </b>mC to 5<b> </b>hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.</p> </div

Preferential binding of GSE to the paternal chromatin.

<p>(A) Brief scheme of two pretreatment procedures before immunocytochemical staining: pretreatment with Triton X-100 before PFA fixation (TP condition) versus conventional PFA fixation (PT condition). (B) Immunocytochemical analysis of GSE (red) in zygotes at PN2 and PN3 after pretreatment under the PT or TP conditions. Histone H3 is shown in green as a control for chromatin-bound factors under the TP condition. Numbers of zygotes analyzed for each group were: PN2 (PT condition), 20; PN2 (TP condition), 25; PN3 (PT condition), 20; PN3 (TP condition), 30. DNA was stained with DAPI (blue). Key: ♀, female pronucleus; ♂, male pronucleus; PB, polar body; scale bars = 50 µm. (C) Immunocytochemical analysis of GSE in parthenogenetic embryos after pretreatment under the PT or TP conditions. H3 is shown in green as a control for chromatin-bound factor under the TP condition. Numbers of zygotes analyzed for each group: PT condition, 15; TP condition, 15. DNA was stained with DAPI (blue). Key: ♀, female pronucleus; PB, polar body; scale bars = 50 µm. (D) Immunoprecipitation with an anti-GSE antibody followed by immunoblotting using anti-H3 or -H4 antibodies in zygotes at PN2 and PN3. Normal rabbit IgG was used as a negative control. Arrows indicate each respective band. Three independent experiments were performed.</p

Localization of GSE in fertilized mouse oocytes.

<p>The dynamic appearance of the pronuclear localization of GSE is illustrated during mouse zygotic development. Shown are representative images of zygotes stained with DAPI (blue) and with anti-GSE antibody immunostaining (red). Key: ♀, female pronucleus; ♂, male pronucleus; PB, polar body; scale bars = 50 µm. The numbers of zygotes analyzed for each stage were: PN1, 11; PN2, 11; PN3, 10; PN4, 10; and PN5, 10.</p

Bisulphite sequencing analysis of LINE1, <i>Lemd1</i>, <i>Nanog</i>, <i>Oct4</i> and H19<i>-</i>DMR in GSE-KD oocytes and zygotes.

<p>(A) Bisulphite sequencing of CpG sites within LINE1 genomic regions in spermatozoa and in untreated and GSE-KD oocytes and zygotes at PN3–4. (B) Bisulphite sequencing of CpG sites within <i>Lemd1</i> genomic regions in spermatozoa and in untreated and GSE-KD oocytes and zygotes at PN3–4. (C) Bisulphite sequencing of CpG sites within <i>Nanog</i> genomic regions in spermatozoa and in untreated and GSE-KD oocytes and zygotes at PN3–4. The methylation profile of the CpG sites in detail is indicated in Figure S8. (D, E, F) Bisulphite sequencing of CpG sites within the upstream distal enhancer (DE) (D), proximal enhancer (PE) (E) and promoter regions (F) of the <i>Oct4</i> gene in spermatozoa and untreated and GSE-KD oocytes and zygotes at PN3–4. (G) Bisulphite sequencing of CpG sites within the H19-DMR in sperm and untreated and GSE-KD oocytes and zygotes at PN3–4.</p

Maternal GSE-knockdown (GSE-KD) zygotes obtained from antisense RNA injection and in vitro fertilization (IVF).

<p>(A) Scheme of the experimental procedures. In immunocytochemistory, bisulphite sequencing, and MeDIP and hMeDIP (methylated and hydroxymethylated DNA immunoprecipitation) analyses, EGFP RNA-uninjected MII oocytes, incubated under similar conditions, were used as untreated controls. (B) Knockdown of GSE expression by an antisense RNA was confirmed by quantitative RT-PCR analysis. The relative ratios were obtained by dividing the expression level of the <i>Gse</i> gene by the expression level of the <i>G3PDH</i> gene. More than 90 oocytes from three independent experiments were analyzed. Shown are statistically significant differences between untreated and GSE-KD oocytes (*<i>p</i><0.05). Bars represent the standard error of the mean. (C) Knockdown of GSE protein expression was confirmed by immunoblot analysis of MII oocytes just before IVF or in 2-cell embryos. Ninety oocytes from three independent experiments were analyzed. Actin was used as a loading control. (D) Densitometric quantification of the immunoblot bands of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060205#pone-0060205-g003" target="_blank">Figure 3C</a> showing statistically significant differences between untreated and GSE-KD oocytes or embryos (*<i>p</i><0.05). Bars represent the standard error of the mean.</p

Carbonyl Sulfide Hydrolase from <i>Thiobacillus thioparus</i> Strain THI115 Is One of the β‑Carbonic Anhydrase Family Enzymes

Carbonyl sulfide (COS) is an atmospheric trace gas leading to sulfate aerosol formation, thereby participating in the global radiation balance and ozone chemistry, but its biological sinks are not well understood. <i>Thiobacillus thioparus</i> strain THI115 can grow on thiocyanate (SCN^–) as its sole energy source. Previously, we showed that SCN^– is first converted to COS by thiocyanate hydrolase in <i>T. thioparus</i> strain THI115. In the present work, we purified, characterized, and determined the crystal structure of carbonyl sulfide hydrolase (COSase), which is responsible for the degradation of COS to H₂S and CO₂, the second step of SCN^– assimilation. COSase is a homotetramer composed of a 23.4 kDa subunit containing a zinc ion in its catalytic site. The amino acid sequence of COSase is homologous to the β-class carbonic anhydrases (β-CAs). Although the crystal structure including the catalytic site resembles those of the β-CAs, CO₂ hydration activity of COSase is negligible compared to those of the β-CAs. The α5 helix and the extra loop (Gly150–Pro158) near the N-terminus of the α6 helix narrow the substrate pathway, which could be responsible for the substrate specificity. The <i>k</i>_cat/<i>K</i>_m value, 9.6 × 10⁵ s^–1 M^–1, is comparable to those of the β-CAs. COSase hydrolyzes COS over a wide concentration range, including the ambient level, <i>in vitro</i> and <i>in vivo</i>. COSase and its structurally related enzymes are distributed in the clade D in the phylogenetic tree of β-CAs, suggesting that COSase and its related enzymes are one of the catalysts responsible for the global sink of COS

Effect of GSE knockdown on the development of early mouse embryos.

*<p>In the untreated group, M II oocytes were cultured for 8.25 h and fertilized <i>in vitro</i>.</p><p>Key: EGFP, enhanced green fluorescent protein; hpi, hours postinsemination; GSE-KD, GSE knockdown.</p

Association of the various aspects of BMI history with type 2 diabetes.

<p>Association of the various aspects of BMI history with type 2 diabetes.</p

Clinical characteristics of the study subjects.

<p>Clinical characteristics of the study subjects.</p

Association results between 2 SNPs and type 2 diabetes.

<p>Association results between 2 SNPs and type 2 diabetes.</p