7 research outputs found

    DNA Demethylation and USF Regulate the Meiosis-Specific Expression of the Mouse Miwi

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    Miwi, a member of the Argonaute family, is required for initiating spermiogenesis; however, the mechanisms that regulate the expression of the Miwi gene remain unknown. By mutation analysis and transgenic models, we identified a 303 bp proximal promoter region of the mouse Miwi gene, which controls specific expression from midpachytene spermatocytes to round spermatids during meiosis. We characterized the binding sites of transcription factors NF-Y (Nuclear Factor Y) and USF (Upstream Stimulatory Factor) within the core promoter and found that both factors specifically bind to and activate the Miwi promoter. Methylation profiling of three CpG islands within the proximal promoter reveals a markedly inverse correlation between the methylation status of the CpG islands and germ cell type–specific expression of Miwi. CpG methylation at the USF–binding site within the E2 box in the promoter inhibits the binding of USF. Transgenic Miwi-EGFP and endogenous Miwi reveal a subcellular co-localization pattern in the germ cells of the Miwi-EGFP transgenic mouse. Furthermore, the DNA methylation profile of the Miwi promoter–driven transgene is consistent with that of the endogenous Miwi promoter, indicating that Miwi transgene is epigenetically modified through methylation in vivo to ensure its spatio-temporal expression. Our findings suggest that USF controls Miwi expression from midpachytene spermatocytes to round spermatids through methylation-mediated regulation. This work identifies an epigenetic regulation mechanism for the spatio-temporal expression of mouse Miwi during spermatogenesis

    Deletion and point mutation analysis of the <i>Miwi</i> promoter.

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    <p>a), Schematic diagram of the CpG islands and E1, E2 and CCAAT boxes in the <i>Miwi</i> promoter. The short vertical bars represent the CpG dinucleotides. b), Promoter activities of a series of deleted constructs determined by luciferase assay. Left panel, schematic representation of the mutants linked with luciferase gene in pGL3 vector. The nucleotides are numbered from the potential transcriptional start site that was assigned +1. Right panel, the relative activities to the 5M4 construct of a series of mutant constructs determined by luciferase assays. c), Point mutation analysis in the E1, E2 and CCAAT boxes of the <i>Miwi</i> promoter by luciferase assay. The 5M4 construct of the promoter (303 bp) was used for these point mutation analyses by linkage to the luciferase gene. The relative activities to the 5M4 construct of the mutant constructs were determined by luciferase assays. The open box and open circle indicate the intact binding site of E1 and E2 or CCAAT box respectively. The filled box or circle indicates the corresponding mutations. Non modified pGL3 was used as a negative control. The firefly luciferase activity was normalized to the Renilla luciferase activity, and the data is shown as the fold increase/decrease over the luciferase activity of pGL3-5M4. The results are the mean ± S.D. of three independent experiments. *, P<0.05; **, P<0.001 compared to the 5M4 wild-type construct.</p

    Methylation profile of the <i>Miwi</i> promoter–driven transgene in <i>Miwi</i>-<i>EGFP</i> transgenic mice.

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    <p>a,c), Flow cytometric analysis of male germ cells from heterozygous <i>Miwi</i>-<i>EGFP</i> transgenic mice (a) and wild type (c). Values on the y-axis represent the number of cells, and values on the x-axis represent EGFP fluorescence intensity. The number in the R2 gate represents the percentage of the cells with EGFP fluorescence. The data is representative of three independent experiments. b,d) Flow cytometric separation and collection of EGFP-positive male germ cells from <i>Miwi</i>-<i>EGFP</i> transgenic mice (b) and wild type mice were used as a control (d). Values on the x-axis represent EGFP fluorescence intensity, and values on the y-axis represent red fluorescence intensity. The cell population in the R4 gate shows clear EGFP fluorescence in panel b. e). The positions of 20 CpG dinucleotides within the promoter region of the <i>Miwi</i>-<i>EGFP</i> transgene. f). Comparison of methylation frequencies along the CpG positions between EGFP-positive cells and EGFP-negative cells from the <i>Miwi</i>-<i>EGFP</i> transgenic mouse testis. The CpG dinucleotides in the E2 box (−80) were hyper-methylated (96%) in the EGFP negative cells, however, the CpG in the same position were completely un-methylated in the EGFP-positive cells.</p

    Over-expression of NF-Y or USF increases <i>Miwi</i>-luciferase activity.

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    <p>a), Over-expression of NF-Y upregulates <i>Miwi</i>-luciferase activity. GC-1 or COS7 cells were transfected with 0.2 µg pGL3-5M4 and 0.2 µg NF-Y expression plasmids (pCX-NFYa, pCX-NFYb, pCX-NFYc) as indicated. b), The mutated CCAAT box construct pGL3-5M4-Cmut (0.2 µg) contransfected with NF-Y expression plasmids (pCX-NFYa, pCX-NFYb, pCX-NFYc) as indicated, does not increase the <i>Miwi</i>-luciferase activity. c), The dominant negative NF-Ya expression plasmid NF-YAM29 (0.1 µg, 0.2 µg, 0.3 µg) was cotransfected with 0.2 µg pGL3-5M4, which inhibits <i>Miwi</i>-luciferase activity. d), Over-expression of USF1/2 upregulates <i>Miwi</i>-luciferase activity. pGL3-5M4 (0.2 µg) was cotransfected with 0.2 µg expression plasmids (pCX-USF1 and/or pCX-USF2) into GC-1 or COS7 cells. e), E2 box mutant pGL3-5M4-E2mut (0.2 µg) was cotransfected with 0.2 µg USF1 and/or USF2 expression plasmids (pCX-USF1 and/or pCX-USF2), indicating no increase in luciferase activity. f), The dominant negative USF expression plasmid A-USF (0.1 µg, 0.2 µg, 0.3 µg) was cotransfected with 0.2 µg pGL3-5M4, which inhibits <i>Miwi</i>-luciferase activity. pGL3-basic was employed as a negative control. pRT-TK served as an inner control for transfection efficiency. The relative luciferase activities to the 5M4 construct were determined by luciferase assay. Cells were harvested 24 hours post-transfection, and luciferase activity was measured and normalized to Renilla luciferase activity. All transfection experiments were repeated at least three times and the data are shown as the fold increase or decrease over the luciferase activity of pGL3-5M4. Mean ± S.D. are shown. *, P<0.05, as compared to the 5M4 wild-type construct.</p

    <i>Miwi</i>-<i>EGFP</i> transgenic construct and expression in transgenic mice.

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    <p>a), Schematic depiction of transgenic <i>Miwi</i>-<i>EGFP</i> construct, which contains a region of the <i>Miwi</i> promoter from −122∼+181 cloned into a pEGFP-1 vector. The <i>Eco</i>RI-<i>Afl</i>II fragment was used for microinjection. b), Western blot analysis of the EGFP protein in the adult testis, heart, liver, spleen, lung and kidney in <i>Miwi</i>-<i>EGFP</i> transgenic mouse. The EGFP expression was only detected in the testis. β-actin served as an internal control.</p

    Electrophoretic mobility shift and ChIP assays of NF-Y and USF binding to the <i>Miwi</i> promoter.

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    <p>a), NF-Y binding to <i>Miwi</i> promoter. The oligo1 corresponding to −102/−80 was γ-<sup>32</sup>P-ATP labeled and incubated with 5 µg of nuclear extract of mouse testis in the absence or presence of 50-fold excess of various competitor DNA oligos (mutant or non-labeled oligo1) or antibodies (NF-Ya, NF-Yb and NF-Yc), as indicated on the top of the gel image. The specific DNA/protein complex and the super-shift bands are indicated by arrows. The DNA/protein complex formation cannot be affected by adding competitor mutated DNA (lane 6). The sequences of oligo1 and corresponding mutant are show under the panel. b), USF binding to <i>Miwi</i> promoter. The oligo2 corresponding to −102/−71 was γ-<sup>32</sup>P-ATP labeled and incubated with 5 µg of nuclear extract of mouse testis in the absence or presence of 50-fold excess of various competitor DNA oligos (mutants or non-labeled oligo2) or antibodies (NF-Ya,USF1/2) as indicated. The specific DNA/protein complex and the super-shift band are indicated by arrows. The sequence of oligo2 and corresponding mutants are indicated under the panel. Two distinct migrating complexes were detected (lane 1). Supershift complex with anti-NFYa showed in lane 2. The addition of anti-USF1 or anti-USF2 antibody to the binding reaction caused the disappearance of the DNA/protein complex (lane 3,4). The DNA/protein complex formation can not be affected by adding mutated DNA competitor (lane 6,7,8). c), ChIP assay of NF-Y and USF binding to the <i>Miwi</i> promoter in testis. The interaction of NF-Y and USF <i>in vivo</i> with the <i>Miwi</i> promoter was determined by chromatin immunoprecipitation analysis. Mouse testis and livers were chopped into small pieces in 1% formaldehyde to cross-link endogenous proteins and DNA. Samples of sonicated chromatin were immunoprecipitated with anti-NFYA, anti-USF1, anti-USF2, no antibody (beads only) or preimmuno IgG (control) respectively. DNA isolated from immunoprecipitated material was amplified by PCR with primers to amplify the 218-bp mouse <i>Miwi</i> promoter sequence corresponding to the −206 to +12 region. Primers for an unrelated part of the sixth exon of <i>Miwi</i> were used as negative controls. The amplified PCR fragments were analyzed on 2% agarose gel. d), The relative positions of the primers in the ChIP assay.</p

    The cytosine methylation profile of the CpG islands in the <i>Miwi</i> promoter, and methylation at the E2 box inhibits USF binding.

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    <p>a), Schematic depiction of the CpG islands and CpG dinucleotides (short verticals) spanning the promoter. The relative positions of the E1, E2 and CCAAT box are indicated. b), Methylation status of each CpG dinucleotide in three CpG islands in various cell types of testis and liver. The spermatogonia, pachytene spermatocytes, round spermatids, epididymal spermatozoa and Sertoli cells were isolated from adult testis by flow cytometry. Bisulfite-treated genomic DNA was amplified and cloned into T-easy vector. Five independent clones from each sample were sequenced. Each row represents the sequence of an individual clone, whereas, each column depicts the position of each CpG site relative to the potential transcription start site. Open squares indicate unmethylated CpG sites; filled squares indicate methylated CpG sites; black triangles represent CpG site in the E2 box. c), The CpG methylation at the E2 box inhibits USF binding. The oligo3 corresponding to −92/−71 was γ-<sup>32</sup>P-ATP labeled and incubated with 5 µg of nuclear extract prepared from mouse testis in the absence or presence of various competitor DNA, as indicated on the top of the gel image. Nuclear extracts were incubated with labeled oligo3 in the absence (lane 1) or in the presence of various DNA competitors: 50-fold or 100-fold excess methylated oligo3 (lanes 2,3), mutant oligo3 (lane 4) or non-labeled oligo3 (lane 5). The specific DNA/protein complexes were indicated by arrows. The sequences of oligo3 and mutant competitor are shown under the panel.</p
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