Meiosis-specific Expression And Regulation Of Mouse Miwi

The mouse Miwi gene is a member of Piwi subclade of Argonaute gene family. It was first cloned by Satomi Kuramochi-Miyagawa in 2001. Miwi is detected from the mid-pachytene stage to eary round spermatids in mouse testis. Recent research showed that Miwi can interact with a group of newly found small RNAs named piRNA, which were exclusively expressed in the testes. The Miwi-piRNA complex may participate in regulating the transcriptional activities of transposons and maintaining the genome during spermatogenesis. We are highly interested in the question of what mechanism controls Miwi’s tissue specific expression.In this report, we cloned the Miwi gene promoter and identified the potential transcriptional initiation site. We also investigated the methylation profile of CpG islands in Miwi gene core promoter and relationship between the methylation profile and Miwi expression pattern. Important transcription factors and their binding sites in core promoter region were tested to elucidate mechanism of testis specific expression of Miwi gene.1. Testis specific expression of mouse Miwi geneReal-time PCR results indicated that Miwi expressed from 12.5 dpp mouse testis to adult mouse testis with high level at 18.5 dpp mouse testis, but not expressed in liver and ovary. We performed immunohistochemistry and immunofluorescence using the adult mouse testis, and found that Miwi protein was detectable from midpachytene stage spermatocytes to round spermatids. In pachytene stage spermatocytes, Miwi protein diffused in the cytoplasm. In round spermatids, it was concentrated into the P body. The characteristic of the expression pattern of Miwi is tissue specificity.2. Methylation profile of CpG islands in promoter of Miwi geneAfter prediction of CpG island in promoter of Miwi gene using "MethPrimer" software, the methylation status of CpG islands in the promoter of Miwi gene was analyzed using bisulfite sequencing PCR(BSP) and sequence assay. The results revealed that the CpG islands in the promoter of Miwi was methylated in liver, spermatogonium, epididymal sperm and sertoli cells but not in pachytene spermatocytes and round spermatids. There was a negative correlation between the methylation status of CpG islands in promoter of Miwi gene and the Miwi expression. Results of this study provide a hint for the relationship of the methylation status of CpG islands in promoter of Miwi gene and its tissue specific expression.3. Identification of the transcriptional initiation site and the core promoter region of the mouse Miwi geneUsing 5’RACE approach, we identified a potential transcriptional initiation site at 2871 nt 5’upstream from the translation start code ATG. To identify the core promoter region of the mouse Miwi gene, a series of luciferase reporter constructs containing various 5’flanking region deletions of Miwi gene were generated by PCR and they were transient transfected into GC-1 and COS 7 cells by Liposome. The Luciferase Assays showed that the core promoter region of the mouse Miwi gene was mapped in the region from nucleotides-106 to+181. And this region was just located in the CpG islands of Miwi promoter region.4. Both NF-Y and USF activate Miwi-LucWe analyzed the Miwi gene core promoter region for putative transcriptional factor binding site (http://www.cbrc.jp/research/db/TFSEARCH.html). The predicted transcription factor binding sites of NFY and USF were located in this region. Mutation analysis showed that both the NFY binding site (CCAAT box) and USF binding site (E2 box) are indispensable for the full transcriptional activity of Miwi.Expression plasmids NFYa, NFYb, NFYc were co-transfected into the GC-1 or COS 7 cells together with the reporter plasmid containing the Miwi promoter-luciferase. The Luciferase Assays showed that each of these transcription factors can upregulate the reporter gene expression. When NFYa, NFYb, NFYc over-expressing pladmids were co-transfected with the Miwi promoter-luciferase reporter containing a ccaat box mutation, it can’t upregulate the reporter gene expression. For the transcription factor USF1 and USF2, co-transfection of USF1 and USF2 over-expressing plasmids with the Miwi promoter-luciferase pladmid showed that both USF1 and USF2 can upregulate the reporter expression, and co-transfection of these over-expressing plasmids with the Miwi promoter-luciferase pladmid containing a E2 box mutation can’t?? upregulate the reporter expression. However, co-transfection of the dominant negative plasmids of NFYa or USF with the Miwi promoter-luciferase pladmid can obviously downregulate the reporter gene expression. The results indicated that NFY can upregulate Miwi promoter-luciferase pladmid expression by interacting with the CCAAT box, and USF can upregulate its expression by interacting with the E2 box.5. Transcription factors NFY and USF interacted with the-102～-71 promoter region of the mouse Miwi gene in vitro and in vivoElectrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) analysis revealed that transcription factor NFY and USF interacted with the-102～-71 promoter region of the mouse Miwi gene in vitro and in vivo.6. The CpG methylation at the E2 box is crucial for the germ-cell specific expression of Miwi geneElectrophoretic mobility shift assay (EMSA) demonstrated that CpG methylation at the E2 box impedes USF binding to this site. There was a negative correlation between the methylation status of CpG islands in promoter of Miwi gene especially the CpG dinucleotide at the E2 box and the Miwi expression. Chromatin immunoprecipitation (ChIP) analysis revealed that transcription factor USF interacted with the Miwi promoter region in testis but not liver. These results suggested that The CpG methylation at the E2 box is crucial for the germ-cell specific expression of Miwi gene.7. A 303-nt Miwi promoter fragment is sufficient for germ-cell specific expression in vivoThe Miwi-GFP transgenic mice demonstrated that a 303-nt Miwi promoter ranging from-122 to+181 relative to the transcription start site, functional CCAAT box(-97～-93) and E2 box(-82～-77) is sufficient for germ-cell specific expression from pachytene spermatocytes to round spermatids.