| 摘要: | TRIM60 屬於Tripartite Motif 鋅指蛋白家族，Trim60基因率先為本研究室所報導，目前已知TRIM60 於睪丸與著床前胚期專一性表現。本計畫為三年期的第二與三年，我們第一年結果顯示 (i) NF-B與TNF調控了Trim60基因表現(已發表於FEBS J 2011, 278:837-50)；(ii) TRIM60蛋白與 kinesin 蛋白KIF3A-KIF3B複合體交互作用。未來兩年之主要研究目標為繼續： 1. 闡釋TRIM60 蛋白於睪丸中精子生成之角色(第二年) 2. 分析Trim60基因睪丸專一性表現，與著床後胚胎中靜默不表現調控(第二/三年) 為闡釋TRIM60蛋白於睪丸中生物功能，我們將於小鼠睪丸分離不同型態的細胞，利用RT-PCR與western blot分析Trim60基因於睪丸組織表現分佈的情況。由Leydig與 Sertoli細胞分別所建立的TM3與TM4細胞株，也將用於實驗中，以進一步瞭解Trim60之表觀變化於精子生成時所扮演的角色。於Trim60基因表現調控機制方面，除了B外，尚有其他重要元件，如低氧反應因子(Hypoxia response element, HRE) 結合位等，為本計畫第二/三年度分析的重點。為探討導致Trim60除睪丸外其他組織不表現的機制，也將利用睪丸與其他組織，及不同發育期胚細胞，分析Trim60起動子甲基化程度，以及microRNA 之參與情況。上述研究主要方法，含報導基因、EMSA, ChIP, 甲基化分析和microRNA等分析。此計畫完成後，預期可瞭解 TRIM60 於睪丸與精子生成過程中所扮演的生物功能，及Trim60 於睪丸中的轉錄調控機制，對男性不孕症或可提供線索。
TRIM60 is a novel mouse tripartite protein (TRIM) composed of a RING and a B-box zinc-finger, a coiled-coil and a B30.2 functional domains, and is a member of the TRIM superfamily. The Trim60 gene was first reported by our laboratory. In adult mice, Trim60 is expressed specifically in the testis; the gene is also expressed in pre-implantation embryos until the blastocyst stage when the gene is silenced until re-expression in adult testis. To date, TRIM60 is the first TRIM protein that is known to be testis- and preimplantation embryo-specific. This proposal is the 2nd and 3rd year of a 3-year project first submitted to NSC in 2010. In the 1st year, we have shown that nuclear factor-kappa B (NF-B) and tumor necrosis factor-TNFmodulate Trim60 expression (published in FEBS J 2000, 278:837-850). We have also demonstrated interactions of TRIM60 protein with the KIF3A-KIF3B kinesin complex. To continue the study in the next two years, the major goals of this study are as follows: 1. Elucidation of biological functions of the TRIM60 protein in the testis, in particular, spermatogenesis (Year 2). 2. Analysis of regulation of testis-specific gene expression (Years 2 & 3). For elucidation of the biological role of the TRIM60 protein in spermatogenesis, various cell types of the mouse testis will be isolated for studies on biological functions will be performed by RT-PCR and western blot analysis. The Leydig- and Sertoli-derived TM3 and TM4 cells will also be used in the study. For gene regulation, our 1st year data have revealed a B site targeted by the NF-B proteins. Besides the B site, another cis sequence, the hypoxia response element (HRE), has also been identified on the promoter of Trim60. Thus, the second specific aim of the project is to determine the role of HRE, and other potential cis-acting elements, in modulating testis-specific Trim60 gene expression by further reporter analysis, electrophoretic mobility shift and chromatin immunoprecipitation assays. To determine Trim60 silencing in other tissues and at post-implantational developmental stages, possible regulation of the Trim60 promoter by methylation and post-transcriptional regulation of microRNA will also be examined It is anticipated that, at the end of this 3-year study, we will have elucidated how the Trim60 gene is regulated in its expression and the biological role of the TRIM60 protein in spermatogenesis in the testis. The data obtained in this work may shed light on spermatogenesis and infertility. |
