小鼠新穎同源框基因goosecoid-2其轉錄作用 及蛋白質定域之調控機制

Abstract

本研究旨在針對特異性表現於早期胚發育之同源框基因進行選殖，並探討其表現之分子調控機制。初始試驗首先經由小鼠囊胚cDNA基因庫之EST株系，初步篩選八個可能涉及早期胚胎發育有關之基因，進一步應用RT-PCR方式分析各基因之mRNA於埋殖前階段之胚、埋殖後之胚胎、及正常組織之表現輪廓(expressioin profile)，經由RT-PCR分析顯示，goosecoid-2基因轉錄物在胚胎發育過程中，特異性表現於埋殖前階段，尤其是雙細胞至囊胚階段，當胚胎進入埋殖後則偵測不到基因轉錄物之表現，正常組織中goosecoid-2基因轉錄物於眼睛之表現量最高，而少部份表現於睪丸。 本研究之後續試驗，進行goosecoid-2全長cDNA之選殖，啟動子區域之選殖、及蛋白質定域特性分析，並針對其分子調控機制加以探討。goosecoid-2同源框基因全長7854bp，編碼為五個表現子，位於第七染色體上。goosecoid-2 編碼序列全長為741bp，經轉譯作用為247個胺基酸，其具有兩個同源框序列，分別位在核苷酸位置46~210及350~542處。另外，核苷酸位置在487~537及658~687序列，具有入核序列(nuclear localization signal, NLS)及出核序列(nuclear export signal, NES)序列。將全長goosecoid-2架構至pEGFP-C1表現載體，並轉染於TM3細胞株，發現Goosecoid-2蛋白質大部份表現於細胞質，此現象和一般轉錄因子的表現不一樣，推測細胞內分佈可能用來調控此轉錄因子之活性。將goosecoid-2的NES及NLS序列各別進行刪除且架構於pEGFP-C1表現載體上，經由轉染至TM3細胞株顯示，刪除NLS序列之Goosecoid-2蛋白質，僅表現於細胞質之部位; 刪除NES序列後之 Goosecoid-2蛋白質僅表現於細胞核。經由以上試驗結果證實，Goosecoid-2藉由NLS序列引導Goosecoid-2蛋白質由細胞質輸入細胞核內，並經由NES序列，引導Goosecoid-2蛋白質輸出細胞核外。 此外，為謀了解goosecoid-2轉錄活性之分子調控機制，進一步選殖及定序全長6Kb之goosecoid-2啟動子區域，且分析goosecoid-2基因上游-1.5Kb之啟動子區域內，具有三個β-catenin/TCF之保守性結合位置。將goosecoid-2基因啟動子與β-catenin同時轉染於TM3細胞株時，goosecoid-2啟動子之作用活性增加16倍。此試驗顯示，β-catenin/TCF直接活化goosecoid-2基因之轉錄活性。 在本篇論文中，除了證實goosecoid-2基因可經由β-catenin/TCF來調控其轉錄活性外，亦證實goosecoid-2同源框基因為同時具有NLS及NES序列之穿梭蛋白(shuttle protein)，其藉由NES序列NLS序列穿梭於細胞核及細胞質之間，藉此特性調節基因之轉錄作用。goosecoid-2基因轉錄物局限表現於早期胚階段，在正常組織中僅表現於眼睛及少部份之睪丸組織，其在生理學功能可能扮演之意義為何，值得未來進一步應用conditional knock-down或是conditional knock-out策略詳加探討。

The purposes of the present studies were to identify those homeobox genes predominantly expressed during early stages of embryonic development in the mice and to elucidate their molecular mechanisms related to regulations of gene expression. To meet the purposes described above, mouse blastocyst cDNA library was firstly constructed and a total of eight candidate genes were successfully screened in silico, based on the EST databases. Each of the candidate genes was subsequently subjected to the RT-PCR analysis for further characterization of their expression profile in mouse embryos varying in stages of early development and adult mouse tissues. From these initially studies, a novel homeobox gene, named goosecoid-2, was identified and characterized to be specifically expressed early in two-cell up to blastocyst stages before the implantation occurred. In those adult mouse tissues analyzed, however, the expression of goosecoid-2 was constantly evidenced in eyes while a much lesser extent of its transcripts could be found in testes. In the subsequent studies, experiments were addressed on the cloning, sequencing, and characterization of the full length cDNA of goosecoid-2 and its potential promoter region. From these series studies, it was confirmed that the complete sequences of goosecoid-2 cDNA is lengthen in 741bp, encoding with a protein comprised 246 amino acids. Two homeoboxes were identified to be located at nucleotides 46~210 and 350~542, respectively. While a surprising result was observed in the initial experimental result, of that the goosecoid-2 protein was primarily presented in the cytoplasm when the fusion gene containing the full coding sequences of goosecoid-2 and EGFP, driven by the CMV promoter, had been transfected into the TM3 cells. However, in the later experiments conducted to verify the effect of goosecoid-2 coding sequences affect the localization of goosecoid-2 protein, it was found that a partial deletion of the goosecoid-2 coding sequences at either 487~537 or 658~687 nucleotides would result in significant discrepancies in their protein localization, showing the former ones only expressed in the cytoplasm whereas the later ones only expressed in the nuclei. These results indicate that the goosecoid-2 coding sequences possess both of a nuclear localization signal (NLS) and a nuclear export signal (NES), located at 487~537 and 658~687 nucleotides region, respectively. For further elucidation the molecular mechanisms related to regulations of transcriptional activity of goosecoid-2, a 6 kb in length of goosecoid-2 promoter was cloned and sequenced. Of these studies, a total of five β-catenin/TCF consensus binding sites were found to be distributed within -1.5 Kb of the goosecoid-2 promoter region. Cotransfection of CMV-β-catenin and goosecoid-2-luciferase into TM3 cells further evidenced that a 16-fold higher of luciferase activity in those TM3 cells harboring both of transfected genes when comparison was made to those cells that had been transfected with goosecoid-2-luciferase only, indicating that the β-catenin does play a vital role in regulating the transcription activity of goosecoid-2. Conclusions came to these studies were that the goosecoid-2 is a transcriptional factor regulated by β-catenin. Moreover, the goosecoid-2 is a shuttle protein equipped with both of the NLS and the NES to take up their responsibilities for the subcellular translocation between nuclei and cytoplasm, respectively. While the expression of goosecoid-2 in the mouse has been found to be limited at early embryonic stages before their implantation occurred and a constant expression in eyes and much lesser extent of expression in testes were both evidenced in the adult tissues, it is anticipated that further studies for elucidation the physiologic significances of this particular gene, based on either conditional knock-down or knock-out strategy, can be conducted in the coming future.