Regulatory mechanisms related to transcription and protein localization of goosecoid-2, a novel homeobox gene,expressed in the mouse

• Main Authors: Hung-Yi Hsu, 許虹宜
• Other Authors: 鄭登貴
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/34717618477873181082

碩士 === 國立臺灣大學 === 畜產學研究所 === 93 === 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.