The Expression of Both Murine Cetn2 and Nsdhl Genes Appears to be Regulated by a Bidirectional Promoter

• Main Authors: Hsiang-ju Lin, 林相汝
• Other Authors: 鄭登貴
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/30314050567555801697

碩士 === 國立臺灣大學 === 畜產學研究所 === 93 === Mouse centrin 2 (Cetn2) has been characterized as a small, calcium-binding protein that is expressed ubiquitously in centriole. Even the Cetn2 is known as an X-linked gene and is universally expressed in the tissues of adult mice, it should be noted that its transcript level is relatively low in adult testis. Therefore, the expression of Cetn1, a gene shares high percentage of similarity with Cent2, is only in adult testes and might serve as a compensatory mechanism. The Cetn2 and its adjacent gene, NAD(P)H steroid dehydrogenase-like (Nsdhl), are arranged head-to-head in the genome and separated by less than 300 bases. The Nsdhl, a 3b-hydroxysterol dehydrogenase, is involved in one of the later steps of cholesterol biosynthesis. The objectives of this study were to examine the (1) expression profiles of Cetn1, Cetn2 and Nsdhl in the testes during neonatal development, (2) to localize the transcripts of described genes in adult testes, (3) the promoter activity and regulator of the Cetn2 and Nsdhl; to clarify the complementary mechanism of Cetn1 and Cetn2, and the regulatory mechanism(s) of the bidirectional promoter region shared by Cetn2 and Nsdhl which presumably controlling the expression of both genes. As shown in RT-PCR and real-time PCR, Cetn2 was expressed high levels in liver, lung, spleen, brian, heart and neonatal testes but was low in adult. The decrease of mRNA level of Cetn2in testes was started at 17 day postpartum (dpp). Surprisingly, Cetn1, the retroposon of Cetn2, transcript was observed at high levels only in adult testes and was not detectable in neonatal counterparts until 15 days postpartum. Cetn1 transcripts were not found in mature sperms in adult mice, but the In situ hybridization results indicateded that Cetn1 expressed in spermatocytes and spermatids. The expression pattern of Nsdhl was almost the same as Cetn2 in mice. We also analyzed the characteristics of promoter region shared by Cetn2 and Nsdhl using the Genomatix software and followed by making serious deletions of this 1080 bp promoter, which originally included a CpG island. The bidirectional promoter activity was confirmed by a dual expressing fluorochromes vector (pBI-DE-1), which give out the red and green fluorescent light at the same time. This result shown that the Cetn2 and Nsdhl share a typical bidirectional promoter. The core promoter region and the regulatory regions were analysed by promoter deletion through another dual expression plasmid pRF. Our result shown that there was a core promoter shared by Cetn2 and Nsdhl, and the transcription at levels were regulated by some activator or repressor elements. To understand the role of the CpG island in the down regulating pattern of the two genes in adult testes, we studied its methylation patterns in five cell lines, which included NIH/3T3, and other four testes cell lines (GC-1, GC-2, TM3 and TM4). However, the CpG island located inside this bidirectional promoter might not be the decisive regulator of this bidirectional promoter in those cell lines. In conclusion, theses studies confirmed that the expression of Cetn2 and Nsdhl is coexpressed in many tissues and during neonatal testes development and regulated by a shared bidirectional promoter, which included a CpG island. The Cetn1 might play a complementary role for Cetn2 during the spermatogenesis. The competition between two genes for exression which might be regulated by the regulatory element(s) located inside the bidirectional promoter.