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

Date Issued

2005

Date

2005

Author(s)

Lin, Hsiang-ju

DOI

zh-TW

URI

http://ntur.lib.ntu.edu.tw//handle/246246/63613

Abstract

小鼠之Cetn2為表現於中心粒之小型鈣結合蛋白，Cetn2為X聯鎖基因且廣泛表現於各種成年小鼠組織中，值得注意的是其轉錄量於成年小鼠睪丸中較低。而Cetn2之反轉錄子Cetn1則是僅於表現於成年小鼠睪丸中，可能係藉此彌足Cetn2表現降低之情況。而Cetn2與其毗鄰基因Nsdhl則係以頭對頭排列方式呈現，而其間隔少於300鹼基對；Nsdhl為一種3b-氫氧類醇脫氫酶，作用於膽固醇生成之晚期步驟中。本研究之目的在於探討(1)分析Cetn1、Cetn2與Nsdhl三基因於小鼠睪丸發育過程中的轉錄表現，(2)性成熟成年小鼠睪丸中之轉錄位置，(3)Cetn2及Nsdhl共用之啟動子活性與調控等，冀能藉而釐清Cetn1及Cetn2之互補表現與Cetn2與Nsdhl之雙向性啟動子調控機制。由RT-PCR與real-time PCR之結果顯示，Cetn2表現於肝、肺、脾、腦、心與新生小鼠睪丸，卻於成年小鼠睪丸中具較低之表現。而其睪丸轉錄作用減少約起自於出生後17日。相對而言，Cetn2之反轉錄子Cetn1則僅於成年小鼠睪丸中有大量轉錄物，可被測得，且其轉錄產物僅在出生後第15日始克見及；然該基因之表現不見於成熟精子，經原位雜合法試驗結果顯示，該基因表現於精母細胞與精細胞中。而Nsdhl之表現圖譜則與Cetn2相似。以Genomatix軟體預測Cetn2與Nsdhl之啟動子區域，爾後構築包含單一CpG島，長度為1080bp之啟動子為研究基礎。試驗利用雙螢光表現質體系統(pBI-DE-1)，証實該啟動子確具有雙向啟動基因轉錄之功能，進而証實Cetn2與Nsdhl係共用一典型雙向性啟動子；而該啟動子之核心區域與調控序列則利用啟動子缺失與另一雙表現質體pRF進行分析；結果發現兩基因共用一段核心啟動子，且其轉錄量由不同活化及抑制因子所調控。為了解CpG島於成熟睪丸造成兩基因表現下降時扮演之角色，故分析四種睪丸細胞株(GC-1, GC-2, TM3, TM4)與NIH/3T3細胞株中CpG島之甲基化圖譜。試驗結果證實，該CpG島於五種細胞株中皆不受甲基化修飾，暗示CpG島在此雙向啟動子之調控可能不具決定性之影響。由本試驗結果顯示，Cetn2與Nsdhl於多數組織及睪丸發育過程中呈共表現；Cetn1可能於精子發生過程中扮演補足Cetn2之角色。Cetn2與Nsdhl由一含有CpG島之雙向性啟動子調控，並共用相同核心啟動子片段，而競爭強度以決定轉錄方向者，可能為雙向啟動子上調控因子。

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.

Subjects

雙向性啟動子

　中心體蛋白

bidirectional promoter

centrin

Type

thesis