腦疾病相關之頭對頭基因對SERPINI 1及PDCD10其轉錄機轉之研究

A Fine-Tuned Transcriptional Regulation of a Brain Diseases-Related Head to Head Gene Pair: SERPINI1 and PDCD10

陳炳言(Ping-Yen Chen)

指導教授 ： 黎耀基 ； 吳成文

國立清華大學/生命科學院/生命科學系/博士(2008年)

https://doi.org/10.6843/NTHU.2008.00067

摘要

不論原核生物及低等真核生物，為了能有效調節不同基因之表現層次，常可見其基因群以非常靠近彼此的方式緊密排列。然而，近幾年來逐漸有證據顯示，在哺乳動物體內的某些基因，在染色體上也能如原核生物的基因般彼此緊密排列在一起。本論文針對一對和腦疾病相關的異源基因對進行研究。一個是編號SERPINI1的絲氨酸蛋白酵素抑制劑基因 (serine protease inhibitor gene)，它的表現量在腦癌病患中特別低；另一個是編號PDCD10的計畫性細胞死亡基因 (programmed cell death gene)，此基因的突變會造成大腦海綿狀畸形(cerebral cavernous malformation, CCM) 的發生。我們發現，這二條基因以「頭對頭」的方式緊密排列於人類染色體3q26.1的位置，且其基因間區 (intergenic region) 只有851個鹼基對。不像SERPINI1只在正常腦組織大量表現，而在腦細胞癌化後表現量大幅降低；PDCD10則是大量表現於人體各個不同組織器官，且其基因表現量在許多種腫瘤細胞中顯示異常。經由功能性報導基因分析實驗，我們發現緊鄰PDCD10方向的一段175個鹼基對序列，此序列本身G及C鹼基含量豐富，並且具備著可雙向啟動基因表現 (bidirectional promoter) 的特殊機能。此外，我們發現在這個175個鹼基對序列中包含一個非典型的E-box序列 (5’-CATGmCG-3’)，此序列對於下游兩基因的表現非常重要。當我們把這個小片段在175個鹼基對中或是851個鹼基對中去除掉，再去執行報導基因分析實驗，結果顯示不論是那個基因方向，啟動子都呈現失去活性的狀態。我們藉由in vitro的super shift 分析和in vivo 的染色質沈澱試驗發現：著名的c-Myc 轉錄因子能夠和這個非典型的E-box序列結合。雙向調控子的活性以及其下游基因的mRNA表現量也都會因為c-Myc 轉錄因子在細胞中的大量表現而增加。再則，我們發現在非典型的E-box序列上特定鹼基的甲基化可以有效率的阻止c-Myc 轉錄因子和它的結合。這些數據告訴我們，c-Myc 轉錄因子扮演一個重要的活化子的角色來啟動SERPINI1和PDCD10的表現，而且也可能參與調控這兩個基因造成 它們在腦癌細胞中表現量的不同。另一方面。我們發現此基因間區內的第176至473個鹼基對序列，分別具有促進PDCD10與阻遏SERPINI1基因表現的特殊調控功能。在這個片段中的兩個不同位置的CAAT box似乎也參與兩基因表現量之調控。
以上這些實驗成果，加上我們在不同物種之間所觀察到的基因間區序列的高度保留性，我們推論有一個精密調控的分子機制存在於這851個鹼基對中。這個機制必定也是造成兩基因啟動子強弱不一致以及其組織專一性不同的原因。

關鍵字

雙向調控之轉錄子 ； 絲氨酸蛋白酵素抑制劑基因 ； 計畫性細胞死亡基因 ； 轉錄調控 ； 去氧核醣核酸之甲基化

並列摘要

In prokaryotic and lower eukaryotic genomes, genes are often organized in a proximal pattern to ensure effective gene co-expression and co-regulation. Increasing evidences, however, suggest that in mammalian genomes some adjacent genes also tend to be in close physical vicinity. Two human brain disease-related genes, one coding for a serine protease inhibitor (SERPINI1) which is down-regulated in brain tumors, and the other coding for a programmed cell death gene (PDCD10) which is mutated in cerebral cavernous malformation (CCM), are adjacently located in a head-to-head configuration on chromosome 3q26.1. The two genes are only separated by as little as 851 bp. Unlike SERPINI1, which is expressed mainly in normal brain tissues but is down-regulated in brain tumors, PDCD10 is ubiquitously expressed in all normal tissues while its transcription becomes aberrant in different types of cancers. By performing the functional reporter gene analysis, a GC-rich 175-bp fragment within the intergenic region was shown to function as a bidirectional promoter to drive the transcription of the two flanking genes. In addition, we found that a non-canonical E-box element (5’-CATGCG-3’) identified within the minimal bidirectional promoter is crucial for the expression of these two genes. Deletions of this E-box either in the 175-bp minimal promoter or in the 851-bp full-length promoter of these two genes totally abolish the promoter activities in the functional reporter assays. Through the in vitro supershift experiment and the in vivo chromatin immunoprecipitation assay, we discovered that c-Myc transcription factor is able to bind to this E-box. The bidirectional promoter activities and mRNA expressions of the two genes were both affected while varying the amounts of cellular c-Myc protein. Furthermore, we discovered that the methylation of the specific C nucleotide within the E-box sequence (5’-CATGmCG-3’) would effectively interfere the binding of c-Myc to E-box. Taken together, we propose that c-Myc plays an important activator in turning on the transcription of SERPINI1 and PDCD10 and is possibly involved in differential expressions of these two flanking genes in brain tumors. On the other hand, one regulatory fragment which is from nt 176 to 473 of the intergenic region between SERPINI1 and PDCD10 possessing both the repressive activity for SERPINI1 and the enhancing activity for PDCD10 was found to coordinate the expression and regulation of both genes. Two CAAT boxes within this fragment seem to be involved in modulating the expression amount of two flanking genes.
These data, along with a high degree of sequence conservation found in other species, suggest that a fine regulatory mechanism exists within this short 851-bp intergenic region. Such a mechanism should result in the asymmetrical gene expression pattern as well as the tissue specificity of SERPINI1 and PDCD10.

並列關鍵字

bidirectional promoter ； SERPINI1 ； PDCD10 ； transcriptional regulation ； c-Myc ； DNA methylation

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