SPOP基因起動子甲基化與轉錄調控

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Title:	SPOP基因起動子甲基化與轉錄調控
Authors:	李奕儂
Contributors:	生物科技研究所
Keywords:	SPOP 甲基化起動子
Date:	2010
Issue Date:	2011-10-31 15:20:29 (UTC+8)
Abstract:	SPOP, speckle-type POZ蛋白質，是一在人體組織內廣泛表現之蛋白質。SPOP的胺基酸序列在物種之間有高度保留性，在人類、大鼠與小鼠之中胺基酸序列100%相同，顯示SPOP的重要功能。SPOP 含TD 及POZ蛋白結構，屬於TD/POZ 蛋白家族，SPOP 蛋白的功能為參與蛋白泛素化及降解。本研究目的為分析物種之間SPOP基因起動子序列關聯性，與SPOP基因起動子甲基化對表現量造成之差異性，並利用生物資訊分析及實驗方法，了解SPOP基因mRNA的表現及其可能調控方式。根據NCBI網站提供之資料，我們針對在各物種之間(1) 轉錄起始點分布型態，與(2) 起動子序列進行分析比對，以評估物種間SPOP起動子的保留程度。利用RACE (rapid amplification of cDNA ends)，以得到完整SPOP cDNA之5’端，並分析轉錄起始點的位置。由生物資訊與實驗分析結果得知，人類SPOP基因轉錄起始點應屬於廣泛分布。根據NCBI中EST分析圖譜的資料，可以了解不同組織之間SPOP基因表現量的差異，用及時定量PCR技術驗證小鼠各組織與細胞株間SPOP mRNA表現量的差異性，實驗結果發現小鼠各組織與細胞株之間SPOP表現量的差異性，與NCBI上所收集到資訊相似。為瞭解SPOP RNA表現量的差異性是否由於起動子甲基化所造成，分別利用MSP (Methylation- Specific PCR)與BSP (Bisulfite sequencing PCR)技術分析SPOP起動子的甲基化情形。結果顯示於SPOP起動子上游序列並無甲基化現象， SPOP起動子下游延伸序列才有部分甲基化之現象。此結果顯示SPOP RNA表現量於各組織或細胞株中的差異性，並非由於起動子甲基化所造成，而造成SPOP表現差異的原因，則有待進一步探討。 SPOP is a speckle-type POZ protein which is expressed ubiquitously in human tissues. The amino acid sequences of the SPOP proteins in different species are highly conserved and are 100% identical between human, mouse and rat indicating that SPOP has essential biological functions. SPOP is a TD- and POZ-domain containing protein and a member of the TD/POZ protein family. SPOP participates in protein ubiqitination and degradation. This study aimed to analyze the sequences of Spop gene promoter between species, the expression levels of Spop and the methylation status of Spop gene promoter that may correlate with the expression of Spop. Based on NCBI information, we analyzed (1) the distribution of transcription start sites (TSS’s) and (2) alignment of the Spop promoter sequences between in different species. The 5’ RACE (rapid amplification of cDNA ends) method was used to get complete Spop cDNA 5’-end sequences, and the transcription start sites were analyzed. Four TSS’s were obtained, the results were consistent with the data from the NCBI database. According to the information from on NCBI EST database, the expression levels of the SPOP gene are different in different tissues in the mouse. Real-time PCR was used to quantify the expression levels of Spop mRNA in different tissues and cell lines in the mouse. The results showed that the expression levels of Spop mRNA were varied in different tissues and cell lines, consistent the data from the NCBI database. To address the mechanisms of different expression, MSP (Methylation- Specific PCR) and BSP (Bisulfite Sequencing PCR) were used to analyze the methylation status of the Spop promoter. The results showed that the CpG islands were unmethylated in the Spop promoter region in mouse tissues. The results indicate that differential expression levels of Spop between tissues and cell lines may not be due to the methylation status of the Spop promoter region. The mechanism that regulates the Spop gene expression needs to be further studied..
Appears in Collections:	[Graduate Institute of Biotechnology ] thesis

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