LRWD1基因啟動子NF-κB的轉錄活性及甲基化分析

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Title:	LRWD1基因啟動子NF-κB的轉錄活性及甲基化分析 Promoter activity of NF-κB and methylation analysis of LRWD1
Authors:	陳幸彣
Contributors:	嘉南藥理科技大學：生物科技系暨研究所張竣凱
Keywords:	啟動子分析定點突變基因甲基化 DNA methylation site-directed mutagenesis Promoter analysis
Date:	2011
Issue Date:	2011-10-25 15:30:02 (UTC+8)
Abstract:	LRWD1（Leucine-Rich repeats and WD repeat domain containing 1）是睪丸組織大量表現的基因。人類的LRWD1基因位於染色體7q22.1，整個基因體含有15個表現子（exon），蛋白質分子量約70kDa。本研究主要是探討人類LRWD1基因的轉錄調節機制，從轉錄起始點附近約1.3 kb（-1270/ +53）片段由5’端進行序列刪除，分析不同片段（-1270/+53、-1270/-1、-827/+53、-827/-1、-515/+53、-515/-1、-198/+53、-198/-1及-198/+100）於NT2D1細胞株分析啟動子活性表現情形。分析結果顯示-198/+100片段啟動子活性表現最強。因此，以此片段質體DNA作為模板，加入經過設計的突變引子，改變轉錄因子NF-κB其接合位點，使其片段質體喪失轉錄因子NF-κB結合的功能，利用firefly/Renilla luciferase活性的測定，結果發現於NT2D1細胞株中被定位點突變後的-198/+100的片段對於轉錄調控能力有明顯下降的情況，顯示NF-κB對於人類LRWD1啟動子的轉錄活性調控中，扮演著重要角色。進一步利用電泳遷移率分析（electrophoretic mobility shift assay，EMSA)及競爭性結合分析（competitive binding assay）於NT2D1細胞中證實NF-κB與LRWD1結合。另一方面為證實啟動子對於LRWD1轉錄調控作用，本研究進一步分析上述LRWD1啟動子位子的甲基化情形，藉由分析50位精蟲品質不同程度病人的LRWD1啟動子甲基化情況的分析，藉由甲基化程度與病人精蟲品質等臨床表徵的相關性分析，期能對於LRWD1活性及其對於LRWD1轉錄調控之影響有更深入的瞭解。 LRWD1（Leucine-Rich repeats and WD repeat domain containing 1）is a testis-enriched gene. The human LRWD1 gene contains 15 exons and is mapped to chromosome 7q22.1.To study the mechanisms of human LRWD1 transcriptional regulation, we used 5’-RACE to identify transcription start site. The prediction promoter region of human LRWD1 gene in -198 to +53 that the GC-rich region; lacks a TATA box（TATA-less promoter）and CCAAT box, containing putative transcription factor binding site of NF-κB, AP-2, GCF, T-Ag and Sp1. Luciferase reporter analysis of a 1.3 kb 5’-flanking sequence（-1270 to +53 with respect to the transcription start site）. Serial deletions analysis promoter activity in NT2D1 cell that -198 to +100 fragment has strong promoter activity. In mutagenesis analysis confirmed that NF-κB may human LRWD1 gene transcription factor. This results be authentication by EMSA. In addition, use EMBOSS CpG Plot forecast human LRWD1 gene upstream 500 bps CpG island. Predictions, LRWD1 upstream position in the 187-445 bps a CpG island. Methylation analysis may be help us to understand the correlation between the methylation pattern and LRWD1 transcription activity.
Relation:	校內校外均不公開，學年度：99，71頁
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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