Bhlhe40轉錄因子在肌肉細胞中調控過氧化氫體功能及數量的分子機制;Insights into the Molecular Mechanisms by Which Bhlhe40 Regulates Peroxisome Functions and Number in Myogenic Cells.

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題名:	Bhlhe40轉錄因子在肌肉細胞中調控過氧化氫體功能及數量的分子機制;Insights into the Molecular Mechanisms by Which Bhlhe40 Regulates Peroxisome Functions and Number in Myogenic Cells.
作者:	陳盛良
貢獻者:	國立中央大學生命科學系
關鍵詞:	骨骼肌;Bhlhe40;PGC-1α;活性氧化物質;過氧化氫體;肌肉生成;skeletal muscle;Bhlhe40;PGC-1α;ROS;peroxisome;myogenesis
日期:	2018-12-19
上傳時間:	2018-12-20 11:28:30 (UTC+8)
出版者:	科技部
摘要:	Bhlhe40為一個轉錄因子，在骨骼肌中會因為genotoxic stress與缺氧而被高度誘導，以保護肌肉免於活性氧化物質(ROS)的傷害。過氧化氫體(peroxisome)為重要的代謝胞器，參與各式長鏈、不飽和及支鏈脂肪酸的β-氧化，並且藉由Catalase等抗氧化酵素以移除因有氧呼吸所產生的ROS。過氧化氫體的數量及功能皆會被轉錄活化輔因子PGC-1α透過活化許多結合在DNA上的轉錄因子所密切控制。我們近年的研究發現PGC-1α能直接與Bhlhe40結合，並透過共同佔據在PGC-1α目標基因上的啟動子(promoter)或強化子(enhancer)，轉而抑制PGC-1α的激活活性。若抑制Bhlhe40基因表現則能緩解它對PGC-1α的抑制，並能觀察到ROS、脂肪酸氧化、粒線體DNA及PGC-1α的目標基因表現都有所提升。我們初步的結果已更進一步確認，Bhlhe40在調控過氧化氫體數量及功能確實扮演重要的角色。我們發現在抑制Bhlhe40後，過氧化氫體的數量以及功能皆會提升，但過度表現Bhlhe40反而會抑制此效果。當持續性活化的PGC-1α相互作用區域(簡稱VBH135)被過度表現時，過氧化氫體的功能與數量皆增加，但同時卻降低了ROS的量。分析參與過氧化氫體的功能和生物合成的重要基因之表現後，我們發現Bhlhe40藉由調控這些標的基因的表現來密切調控過氧化氫體的功能與數量。在這次研究中，我們將透過分析Bhlhe40在遺傳學(genetics)及表觀遺傳學(epigenetics)的層面上對這些目標基因的調控，以便詳細探討Bhlhe40在分子層面上對過氧化氫體的功能和數量動態平衡的調控機制。因此我們提出以下實驗，以探討Bhlhe40對參與過氧化氫體的功能和數量動態平衡的目標基因之分子調控機制：(一)目標基因功能獲得(gain)或喪失(loss)對過氧化氫體和肌肉生成(myogenesis)的影響。(二)構築目標基因的啟動子和cis-elements報導載體，並且研究它們被Bhlhe40調控的機制。(三)探討在Bhlhe40及輔因子(cofactors)在目標基因啟動子和cis-elements上的recruitment(四)分析被Bhlhe40所影響的表觀遺傳(epigenetic)修飾及染色質(chromatin)結構。 ;Bhlhe40 (also known as Stra13, Dec1, Sharp2, or BHLHB2) is a transcription factor that is highly induced by genotoxic stress and hypoxia, two conditions that are closely associated with exercise, in skeletal muscle (SKM) to protect this tissue from the harmful reactive oxygen species (ROS) associated with exercise. Unfortunately, the detailed mechanisms by which Bhlhe40 protect SKM from ROS induced damages have not been revealed yet. Peroxisomes are important metabolic organelles that participate in the β-oxidation of vary long chain, unsaturated, and branched fatty acids. Besides, antioxidant enzymes, including Catalase, SOD1, and PRDX5, localized in the peroxisomes are critical to the removal of the harmful ROS concomitantly generated from the oxidation of substrates in mitochondria (MITO) and peroxisomes. Both the number and functions of peroxisomes are highly regulated by the transcriptional coactivator PGC-1α. Both the number and functions of peroxisomes are highly regulated by the transcriptional coactivator PGC-1α. It has been shown that PGC-1α plays critical roles in the regulation of the biogenesis and function of mitochondria and peroxisomes through coactivating the activity of various DNA-binding transcription factors, such as PPARγ, ERRα, and NRF1. Our recent discovery showed that PGC-1α directly interacted with Bhlhe40 and they co-occupied PGC-1α targeted gene promoters/enhancers, which in turn repressed PGC-1α transactivational activity. Bhlhe40 repressed PGC-1α activity through HDACs recruitment and preventing the relief of PGC-1α intra-molecular repression. Unleashing this repression by knockdown of Bhlhe40 expression increased levels of ROS, fatty acid oxidation, mitochondria DNA, and the expression of PGC-1α target genes. Our preliminary results have further confirmed that Bhlhe40 does play important roles in regulating peroxisome functions and number. We found that both the number and activity of peroxisomes were increased upon knockdown of Bhlhe40 expression but were repressed by its over-expression. Over-expression of a constitutively active PGC-1α-interactive domain (named as VBH135) of Bhlhe40 mimicked the effects of its knockdown on peroxisomes but simultaneously reduced ROS level. Unsaturated fatty acid oxidation, insulin response, and oxidative respiration were highly enhanced in Bhlhe40 knockdown or VBH135 over-expressed cells, suggesting the importance of Bhlhe40 in the regulation of unsaturated fatty acid and glucose oxidative metabolism. Expression profiling of genes important for peroxisome function and biogenesis also supports the intimate regulation of peroxisomes by Bhlhe40. In this study, we will focus on delineating the regulatory mechanisms of peroxisome function and homeostasis by Bhlhe40 at molecular level in detail via dissecting the regulation of target genes by Bhlhe40 at genetic and epigenetic levels. The following experiments on Bhlhe40 targeted genes involved in peroxisome functions and homeostasis are proposed:1. Gain and loss of function effects on peroxisomes and myogenesis. 2. Cloning their promoters and cis-elements and characterizing their regulation by Bhlhe40.3. Examining the recruitment of Bhlhe40 and cofactors to these genes in vitro and in vivo.4. Analyzing their epigenetic modification and chromatin structure affected by Bhlhe40.
關聯:	財團法人國家實驗研究院科技政策研究與資訊中心
顯示於類別:	[Department of Life Science] Research Project

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