| 摘要: | 骨骼肌的分化是經由一連串的複雜的生理反應以及機制所建構而成，成熟的骨骼肌必須具有肌肉收縮調節組織，肌原母細胞的細胞週期停止以及細胞間的融和，而進一步形成具有自主性收縮以及多核的肌肉細胞。這些步驟都受到肌肉特化性因子的調控，而肌肉特化性因子包含MyoD、Myf-5、Myogenin 和MRF4這四個成員。然而在肌肉分化過程中肌肉特化性因子會和 MEF2s 共同活化分化過程中所需的肌肉特化性基因的表現。在本研究中，我們發現Bhlhe40這個基因的表現量會隨著肌肉細胞進行終極分化的過程而逐漸上升，而在之前的研究指出Bhlhe40會廣泛表現於各器官組織中並扮演著轉錄抑制因子的角色，我們同時發現不管是在in vitro 亦或是 in vivo 狀態下Bhlhe40 都會直接結合到 PGC-1α, M-cadherin 和 Myogenin 的啟動子上面並抑制它們的轉錄作用。而且我們發現，當我們加入P/CAF這個重要的肌肉特化性因子共同活化子之後，Bhlhe40 對於肌肉特化性因子轉錄活性的抑制可以被回復。我們更證明 Bhlhe40 抑制肌肉特化性因子轉錄活化 M-cadherin 的表現是透過 HDAC 非依賴型的機制，在探討Bhlhe40 對於 M-cadherin 的轉錄抑制的同時我們發現 Bhlhe40 的 DNA 結合能力對於抑制M-cadherin 的轉錄機制是必須的但是卻還不足夠。然而 Bhlhe40 抑制 M-cadherin 啟動子的活性可能不僅僅只透過Bhlhe40直接結合到 M-cadherin 啟動子上的 E3-box，也有可能是藉由先結合到未知的bHLH 蛋白在進一步傳導抑制的能力。我們現在正嘗試分析這個未知的蛋白，並且未來希望更進一步觀察這些未知的蛋白跟肌肉特化性因子、Bhlhe40、P/CAF 或是其他的共同活化或抑制因子們在肌肉特化性基因啟動子上的相互作用為何? 基於我們對PGC-1α 和 M-cadherin 這兩個基因在轉錄調控機制上的了解，我們猜測MyoD 對於結合序列的專一性以及轉錄活性可能會受到專一結合序列旁的E-boxes 或E-boxes 上的結合蛋白所調節。我們目前正在分析 MyoD 和 Bhlhe40 在整個 genome 的結合情形並試著證明我們的假說。 The differentiation of functional skeletal muscle cells is characterized by the acquirement of contractile filaments, cell cycle exit, and fusion of myoblasts to form voluntarily contractible multinucleated myotubes. This process is critically regulated by a family of basic helix-loop-helix (bHLH) transcription factors, including MyoD, Myf-5, Myogenin and MRF4, called as myogenic regulatory factors (MRFs) that work together with myocyte enhancer factors (MEF2s) to activate the expression of muscle-specific genes required for myogenic differentiation. In this study, we found that the expression level of Bhlhe40, a ubiquitously expressed bHLH transcriptional repressor, was up-regulated during terminal myogenic differentiation and it could repress the MRF-activated transcription of PGC-1α, M-cadherin, and Myogenin by directly binding to their promoters in vitro and in vivo. Furthermore, we found that Bhlhe40-mediated repression of MRF transactivational activity could be relieved by over-expression of P/CAF, an essential coactivator of MRFs. We demonstrated that Bhlhe40 repressed MRF-activated M-cadherin transcription through a HDAC independent pathway and the DNA binding activity of Bhlhe40 was required but not sufficient to repress the M-cadherin expression. We also found that Bhlhe40-mediated repression of M-cadherin promoter activity may not only through its direct binding to the E3-box in the M-cadherin proximal promoter but also could be mediated by unknown bHLH proteins. We are currently identifying the unknown proteins and observing the interaction among MRFs, Bhlhe40, P/CAF, and other co-factors (co-activators or co-repressors) acting on muscle-specific gene promoters. Based on our observations on the regulation of PGC-1α and M-cadherin transcription, we speculate that the specificity of MyoD targeting and transactivation can be significantly determined by the E-boxes and their binding proteins adjacent to the MyoD binding sites. We are currently identifying genome-wide targeting sites of both MyoD and Bhlhe40 to prove this hypothesis. |
