In vitro transcription system delineates the distinct roles of the coactivators pCAF and p300 during MyoD/E47-dependent transactivation
In vitro transcription system delineates the distinct roles of the coactivators pCAF and p300 during MyoD/E47-dependent transactivation
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DOI：
10.1073/pnas.0404192101
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发表时间：
2004-08-10
影响因子：
11.1
通讯作者：
Tapscott, SJ
中科院分区：
文献类型：
--
作者：
Dilworth, FJ;Seaver, KJ;Tapscott, SJ
转录辅激活因子p300和pCAF是肌生成因子MyoD启动骨骼肌基因表达所必需的。除了介导组蛋白乙酰化，这两种因子都可以乙酰化MyoD;然而，用于研究MyoD的细胞系统的复杂性阻碍了这两种乙酰转移酶的具体作用的描绘。因此，我们建立了一个MyoD依赖的体外转录系统，使我们能够确定在MyoD依赖的转录激活p300和pCAF的作用。与细胞系统的结果一致，我们证明了体外最大水平的反式激活需要p300和pCAF，以及辅因子乙酰辅酶A。对导致转录起始的步骤的剖析揭示了p300和pCAF的活性不是冗余的。在反式激活的初始阶段，p300乙酰化启动子区域内的组蛋白H3和H4，然后将pCAF募集到MyoD。一旦与启动子连接，pCAF乙酰化MyoD以促进反式激活过程。因此，我们已经确定pCAF和p300在启动子上进行顺序和功能上不同的事件，导致转录激活。进一步解剖这种体外转录系统应该是非常有用的，以阐明的机制，辅激活因子促进差异基因表达的MyoD。
The transcriptional coactivators p300 and pCAF are necessary for the myogenic factor MyoD to initiate the expression of skeletal muscle genes. In addition to mediating histone acetylation, both of these factors can acetylate MyoD; however, the complexity of cellular systems used to study MyoD has impeded delineation of the specific roles of these two acetyltransferases. Therefore, we established a MyoD-dependent in vitro transcription system that permits us to determine the roles of p300 and pCAF during MyoD-dependent transcriptional activation. Consistent with results from cellular systems, we demonstrate that maximal levels of transactivation in vitro require both p300 and pCAF, as well as the cofactor acetyl CoA. Dissection of the steps leading to transcription initiation revealed that the activities of p300 and pCAF are not redundant. During the initial stages of transactivation, p300 acetylates histone H3 and H4 within the promoter region and then recruits pCAF to MyoD. Once tethered to the promoter, pCAF acetylates MyoD to facilitate the transactivation process. Thus, we have established that pCAF and p300 carry out sequential and functionally distinct events on a promoter leading to transcriptional activation. Further dissection of this in vitro transcription system should be highly useful toward elucidating the mechanism by which coactivators facilitate differential gene expression by MyoD.