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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >In vitro transcription system delineates the distinct roles of the coactivators pCAF and p300 during MyoD/E47-dependent transactivation.
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In vitro transcription system delineates the distinct roles of the coactivators pCAF and p300 during MyoD/E47-dependent transactivation.

机译：体外转录系统描绘了在MyoD / E47依赖性反式激活过程中，共激活因子pCAF和p300的独特作用。

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摘要

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.
机译：转录共激活因子p300和pCAF对于肌源因子MyoD启动骨骼肌基因的表达是必需的。除了介导组蛋白乙酰化以外，这两个因素都可以使MyoD乙酰化。然而，用于研究MyoD的细胞系统的复杂性阻碍了对这两种乙酰基转移酶的特定作用的描述。因此，我们建立了一个MyoD依赖的体外转录系统，该系统使我们能够确定在MyoD依赖的转录激活过程中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。解剖导致转录起始的步骤揭示了p300和pCAF的活性不是多余的。在反式激活的初始阶段，p300将启动子区域内的组蛋白H3和H4乙酰化，然后将pCAF募集到MyoD。一旦与启动子拴在一起，pCAF会乙酰化MyoD，以促进反式激活过程。因此，我们已经建立了pCAF和p300在启动子上进行顺序和功能上不同的事件，从而导致转录激活。进一步剖析该体外转录系统对于阐明共激活因子促进MyoD差异基因表达的机制非常有用。

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