Cooperative activity of cdk8 and GCN5L within Mediator directs tandem phosphoacetylation of histone H3

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Abstract
The human Mediator complex is generally required for expression of protein‐coding genes. Here, we show that the GCN5L acetyltransferase stably associates with Mediator together with the TRRAP polypeptide. Yet, contrary to expectations, TRRAP/GCN5L does not associate with the transcriptionally active core Mediator but rather with Mediator that contains the cdk8 subcomplex. Consequently, this derivative ‘T/G‐Mediator’ complex does not directly activate transcription in a reconstituted human transcription system. However, within T/G‐Mediator, cdk8 phosphorylates serine‐10 on histone H3, which in turn stimulates H3K14 acetylation by GCN5L within the complex. Tandem phosphoacetylation of H3 correlates with transcriptional activation, and ChIP assays demonstrate co‐occupancy of T/G‐Mediator components at several activated genes in vivo. Moreover, cdk8 knockdown causes substantial reduction of global H3 phosphoacetylation, suggesting that T/G‐Mediator is a major regulator of this H3 mark. Cooperative H3 modification provides a mechanistic basis for GCN5L association with cdk8‐Mediator and also identifies a biochemical means by which cdk8 can indirectly activate gene expression. Indeed our results suggest that T/G‐Mediator directs early events—such as modification of chromatin templates—in transcriptional activation.