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The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator.

ABSTRACT: The human activator-recruited cofactor (ARC), a family of large transcriptional coactivator complexes related to the yeast Mediator, was recently identified based on functional association with the activation domains of multiple cellular and viral transcriptional activators, including the herpes simplex viral activator VP16, sterol regulatory element binding protein, and NF-kappaB. Here we describe the biochemical purification and cloning of the 92-kDa ARC/Mediator subunit, ARC92, that is specifically targeted by the activation domain of the VP16 transactivator. Affinity chromatography using the VP16 activation domain followed by peptide microsequencing led to the identification of ARC92 as a specific cellular interaction partner of the VP16 activation domain. ARC92 associates with the VP16 activation domain in vitro and in vivo, and the VP16 binding domain of ARC92 is a strong competitive inhibitor of Gal4-VP16 in vivo. Moreover, small interfering RNA-mediated knockdown of ARC92 in human cells results in selective inhibition of Gal4-VP16 gene activation. Taken together, our results suggest that ARC92 is a direct and specific target of the VP16 transactivator that serves in the context of the ARC/Mediator coactivator as an important transducer of transcription activating signals from the VP16 activation domain to the RNA polymerase II transcriptional machinery.

SUBMITTER: Yang F

PROVIDER: S-EPMC356952 | biostudies-literature | 2004 Feb

REPOSITORIES: biostudies-literature

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The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator.

Yang Fajun F DeBeaumont Rosalie R Zhou Sharleen S Näär Anders M AM

Proceedings of the National Academy of Sciences of the United States of America 20040201 8

The human activator-recruited cofactor (ARC), a family of large transcriptional coactivator complexes related to the yeast Mediator, was recently identified based on functional association with the activation domains of multiple cellular and viral transcriptional activators, including the herpes simplex viral activator VP16, sterol regulatory element binding protein, and NF-kappaB. Here we describe the biochemical purification and cloning of the 92-kDa ARC/Mediator subunit, ARC92, that is specif ...[more]

PMID: 14983011

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Project description:The proteasome inhibitor MG132 had been shown to prevent galactose induction of the S. cerevisiae GAL1 gene, demonstrating that ubiquitin proteasome-dependent degradation of transcription factors plays an important role in the regulation of gene expression. The deletion of the gene encoding the F-box protein Mdm30 had been reported to stabilize the transcriptional activator Gal4 under inducing conditions and to lead to defects in galactose utilization, suggesting that recycling of Gal4 is required for its function. Subsequently, however, it was argued that Gal4 remains stably bound to the enhancer under inducing conditions, suggesting that proteolytic turnover of Gal4 might not be required for its function. We have performed an alanine-scanning mutagenesis of ubiquitin and isolated a galactose utilization-defective ubiquitin mutant. We have used it for an unbiased suppressor screen and identified the inhibitor Gal80 as a suppressor of the transcriptional defects of the ubiquitin mutant, indicating that the protein degradation of the inhibitor Gal80, and not of the activator Gal4, is required for galactose induction of the GAL genes. We also show that in the absence of Gal80, Mdm30 is not required for Gal4 function, strongly supporting this hypothesis. Furthermore, we have found that Mediator controls the galactose-induced protein degradation of Gal80, which places Mediator genetically upstream of the activator Gal4. Mediator had originally been isolated by its ability to respond to transcriptional activators, and here we have discovered a leading role for Mediator in the process of transcription. The protein kinase Snf1 senses the inducing conditions and transduces the signal to Mediator, which initiates the degradation of the inhibitor Gal80 with the help of the E3 ubiquitin ligase SCF(Mdm30). The ability of Mediator to control the protein degradation of transcriptional inhibitors indicates that Mediator is actually able to direct its own recruitment to gene promoters.

Dataset Information

The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator.

Publications

The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator.

Similar Datasets

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