The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator.
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.
Massachusetts General Hospital Cancer Center, Department of Cell Biology, Harvard Medical School, Building 149, 13th Street, Charlestown, MA 02129, USA.
Fajun
F
Yang
Rosalie
R
DeBeaumont
Sharleen
S
Zhou
Anders M
AM
Näär
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D056892:Mediator Complex; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012319:RNA Polymerase II; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; C483045:MED25 protein, human; D056892:Mediator Complex; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D012319:RNA Polymerase II