Interaction of Protein Inhibitor of Activated STAT (PIAS) Proteins with the TATA-binding Protein, TBP

Abstract

Transcription activators often recruit promoter-targeted assembly of a pre-initiation complex; many repressors antagonize recruitment. These activities can involve direct interactions with proteins in the pre-initiation complex. We used an optimized yeast two-hybrid system to screen mouse pregnancy-associated libraries for proteins that interact with TATA-binding protein (TBP). Screens revealed an interaction between TBP and a single member of the zinc finger family of transcription factors, ZFP523. Two members of the protein inhibitor of activated STAT (PIAS) family, PIAS1 and PIAS3, also interacted with TBP in screens. Endogenous PIAS1 and TBP co-immunoprecipitated from nuclear extracts, suggesting the interaction occurred in vivo. In vitro-translated PIAS1 and TBP coimmunopreciptated, which indicated that other nuclear proteins were not required for the interaction. Deletion analysis mapped the PIAS-interacting domain of TBP to the conserved TBP CORE and the TBP-interacting domain on PIAS1 to a 39-amino acid C-terminal region. Mammals issue seven known PIAS proteins from four pias genes, pias1, pias3, piasx, and piasy, each with different cell typespecific expression patterns; the TBP-interacting domain reported here is the only part of the PIAS C-terminal region shared by all seven PIAS proteins. Direct analyses indicated that PIASx and PIASy also interacted with TBP. Our results suggest that all PIAS proteins might mediate situation-specific regulatory signaling at the TBP interface and that previously unknown levels of complexity could exist in the gene regulatory interplay between TBP, PIAS proteins, ZFP523, and other transcription factors. TATA-binding protein (TBP)2 functions in transcription initiation by all three nuclear eukaryotic RNA polymerases (1, 2). TBP-containing complexes include SL1, TFIID, and TFIIIB, which function with RNA polymerase I (RNAPI), RNAPII, and RNAPIII, respectively. RNAPII requires TFIID for promoter-targeted assembly of the pre-initiation complex (3). TBP is also an essential component of the human SNAPc complex, which functions in transcription initiation at small nuclear RNA genes by both RNAPII and RNAPIII (4), and the yeast SAGA complex (5).Recruitment of TBP to the core promoter is regulated by both positive and negative factors (3). Some activators of transcription bind TBP or the TBP-associated factor components of the TFIID complex and direct TFIID to the promoter (6, 7). TBP function can also be up-or down-regulated through interactions with BTAF1/MOT1 and the NC2␣/␤ subunits of the NC2 complex (8, 9). Recently, ZNF76, the human ortholog of mouse zinc finger protein 523 (ZFP523) and of frog Staf (10), was shown to function via direct interaction with TBP (11). The interaction of ZNF76 with TBP is blocked by PIAS1-dependent sumoylation of ZNF76 (11).PIAS proteins are found in all eukaryotes. The human and mouse family of PIAS proteins consists of PIAS1, PIAS3, PIASx, and PIASy proteins (12). The piasx gene encodes two splice variants, PIASx␣/ ARIP3 (androgen receptor...

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