Proteomics Reveals a Physical and Functional Link between Hepatocyte Nuclear Factor 4α and Transcription Factor IID

Abstract

Proteomic analyses have contributed substantially to our understanding of diverse cellular processes. Improvements in the sensitivity of mass spectrometry approaches are enabling more in-depth analyses of protein-protein networks and, in some cases, are providing surprising new insights into well established, longstanding problems. Here, we describe such a proteomic analysis that exploits MudPIT mass spectrometry and has led to the discovery of a physical and functional link between the orphan nuclear receptor hepatocyte nuclear factor 4␣ (HNF4␣) and transcription factor IID (TFIID). A systematic characterization of the HNF4␣-TFIID link revealed that the HNF4␣ DNA-binding domain binds directly to the TATA boxbinding protein (TBP) and, through this interaction, can target TBP or TFIID to promoters containing HNF4␣-binding sites in vitro. Supporting the functional significance of this interaction, an HNF4␣ mutation that blocks binding of TBP to HNF4␣ interferes with HNF4␣ transactivation activity in cells. These findings identify an unexpected role for the HNF4␣ DNA-binding domain in mediating key regulatory interactions and provide new insights into the roles of HNF4␣ and TFIID in RNA polymerase II transcription.Promoter-specific transcription by RNA polymerase II depends on the general initiation factors TFIIA, 2 -B, -D, -E, -F, and -H, which are the minimal set of factors needed for assembly of the preinitiation complex and subsequent initiation of transcription (1-4). The activity of RNA polymerase II and the general transcription factors is controlled by a host of DNAbinding transcription factors, the Mediator of RNA polymerase II transcription, and other coactivators and corepressors (5-7).Preinitiation complex assembly begins with DNA sequencespecific binding of TFIID to the core promoter to provide a nucleoprotein recognition site for subsequent binding of polymerase II and the remaining general transcription factors. The TATA box-binding protein (TBP) subunit of TFIID recognizes the TATA box element and can substitute for TFIID in transcription in vitro. Additional TBP-associated factor (TAF) subunits of TFIID recognize the initiator (Inr) and other core promoter elements and contribute to functional interactions between the general transcription machinery and DNA-binding transcription factors and coregulators (1,8,9). TBP has also been shown to be an essential component of SL1 and TFIIIB, which are multisubunit general transcription factors for RNA polymerases I and III, respectively (10 -16).Because of its central role in transcriptional regulation, there has been considerable interest in defining the repertoire of TBP-interacting proteins. Biochemical studies that led to definition of TFIID, SL1, TFIIIB, and other TBP-associated proteins have typically used conventional chromatography and/or immunoaffinity purification methods to purify TBP-containing complexes (17), followed by protein isolation by reverse-phase chromatography or SDS-PAGE and identification by Edman sequencing or mass spectrometry (e....

Direct Transactivator-Transcription Factor IID (TFIID) Contacts Drive Yeast Ribosomal Protein Gene Transcription

“…By contrast, removal of residues 253-344 (Fig. 1A, green-shaded domain) completely abolished Rap1 binding (lanes [11][12][13][14][15][16][17][18][19][20]. To test whether the Taf4 RBD is essential, we scored the ability of the TAF4 deletion family to support viability via a plasmid shuffle assay.…”

“…The structures of yeast and human TFIID in isolation and in complex with DNA, a subset of general transcription factors, or with activators have been determined using electron microscopy (EM) methods (10 -17). The derived structures provide insights into the overall organization of the complex as well as possible modes of interaction of TFIID with a small sampling of possible binding partners (18,19). However, the functional consequences of these interactions are still not understood.…”

Two novel cis-elements involved in hepatocyte nuclear factor 4α regulation of acyl-coenzyme A:cholesterol acyltransferase 2 expression

“…Site II (238 to 229) is adjacent to the transcriptional start site. HNF4a binding to site II could easily recruit Transcription Factor IID (TFIID) or Transcription Factor IIB (TFIIB) and other protein components to initiate transcription [42,43]. We suggest that site II plays a fundamental role in maintaining liver-specific ACAT2 gene promoter activity, while other sites may cooperate with site II to synergistically regulate ACAT2 expression.…”

Identifying a TFIID interactome via integrated biochemical and high-throughput proteomic studies

“…A number of previous studies have used global mass spectrometry to identify factors interacting with the TBP subunit of TFIID [3,[32][33][34]. Alternative proteomic approaches utilizing the intact native human TFIID complex as bait to find direct interactors had not been assessed.…”