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

Hidehisa Takahashi,Skylar Martin-Brown,Michael P Washburn,Laurence Florens,Joan W Conaway,Ronald C Conaway

doi:10.1074/jbc.m109.017954

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 4alpha (HNF4alpha) and transcription factor IID (TFIID). A systematic characterization of the HNF4alpha-TFIID link revealed that the HNF4alpha DNA-binding domain binds directly to the TATA box-binding protein (TBP) and, through this interaction, can target TBP or TFIID to promoters containing HNF4alpha-binding sites in vitro. Supporting the functional significance of this interaction, an HNF4alpha mutation that blocks binding of TBP to HNF4alpha interferes with HNF4alpha transactivation activity in cells. These findings identify an unexpected role for the HNF4alpha DNA-binding domain in mediating key regulatory interactions and provide new insights into the roles of HNF4alpha and TFIID in RNA polymerase II transcription.

Highlights

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 [1,2,3,4]
MudPIT Analysis of TATA box-binding protein (TBP)-associated Proteins—To define TBP-interacting proteins, HA epitope-tagged TBP and associated proteins were subjected to anti-HA immunopurification from a cell line stably expressing HA-TBP and analyzed by MudPIT mass spectrometry
The results of a recent study indicate that HNF4␣ can stimulate basal transcription via a Mediator-independent mechanism in a highly purified, reconstituted transcription system containing RNA polymerase II, general transcription factors, and PC4 [65], a finding we have confirmed in our laboratory

Summary

Introduction

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 [1,2,3,4]. 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].

Results

Conclusion