A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation.

Li Li,Robert Tjian,Silvia Sanchez Martinez,Zhe Liu,Wenxin Hu

doi:10.7554/elife.08536

Abstract

TFIID-a complex of TATA-binding protein (TBP) and TBP-associated factors (TAFs)-is a central component of the Pol II promoter recognition apparatus. Recent studies have revealed significant downregulation of TFIID subunits in terminally differentiated myocytes, hepatocytes and adipocytes. Here, we report that TBP protein levels are tightly regulated by the ubiquitin-proteasome system. Using an in vitro ubiquitination assay coupled with biochemical fractionation, we identified Huwe1 as an E3 ligase targeting TBP for K48-linked ubiquitination and proteasome-mediated degradation. Upregulation of Huwe1 expression during myogenesis induces TBP degradation and myotube differentiation. We found that Huwe1 activity on TBP is antagonized by the deubiquitinase USP10, which protects TBP from degradation. Thus, modulating the levels of both Huwe1 and USP10 appears to fine-tune the requisite degradation of TBP during myogenesis. Together, our study unmasks a previously unknown interplay between an E3 ligase and a deubiquitinating enzyme regulating TBP levels during cellular differentiation.

Highlights

The TATA-box binding protein (TBP) is one of the central players in eukaryotic transcription
Consistent with several earlier observations from our lab and other studies (Perletti et al, 1999; Deato and Tjian, 2007), we found that upon myogenic differentiation of C2C12 cells TBP protein levels become significantly downregulated, while its mRNA levels remained largely unchanged (Figure 1—figure supplement 1), suggesting that TBP downregulation mainly occurs via modulation of its protein levels
TBP protein levels were partially restored within 4 hr after MG132 treatment (Figure 1A), suggesting that the ubiquitin/proteasome system (UPS) likely plays a dominant role in regulating TBP levels during terminal differentiation

Summary

Introduction

The TATA-box binding protein (TBP) is one of the central players in eukaryotic transcription. TBP serves as a key subunit to facilitate transcription initiation by all three RNA polymerases in eukaryotic cells. TBP and its associated factors (TAFs) that make up the TFIID complex are specific for Pol II-mediated mRNA transcription (Dynlacht et al, 1991). The TBP/B/BRF complex TFIIIB drives the transcription of small nuclear RNAs by Pol III (Taggart et al, 1992). Given this pivotal role in transcription, TBP-mediated transcriptional regulation has been extensively studied by biochemical and genetic approaches in past decades(Hernandez, 1993). Only limited studies have interrogated the regulation of TBP during cell-type specification. The main reason is that cell-type specific transcription programs have generally been considered to be dictated by classic sequence-specific transcription factors (Farnham, 2009), while the components of the core promoter recognition machinery such as TBP were thought to be largely invariant across different cell types (Thomas and Chiang, 2006)

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