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1. Heterologous SUMO-2/3-ubiquitin chains optimize IκBα degradation and NF-κB activity.

Author

Fabienne Aillet, Fernando Lopitz-Otsoa, Isabel Egaña, Roland Hjerpe, Paul Fraser, Ron T Hay, Manuel S Rodriguez, and Valérie Lang

Subjects
Medicine, Science
Abstract

The NF-κB pathway is regulated by SUMOylation at least at three levels: the inhibitory molecule IκBα, the IKK subunit γ/NEMO and the p52 precursor p100. Here we investigate the role of SUMO-2/3 in the degradation of IκBα and activation of NF-κB mediated by TNFα. We found that under conditions of deficient SUMOylation, an important delay in both TNFα-mediated proteolysis of IκBα and NF-κB dependent transcription occurs. In vitro and ex vivo approaches, including the use of ubiquitin-traps (TUBEs), revealed the formation of chains on IκBα containing SUMO-2/3 and ubiquitin after TNFα stimulation. The integration of SUMO-2/3 appears to promote the formation of ubiquitin chains on IκBα after activation of the TNFα signalling pathway. Furthermore, heterologous chains of SUMO-2/3 and ubiquitin promote a more efficient degradation of IκBα by the 26S proteasome in vitro compared to chains of either SUMO-2/3 or ubiquitin alone. Consistently, Ubc9 silencing reduced the capture of IκBα modified with SUMO-ubiquitin hybrid chains that display a defective proteasome-mediated degradation. Thus, hybrid SUMO-2/3-ubiquitin chains increase the susceptibility of modified IκBα to the action of 26S proteasome, contributing to the optimal control of NF-κB activity after TNFα-stimulation.

Published
2012
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2. SUMO-1 modification of human transcription factor (TF) IID complex subunits: inhibition of TFIID promoter-binding activity through SUMO-1 modification of hsTAF5

Author

Michaël, Boyer-Guittaut, Kivanç, Birsoy, Corinne, Potel, Gill, Elliott, Ellis, Jaffray, Joanna M, Desterro, Ron T, Hay, and Thomas, Oelgeschläger

Subjects
TATA-Binding Protein Associated Factors, Binding Sites, Models, Genetic, Sequence Homology, Amino Acid, Transcription, Genetic, Protein Conformation, Lysine, Immunoblotting, Molecular Sequence Data, SUMO-1 Protein, DNA, Transfection, Models, Biological, Recombinant Proteins, Protein Structure, Tertiary, Epitopes, Catalytic Domain, Humans, Electrophoresis, Polyacrylamide Gel, Transcription Factor TFIID, Amino Acid Sequence, RNA Polymerase II, Promoter Regions, Genetic, HeLa Cells, Protein Binding
Abstract

The TFIID complex is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFs) and is the only component of the general RNA polymerase II (RNAP II) transcription machinery with intrinsic sequence-specific DNA-binding activity. Binding of transcription factor (TF) IID to the core promoter region of protein-coding genes is a key event in RNAP II transcription activation and is the first and rate-limiting step of transcription initiation complex assembly. Intense research efforts in the past have established that TFIID promoter-binding activity as well as the function of TFIID-promoter complexes is tightly regulated through dynamic TFIID interactions with positive- and negative-acting transcription regulatory proteins. However, very little is known about the role of post-translational modifications in the regulation of TFIID. Here we show that the human TFIID subunits hsTAF5 and hsTAF12 are modified by the small ubiquitin-related modifier SUMO-1 in vitro and in human cells. We identify Lys-14 in hsTAF5 and Lys-19 in hsTAF12 as the primary SUMO-1 acceptor sites and show that SUMO conjugation has no detectable effect on nuclear import or intranuclear distribution of hsTAF5 and hsTAF12. Finally, we demonstrate that purified human TFIID complex can be SUMO-1-modified in vitro at both hsTAF5 and hsTAF12. We find that SUMO-1 conjugation at hsTAF5 interferes with binding of TFIID to promoter DNA, whereas modification of hsTAF12 has no detectable effect on TFIID promoter-binding activity. Our observations suggest that reversible SUMO modification at hsTAF5 contributes to the dynamic regulation of TFIID promoter-binding activity in human cells.

Published
2005

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