Ubc9 interacts with chicken ovalbumin upstream promoter-transcription factor I and represses receptor-dependent transcription.
Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors involved in regulation of neurogenesis and organogenesis. COUP-TF family members are generally considered to be transcriptional repressors and several mechanisms have been proposed to underlie this activity. To explore novel transcriptional coregulators for COUP-TFs, we used the COUP-TFI as bait in a yeast two-hybrid screen of an adrenocortical adenoma cDNA library. We have identified Ubc9, a class E2 conjugating enzyme of small ubiquitin-related modifier (SUMO)-1 as a COUP-TFI corepressor. Ubc9 interacts with COUP-TFI in yeast and in glutathione S-transferase pulldown and coimmunoprecipitation assays. Fluorescence imaging studies show that both Ubc9 and COUP-TFI are colocalized in the nuclei of transfected COS-1 cells. The C-terminal region of Ubc9 encoding amino acids 59-158 interacts with the C-terminus of COUP-TFI encoding amino acids 383-403, in which transcriptional repression domains are located. Mammalian one-hybrid assays utilizing a variety of Ubc9 fragments fused to Gal4 DNA-binding domain show that a Ubc9 fragment encoding amino acids 1-89 contains autonomous transferrable repression domain. Transfection of Ubc9 into COS-1 cells markedly enhances transcriptional repression by Gal4 DNA-binding domain-fused to COUP-TFI(155-423), but not by Gal4-COUP-TFI(155-388) which lacks a repressor domain. Coexpression of a C-terminal deletion mutant of Ubc9(1-58), which fails to interact with COUP-TFI, but retains a transcriptional repression domain, has no effect on Gal4-COUP-TFI-mediated repression activity. These findings indicate that interaction of Ubc9 with COUP-TFI is crucial for the corepressor function of Ubc9. Overexpression of Ubc9 similarly enhances COUP-TFI-dependent repression of the promoter activity of the bovine CYP17 gene encoding steroid 17alpha-hydroxylase. In addition, the C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, continues to function as a COUP-TFI corepressor. Our studies indicate that Ubc9 functions as a novel COUP-TFI corepressor, the function of which is distinct from its SUMO-1 conjugating enzyme activity.
Kobayashi
S
S
Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Shibata
H
H
Kurihara
I
I
Yokota
K
K
Suda
N
N
Saito
I
I
Saruta
T
T
D000818:Animals; D019556:COS Cells; D051836:COUP Transcription Factors; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011987:Receptors, Steroid; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D013254:Steroid 17-alpha-Hydroxylase / Q000378:metabolism*; D014018:Tissue Distribution; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D051836:COUP Transcription Factors; D004268:DNA-Binding Proteins; D011987:Receptors, Steroid; D025842:SUMO-1 Protein; D014157:Transcription Factors; D013254:Steroid 17-alpha-Hydroxylase; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9