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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.

In addition to the known four alternative first exons E1₁, E1₂, E1₃ and E1₄ of the rat prolactin receptor (PRL-R) gene, a novel first exon, E1₅, was identified by cDNA cloning of the 5′-end region of PRL-R mRNA in the rat liver. Genomic fragments containing E1₅ and its 5′- or 3′-flanking regions were also cloned from rat kidney genomic DNA. A sequence search for E1₅ revealed that E1₅ is located 49 kb upstream of exon 2 of the PRL-R gene in rat chromosome 2q16. RT-PCR analysis revealed that E1₅ was preferentially expressed in the liver, brain and kidney. Expression profiles of E1₂-, E1₃- and E1₅-PRL-R mRNAs in the liver of male and female rats at 5 days of age and those at 8 weeks of age were examined by RT-PCR. The levels of E1₂-PRL-R mRNA in the female rat increased remarkably in rats at 8 weeks of age compared with those at 5 days of age, and the levels of E1₅-PRL-R mRNA in the male rat decreased markedly at 8 weeks of age compared with those at 5 days of age. In the female rat, the levels of E1₂-PRL-R mRNA at 8 weeks of age decreased with ovariectomy performed at 4 weeks of age and recovered with the administration of β-oestradiol. On the contrary, the levels of E1₅-PRL-R mRNA increased with ovariectomy and decreased with the oestrogen treatment. In the male rat liver, the levels of E1₂-PRL-R mRNA at 8 weeks of age increased strikingly with castration performed at 4 weeks of age and became undetectable with the administration of testosterone. The levels of E1₅-PRL-R mRNA increased slightly with castration and were restored by testosterone treatment. Removal of gonadal tissues and sex steroid hormone treatment had no effect on the expression levels of E1₃-PRL-R mRNA in both female and male rat livers. These results indicated that the expression of the PRL-R gene in the liver is regulated by the differential effects of sex steroid hormones on the transcription of the multiple first exons including the novel one.