Liver receptor homolog‐1 (LRH‐1; NR5A2) is an orphan member of the nuclear receptor superfamily, mainly expressed in endoderm‐derived tissues and in the ovary. In ovarian granulosa and luteal cells, LRH‐1 regulates the expression of genes associated with ovarian steroidogenesis. LRH‐1 can be transported to transcriptionally inactive nuclear bodies after conjugation with small ubiquitin‐related modifier (SUMO). In the present study, we investigated the effects of SUMO modification at five lysine residues of LRH‐1 in rat granulosa cells. Lysine 289 could be conjugated with SUMO‐1 in vitro, and the mutation K289R increased transcriptional activity of LRH‐1, suggesting that SUMO conjugation is associated with transcription repression. Coexpression of SUMO‐1 targets LRH‐1 to the dot‐like nuclear bodies, but the effect of lysine mutations on blocking subnuclear localization depended on the cell type. In COS‐7 cells, mutation of either K173 or K289 prevented SUMO‐1‐mediated translocation of LRH‐1 into nuclear bodies and also reduced the conjugation by SUMO‐1, suggesting that K289 and K173 are two important sites involved in SUMO‐1 modification. In granulosa cells, three or more altered lysine residues were required for nucleoplasm retention. This result suggests that multiple lysine residues are targets for SUMO conjugation in vivo and granulosa cells are more sensitive to SUMO‐1‐mediated LRH‐1 localization to nuclear bodies. Nuclear body localization of LRH‐1 was suppressed by forskolin and cholera toxin. Forskolin treatment obviously influences the expression of members involved in the SUMO pathway. The results obtained in the present study suggest that cAMP signaling could change the dynamic process of sumoylation and repress LRH‐1 targeting to nuclear speckles in rat granulosa cells.
We have investigated the functional elements involved in cAMP-stimulated transcription of the human ferredoxin gene. Unlike the bovine gene, the human gene lacked a second upstream RNA initiation site as demonstrated by sequence analysis of the exon boundary, lack of upstream RNA, and analysis of the promoter. The presence of a single promoter was determined by testing the ability of various gene segments to drive the expression of the chloramphenicol acetyltransferase gene after transfection into a mouse adrenal cell line Y1. Full promoter activity was conferred by a DNA fragment spanning -209 to +55, although the -94 to +55 fragment already provided some promoter activity. Transcription from the -94 to +55 segment was stimulated by 2-fold when 8-bromo-cAMP was added to the cell. Footprinting analyses showed two GC boxes at -50 to -70 and -87 to -108 were protected by proteins from both Y1 and HeLa cells. Competition experiments showed that a protein with a recognition sequence indistinguishable from Sp1 bound to these sites. When connected to a heterologous TATA box, the sequence at -76 to -42, which contained the proximal GC box, was able to confer a high level of basal transcription and cAMP stimulation. This sequence does not show sequence homology with the known cAMP-responsive element. Mutations or deletion of the Sp 1-binding site showed diminished basal transcription and defined the cAMP responsive sequence to be from -76 to -62. Therefore the cAMP-responsive sequence of the human ferredoxin gene was located at -76 to -62, which was adjacent to the Sp 1-binding site.
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