The expression of estrogen-related receptor-(ERR ) is stimulated by estrogen in selective tissues. Recently, a correlation between ERR expression and the induction of peroxisome proliferator-activated receptor-coactivator-1 (PGC-1 ) in the liver of fasting animals and in cold-stressed brown-fat tissues and skeletal muscle was shown. To explore the molecular mechanisms of ERR regulation by diverse signals, the promoter of the human ERR gene was cloned and characterized. Mutation and deletion analyses revealed that a 53 bp region containing repeated core element AGGTCA motifs of the ERR gene serves as a multi-hormone response element (MHRE) for several nuclear receptors in transient co-transfection studies of human endometrial carcinoma (HEC-1B) cells. Among the nuclear receptors tested, ERR bound to and robustly stimulated the transcription of reporters containing at least two AGGTCA motifs. Ectopic expression of PGC-1 in HEC-1B cells strongly activated the reporter containing the MHRE, presumably via the endogenous nuclear receptor binding to the element. Reducing the endogenous level of ERR by small interfering RNA, and increasing the ERR level by ectopic expression, substantially decreased and increased respectively the transactivation capability of PGC-1 . The activation function 2 domain of the ERR and the L2 and L3 motifs of PGC-1 were essential to transactivate the MHRE. Additionally, PGC-1 increases the amount of endogenous ERR bound to the MHRE region as determined by a chromatin immunoprecipitation assay. The present study demonstrates that the MHRE of the ERR gene is a target for ERR transactivation, which is enhanced by PGC-1 .
In the present study, we report that, despite the presence of one perfect p53 consensus sequence homology (designated SCL CS) and four half-sites within the 3'-untranslated region of the stem cell leukemia (SCL) gene, the native endogenous gene is not regulated by p53. We employ a tet-repressible system to show that, under conditions in which the WAF1 mRNA steady-state level is upregulated fourfold by p53, the SCL mRNA level is not altered. In a previous report, we demonstrated that p53 interactions with the SCL CS can upregulate downstream reporter gene activity 43-fold in transient reporter assays. This disparity prompted us to explore the dierences between p53 regulation of SCL CS activity in organized (chromosomally integrated) and disorganized (non-replicating episomal plasmid) chromatin. We show that p53 can increase (between 3 ± 80-fold), decrease (between 5 ± 33-fold) or have no eect upon transactivation of an SCL CS/reporter fusion gene depending upon chromosomal integration site. Most studies used to characterize p53 binding sites employ transient transfection assays. Our results suggest that characterization of consensus sequence homologies by assay of transiently transfected cells may be inaccurate.
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