Abstract
StAR (steroidogenic acute regulatory protein) mediates the transport of cholesterol from the outer to the inner mitochondrial membrane, the process of which is the rate-limiting step for steroidogenesis. Transcriptional regulation of the proximal promoter of the human StAR gene has been well characterized, whereas analysis of its distal control region has not. Recently, we found that SF-1 (steroidogenic factor 1) induced the differentiation of mesenchymal stem cells (MSCs) into steroidogenic cells with the concomitant strong induction of StAR expression. Here, we show, using differentiated MSCs, that StAR expression is regulated by a novel distal control region. Using electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays, we identified novel SF-1 binding sites between 3,000 and 3,400 bp upstream of StAR. A luciferase reporter assay revealed that the region worked as a strong regulator to exert maximal transcription of StAR. ChIP analysis of histone H3 revealed that upon SF-1 expression, nucleosome eviction took place at the SF-1 binding sites, not only in the promoter but also in the distal SF-1 binding sites. Chromosome conformation capture analysis revealed that the region upstream of StAR formed a chromatin loop both in the differentiated MSCs and in KGN cells, a human granulosa cell tumor cell line, where SF-1 is endogenously expressed. Finally, SF-1 knockdown resulted in disrupted formation of this chromatin loop in KGN cells. These results indicate that the novel distal control region participate in StAR activation through SF-1 dependent alterations of chromatin structure, including histone eviction and chromatin loop formation.
Highlights
Drial membrane, the process of which is the rate-limiting step for steroidogenesis
These results suggest that novel ment with 8-Br-cAMP augmented luciferase activities of all distal control regions may exist within the 15 kb region reporters
We propose that SF-1 simultaneously binds to the proximal promoter site and the Ϫ3 kb distal control region to form a DNA loop and a protein-DNA complex, which promotes histone eviction from these sites as well as transcription of StAR
Summary
These results suggest that novel ment with 8-Br-cAMP augmented luciferase activities of all distal control regions may exist within the 15 kb region reporters (supplemental Fig. 3). This resulted in a significant decrease of StAR expression under both cAMP-stimulated and unstimulated conditions (Fig. 4C) This was confirmed by the reporter assay where co-transfection of siRNA with luciferase reporter constructs (Ϫ244/ϩ33 and Ϫ3,402/ϩ33) resulted in a dramatic decrease of reporter activities compared with the control (Fig. 4D). Depletion of SF-1 resulted in the abolishment of the 3C assay PCR product, indicating that SF-1 is essential for the loop formation between the proximal promoter and the Ϫ3,400/Ϫ3,000 bp distal control region These results strongly suggest that endogenously expressed SF-1 plays a critical role in the transcriptional activation of StAR through the formation of a DNA loop between its distal control region (Ϫ3,494/Ϫ3,009 bp) and the proximal promoter by altering chromatin structure. The entire distal control regions among human, rat, and mouse were highly conserved (Fig. 5B), suggesting that the region may be important for achieving maximum transcription of StAR in other mammals
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