Sp1 can interact with ERa and contribute to transcriptional outcomes. As mentioned above, reports have documented that MGARP participates in steroid synthesis, and steroids also regulate MGARP expression. However, the detailed regulatory mechanisms of MGARP gene expression remain unknown. In the present study, we have carried out a characterization study of the MGARP promoter. Using bioinformatics, we identify two classic Sp1-binding GC-rich motifs proximal to the transcription start site. We demonstrate that reporters driven by the MGARP promoters containing the specific GC-rich motifs are activated by Sp1, and are shown by EMSA and ChIP to also bind Sp1. We also determine that ERa could further enhance the activity of the MGARP promoter that is activated by endogenous or exogenous Sp1 in a dominant manner. Collectively, our findings suggest a Sp1 regulatory mechanism in MGARP transcriptional regulation, with ERa functioning cooperatively with Sp1. We demonstrated that the region spanning -150 to 0 bp of the MGARP promoter fragment has basic promoter properties and contains multiple Sp1 binding sites that converge into two GC-Boxes. Indeed, using a reporter assay we found that the MGARP promoter could be stimulated by Sp1 in a dose-dependent manner, suggesting that Sp1 functions as a limiting factor. In addition, integration of each GC-Box into basic reporters resulted in minimally active transcription and combining two GC-Boxes resulted in full activation of the promoter, indicating a synergistic mechanism between these two motifs. The findings that each individual GC-Box carries Sp1- activated promoter function and that a 2150 bp proximal region is responsible for a significant part of MGARP promoter activity demonstrate that Sp1 is a dominant transactivator for MGARP expression. Comparing these two specific GC-rich Boxes, we propose that Box1 plays a major role in Sp1 transcriptional activity and that Box2 works cooperatively with Box1 to achieve full transactivation. Our previous study showed that MGARP is highly expressed in the ovary, testis, retina and adrenal gland tissues, and its expression is under the PF-4217903 regulation of the HPG axis. MGARP has also been shown to be up-regulated by estrogens and its expression level correlates with the level of estrogens in the ovary during the estrous cycle. These findings imply that MGARP functions in steroidogenesis and that MGARP is modulated by steroids. In our computational promoter analysis, we did not identify classic ERa binding element in the 23 kb proximal region; however, there still exists a possibility for direct ERa engagement with the proximal or distal promoter via non-classical binding site. In any case, here we demonstrate that ERa can stimulate the MGARP promoter in a dose-dependent manner. We further determined that ERa co-expression can stimulate Sp1- mediated promoter activation and this synergy can be further enhanced by estrogens. This suggests the existence of cross-talk between ERa and Sp1 at this gene locus, consistent with the reported findings that estrogens can enhance ERa-Sp1 interactions. Moreover, the critical dependence of ERa stimulatory effects on the GC Boxes and Sp1 indicated that Sp1 plays a dominant role in this synergistic interaction. The magnitude of ERa stimulatory effects on the MGARP promoter may depend on the ratio and sufficiency of each of the components in the systems, the availability of Sp1 and estrogens, and the structural composition of the promoter. The isolated mini MGARP promoter has a higher basal activity and more substantial response to ERa than the full-length 23 kb promoter, indicating that there are other factors in the 23 kb promoter contributing to the transcriptional regulation and the effects of ERa.