S. To verify the above findings in an independent cellular system, Y1 cells were used because they express abundant MGARP protein and may contain a substantial amount of competent cofactors of Sp1 molecules that assist Sp1 in regulation of MGARP transcription. As shown in Figure 4C, a similar purchase AZP-531 observation in binding was made by EMSA using Y1 cell nuclearMGARP Is Regulated via Tandem Sp1 ElementsFigure 2. MGARP AZP-531 promoter activation is mediated by Sp1. A. Luc assay shows that Sp1 mediates the MGARP promoter activity in a dosedependent manner. HEK-293T cells were co-transfected with pGL3-(23 kb) and the increasing doses of Sp1 plasmids for Luc assay. B. Similarly, the pDsRed-MGARP(23 kb) reporter and several doses of Sp1 plasmids were co-transfected into HEK-293T cells to examine the expression of red fluorescent protein at 72 hours post transfection. C. Western blotting shows that knockdown of Sp1 with Sp1-specific RNAi (630-RNAi and 1722-RNAi) reduces the expression of both endogenous and exogenous Sp1. The scramble-RNAi and RNAi targeting GFP were used as control. D. Luc assays to determine the effect of Sp1 knockdown on MGARP promoter activity. *** represents p,0.001 (E) Semiquantitative RT-PCR analysis confirms that knockdown of Sp1 reduces the MGARP gene expression. Similarly, HEK-293T cells were transfected with control and Sp1-specific RNAi and harvested for semiquantitative RT-PCR analysis of MGARP messages expression. doi:10.1371/journal.pone.0050053.gextract, indicating that the endogenous Sp1 proteins can effectively bind the GC boxes of the MGARP promoter in different kinds of cell lines. However, with the addition of the antibody to the reaction mixture of Y1 cell nuclear extract, the super-shifted band disappeared (Figure 4C). This may indicate that an interaction of Sp1 with this antibody changed the molecular behaviors of Sp1, either by structurally altering the Sp1 protein, or by blocking the accessibility of the oligos. In any case, the test with the antibody indicated that the shifted bands specifically depend on Sp1. To further validate the binding between Sp1 and GC-Boxes in vivo, we performed Chromatin Immunoprecipitation (ChIP) assay using HEK-293T cells. The results demonstrated thatSp1specifically bound to the GC-box locus on the endogenous MGARP promoter, but not to the control GAPDH locus (Figure 5). Together, our results suggest that Sp1 proteins directly bind to the proximal GC-rich region of the MGARP promoter.Sp1 and ER Synergistically Stimulate MGARP Promoter ActivityIt was reported that ERa interacts with Sp1 and they act synergistically to activate downstream genes [19,28]. Considering that MGARP protein expression can be up-regulated by estrogens [5], we reasoned that ERa might be able to regulate the transcription of MGARP or coordinate with Sp1 in the activation of the MGARP promoter. This hypothesis was first tested by coMGARP Is Regulated via Tandem Sp1 ElementsFigure 3. GC-box1 plays a major role in MGARP promoter activation and both GC-boxes are required for full transactivation. The Luc reporters driven by the full-length MGARP promoter (23 kb) were transfected into HEK-293T cells, as compared to various promoter truncates either missing the GC-Boxes or expressing the GC-Boxes alone, without or with co-transfection of Sp1 plasmids (10 ng) as indicated. Luc activity was examined at 72 hours post transfection. *** represents p,0.001 and #represents p.0.05 (no significant difference). doi:10.1371/journ.S. To verify the above findings in an independent cellular system, Y1 cells were used because they express abundant MGARP protein and may contain a substantial amount of competent cofactors of Sp1 molecules that assist Sp1 in regulation of MGARP transcription. As shown in Figure 4C, a similar observation in binding was made by EMSA using Y1 cell nuclearMGARP Is Regulated via Tandem Sp1 ElementsFigure 2. MGARP promoter activation is mediated by Sp1. A. Luc assay shows that Sp1 mediates the MGARP promoter activity in a dosedependent manner. HEK-293T cells were co-transfected with pGL3-(23 kb) and the increasing doses of Sp1 plasmids for Luc assay. B. Similarly, the pDsRed-MGARP(23 kb) reporter and several doses of Sp1 plasmids were co-transfected into HEK-293T cells to examine the expression of red fluorescent protein at 72 hours post transfection. C. Western blotting shows that knockdown of Sp1 with Sp1-specific RNAi (630-RNAi and 1722-RNAi) reduces the expression of both endogenous and exogenous Sp1. The scramble-RNAi and RNAi targeting GFP were used as control. D. Luc assays to determine the effect of Sp1 knockdown on MGARP promoter activity. *** represents p,0.001 (E) Semiquantitative RT-PCR analysis confirms that knockdown of Sp1 reduces the MGARP gene expression. Similarly, HEK-293T cells were transfected with control and Sp1-specific RNAi and harvested for semiquantitative RT-PCR analysis of MGARP messages expression. doi:10.1371/journal.pone.0050053.gextract, indicating that the endogenous Sp1 proteins can effectively bind the GC boxes of the MGARP promoter in different kinds of cell lines. However, with the addition of the antibody to the reaction mixture of Y1 cell nuclear extract, the super-shifted band disappeared (Figure 4C). This may indicate that an interaction of Sp1 with this antibody changed the molecular behaviors of Sp1, either by structurally altering the Sp1 protein, or by blocking the accessibility of the oligos. In any case, the test with the antibody indicated that the shifted bands specifically depend on Sp1. To further validate the binding between Sp1 and GC-Boxes in vivo, we performed Chromatin Immunoprecipitation (ChIP) assay using HEK-293T cells. The results demonstrated thatSp1specifically bound to the GC-box locus on the endogenous MGARP promoter, but not to the control GAPDH locus (Figure 5). Together, our results suggest that Sp1 proteins directly bind to the proximal GC-rich region of the MGARP promoter.Sp1 and ER Synergistically Stimulate MGARP Promoter ActivityIt was reported that ERa interacts with Sp1 and they act synergistically to activate downstream genes [19,28]. Considering that MGARP protein expression can be up-regulated by estrogens [5], we reasoned that ERa might be able to regulate the transcription of MGARP or coordinate with Sp1 in the activation of the MGARP promoter. This hypothesis was first tested by coMGARP Is Regulated via Tandem Sp1 ElementsFigure 3. GC-box1 plays a major role in MGARP promoter activation and both GC-boxes are required for full transactivation. The Luc reporters driven by the full-length MGARP promoter (23 kb) were transfected into HEK-293T cells, as compared to various promoter truncates either missing the GC-Boxes or expressing the GC-Boxes alone, without or with co-transfection of Sp1 plasmids (10 ng) as indicated. Luc activity was examined at 72 hours post transfection. *** represents p,0.001 and #represents p.0.05 (no significant difference). doi:10.1371/journ.
