In a variety of biological processes, such as early embryonic development, the G1 phase of the cell cycle, and importantly, steroid receptor-mediated transcription [19,21,22]. Sp1 can interact with ERa and contribute to transcriptional outcomes [15,23?6]. As mentioned above, reports have documented that MGARP participates in steroid synthesis, and steroids also regulate MGARP expression [4,5]. 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 (2150 bp/240 bp and 239 12926553 bp/0 bp) proximal to the transcription start site (TSS). 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.Box2. For knockdown of Sp1, four short hairpin oligos targeting the 630 position (upper strand sequence: 59-GAT CCA CCA ACA GAT TAT CAC AAA TTC AAG AGA TTT GTG ATA ATC TGT TGG TTT TTT TGG AAA-39; lower strand sequence: 59AGC TTT TCC AAA AAA ACC AAC AGA TTA TCA CAA ATC TCT TGA ATT TGT GAT AAT CTG TTG GTG-39) and 1722 position (upper strand sequence: 59-GAT CCG TAC ATG ATG ACA CAG CAG GTT CAA GAG ACC TGC TGT GTC ATC ATG TAT TTT TTG GAA A-3; lower strand sequence: 59AGC TTT TCC AAA AAA TAC ATG ATG ACA CAG CAG GTC TCT TGA ACC TGC TGT GTC ATC ATG TAC G-39) of the Sp1 gene were synthesized and cloned into pSilencer, generating two shRNA expression plasmids, 630-RNAi and 1722RNAi, respectively. Their effectiveness was tested by western purchase Licochalcone-A blotting (Text S2). The Sp1 expression plasmid was a kind gift from Dr. Jon Horowitz (Department of Molecular Biomedical Sciences, North Carolina State University, College of Veterinary Medicine) and was sent to us with the ERa plasmid by Dr. Shaoyong Chen (BIDMC, 115103-85-0 Harvard Medical School, USA). The Sp1 antibody was purchased from Millipore (Upstate, MA, USA).Cell Culture, Transfection, Luciferase (Luc) Assay and Red Fluorescence Protein DetectionHEK-293T cells were obtained from the Cell Resource Center (IBMS, CAMS/PUMC, BJ, 1516647 China) and were grown in DMEM (Hyclone, Logan, UT, USA) supplemented with 10 fetal bovine serum (FBS) (ExCell Biology, SH, China) and penicillin/streptomycin. The reporters were transfected, as indicated, into HEK293T cells using Vigofect reagent (Vigorous Biotechnology, BJ, China) according to the manufacturer’s protocol. After 6 hours, the medium was replaced with DMEM containing 10 FBS and antibiotics. 72 hours post transfection, cells were harvested for Luc assay using the Luc assay system (Vigorous Biotechnology, BJ, China), and the activity of Firefly luciferase values were normalized to that of the Renilla luciferase.Materials and Methods Plasmids and ReagentsThe bioinformatics analysis was carried out as described in Text S1. The bacterial artificial chromosome clone bearing the MGARP gene (BAC, RP11-468C4) was purchased from Invitrogen (Carlsbad, CA, US). The MGARP promoter (23 kb) was amplified by PCR using the following primers: 59 GCT AAG CTT ATT CCA CAG AGA GGC TGA GAG-39 and 59-TAT GGA TCC GGA CTT TCT TAA.In a variety of biological processes, such as early embryonic development, the G1 phase of the cell cycle, and importantly, steroid receptor-mediated transcription [19,21,22]. Sp1 can interact with ERa and contribute to transcriptional outcomes [15,23?6]. As mentioned above, reports have documented that MGARP participates in steroid synthesis, and steroids also regulate MGARP expression [4,5]. 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 (2150 bp/240 bp and 239 12926553 bp/0 bp) proximal to the transcription start site (TSS). 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.Box2. For knockdown of Sp1, four short hairpin oligos targeting the 630 position (upper strand sequence: 59-GAT CCA CCA ACA GAT TAT CAC AAA TTC AAG AGA TTT GTG ATA ATC TGT TGG TTT TTT TGG AAA-39; lower strand sequence: 59AGC TTT TCC AAA AAA ACC AAC AGA TTA TCA CAA ATC TCT TGA ATT TGT GAT AAT CTG TTG GTG-39) and 1722 position (upper strand sequence: 59-GAT CCG TAC ATG ATG ACA CAG CAG GTT CAA GAG ACC TGC TGT GTC ATC ATG TAT TTT TTG GAA A-3; lower strand sequence: 59AGC TTT TCC AAA AAA TAC ATG ATG ACA CAG CAG GTC TCT TGA ACC TGC TGT GTC ATC ATG TAC G-39) of the Sp1 gene were synthesized and cloned into pSilencer, generating two shRNA expression plasmids, 630-RNAi and 1722RNAi, respectively. Their effectiveness was tested by western blotting (Text S2). The Sp1 expression plasmid was a kind gift from Dr. Jon Horowitz (Department of Molecular Biomedical Sciences, North Carolina State University, College of Veterinary Medicine) and was sent to us with the ERa plasmid by Dr. Shaoyong Chen (BIDMC, Harvard Medical School, USA). The Sp1 antibody was purchased from Millipore (Upstate, MA, USA).Cell Culture, Transfection, Luciferase (Luc) Assay and Red Fluorescence Protein DetectionHEK-293T cells were obtained from the Cell Resource Center (IBMS, CAMS/PUMC, BJ, 1516647 China) and were grown in DMEM (Hyclone, Logan, UT, USA) supplemented with 10 fetal bovine serum (FBS) (ExCell Biology, SH, China) and penicillin/streptomycin. The reporters were transfected, as indicated, into HEK293T cells using Vigofect reagent (Vigorous Biotechnology, BJ, China) according to the manufacturer’s protocol. After 6 hours, the medium was replaced with DMEM containing 10 FBS and antibiotics. 72 hours post transfection, cells were harvested for Luc assay using the Luc assay system (Vigorous Biotechnology, BJ, China), and the activity of Firefly luciferase values were normalized to that of the Renilla luciferase.Materials and Methods Plasmids and ReagentsThe bioinformatics analysis was carried out as described in Text S1. The bacterial artificial chromosome clone bearing the MGARP gene (BAC, RP11-468C4) was purchased from Invitrogen (Carlsbad, CA, US). The MGARP promoter (23 kb) was amplified by PCR using the following primers: 59 GCT AAG CTT ATT CCA CAG AGA GGC TGA GAG-39 and 59-TAT GGA TCC GGA CTT TCT TAA.