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. 2018 May 21;12(7):1026–1046. doi: 10.1002/1878-0261.12308

Figure 3.

Figure 3

SALL2 binds and represses CCND1 and CCNE1 promoters. Bioinformatic analyses of cyclin promoters to identify putative SALL2 sites were performed using a previously reported binding site matrix (consensus sequence GGG(T/C)GGG) (Gu et al., 2011) in Transcriptional Regulatory Element Database (TRED) (https://cb.utdallas.edu/cgi-bin/TRED/tred.cgi?process=home) (Jiang et al., 2007). Sequences analyzed [−2000 bp from transcription start site (+1)] were obtained from Eukaryotic Promoter Database (EPD) and included mouse Ccnd1 (NM 007631), mouse Ccne1 (NM 007633), human CCND1 (NM 053056), and human CCNE1 (NM 001238). A. Schematic representation of human CCND1 (NM 053056) and CCNE1 (NM 001238) gene promoters. SALL2 putative binding sites are represented by square symbols. Putative SALL2 binding sites are classified as high, middle, and low scores according to their identity with the SALL2 consensus matrix (Gu et al., 2011) The transcription start site (+1) is represented by a flag. (B,C) Repression of CCND1 (B) and CCNE1 (C) promoters’ activities by SALL2. Transient co‐transfections of pGL3‐CCND1 or pGL3‐CCNE1 reporter with or without SALL2 E1 (blue bars) (or E1A, green bars) into HEK293 cells were performed as described under ‘Materials and Methods’. Luciferase activity was measured from cell lysates and normalized to β‐galactosidase activity, and promoter activity was expressed as relative luciferase units (R.L.U). pGL3 vector served as control. Data are expressed as mean ± SD from three independent experiments performed in triplicate. ***P ≤ 0.001, one‐way ANOVA. (D–F) HEK293 cells were transfected with pcDNA3‐SALL2 (E1, blue) or pcDNA3‐SALL2 (E1A, green) vector. Chromatin was immunoprecipitated 24 h after transfection using SALL2 antibody or normal rabbit IgG (control antibody), and specific genomic regions in the human CCND1 (D), CCNE1 (E), proximal promoters, and a nonrelated promoter region (NRR) were analyzed by real‐time PCR. Graphs show quantification of the amplified DNA for each immunoprecipitation relative to IgG. Results are representative of two assays performed in triplicate.