Sp1 participates in the <i>Aldh1a2</i> expression.

<p>(<b>A</b>) The genomic organization of the mouse <i>Aldh1a2</i> gene and its 5′-flanking region is shown. A fragment containing exon 1 and its 5′-flanking region from −2,600 to +182 was inserted into reporter vectors. DNA binding sites (STAT-binding sites, NF-κB binding sites, a SREBP binding site, and putative RARE half-sites (RARE-h)), the TATA box, and the GC-rich region in the fragment are indicated. (<b>B</b>) Flt3L-generated BM-DCs were cultured with 10 ng/ml GM-CSF for 16 h in the presence or absence of 1 µM mithramycin A. After the culture, <i>Aldh1a2</i> mRNA expression was assessed by real-time PCR (<i>Left panel</i>), and protein expression of RALDH2 (ALDH1A2) and α-tubulin was analyzed by Western blotting (<i>Right panel</i>). Relative mRNA expression levels were calculated by defining the <i>Aldh1a2</i> mRNA expression level in the cells incubated with medium alone for 16 h was set to 1 (<i>Left panel</i>). Data are representative of three (<i>Left panel</i>) or two (<i>Right panel</i>) independent experiments. (<b>C</b>) Serial-deletion fragments derived from the 5′-flanking region of the mouse <i>Aldh1a2</i> gene were inserted in the reporter vector, pGL3-basic. COS-7 cells were transfected in triplicate with one of the deletion constructs (1.25 µg) or the pGL3-RALDH2 (−2,600) reporter vector (1.25 µg) in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after the transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL3-basic alone as 1. Data are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's <i>t</i> test (**<i>p</i><0.01, ***<i>p</i><0.001). Data are representative of three independent experiments.