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. 2010 Jul 30;29(18):3170–3183. doi: 10.1038/emboj.2010.172

Figure 4.

Figure 4

Zic2 directly regulates Sert expression. (A) VISTA view of the occurrence of conserved sequence domains upstream of the first sert exon (purple arrow). Light blue arrow indicates the consensus transcription start site of sert (TSS). Shown from top to bottom are human versus mouse (H/M), monkey versus mouse (M/M), dog versus mouse (D/M), horse versus mouse (H/M) and rat versus mouse (R/M) sequences. The coloured peaks (purple: coding, blue: UTR, pink: conserved non-coding regions) indicate regions of at least 100 bp, with a conservation score of at least 75%. The pink arrow indicates a highly conserved peak of 130 bp upstream of the TSS that has been proposed as the basal Sert promoter sequence. The 130 bp mouse sequence of the conserved Sert promoter, including putative binding sites (in different colours) for the indicated transcription factors is graphically represented below the VISTA comparison. (B) Luciferase assays to evaluate the transcriptional activity of Zic2 on the 130 bp Sert promoter region. After transfection of pCAG-Zic2 in 293HEK cells together with luciferase vectors containing the 130-bp Sert promoter region, Zic2 increases the basal transcriptional activity of the 130-bp conserved Sert promoter more than seven-fold compared with the controls. A mutation in the Gli-binding sequence significantly reduces this induction. By contrast, mutations in the CRE-like motif or the GC-rich domain of the sert promoter had no effects on the induction by Zic2. The values represent means±s.e.m. of at least four triplicates, except for pGL3-GC with two triplicates (*P<0.01, Student's unpaired t-test). (C) ChIP assays demonstrate that Zic2 binds to the basal promoter region of Sert in the embryonic E16.5 VT retina in vivo. In the upper panel, primers to amplify the 130-bp Sert basal promoter were used, whereas in the bottom panel, control primers to amplify a 5′upstream non-conserved sequence were used. Zic2 binds to the Sert basal promoter (black arrow) but not to the control sequence (black arrowhead). Anti-AcH3 antibodies were used as positive controls and non-specific rabbit serum as negative control (IgGs).