Fig. 4

Membrane depolarization mediates a decrease in Yy1 phosphorylation and depletes Yy1 from the Senp1 promoter via the PP1/PP2A phosphatases. (a) Okadaic acid (OA) prevents Yy1 de-phosphorylation upon neuronal depolarization. Left: mouse cortical neurons were treated with or without neuronal depolarization with 60 mM KCl for 2 hrs, or pretreated with 100 nM OA for 4 hrs followed with 2 hr treatment with 60 mM KCl. Endogenous Yy1 was pulled down and Phosphorylated Yy1 was detected with anti-phospho-Serine antibodies. Right: quantification of phosphorylated Yy1 levels versus total Yy1. (b) upper panel: a schematic diagram of Yy1 protein and the location of serines S184 and S247 relative to the acidic N-terminus, the glycine-lysine-rich central (GK), and the C-terminal DNA-binding zinc (Zn) finger domains. Lower panel: S184, 247A mutation inhibits Yy1 phosphorylation. (c) Inhibition of Yy1 phosphorylation leads to the reduction of its binding to Senp1 promoter in vitro. Upper panel showed the purified proteins for in vitro binding assay by Western blotting. In vitro binding of Myc-Yy1 wild type (Myc-Yy1-WT) and Myc-Yy1-S184, 247A to the DNA fragments containing Senp1 promoter was assessed with qRT-PCR. Graphs showed the relative enrichment after normalization with the enrichment in the control (Myc alone). (d) Yy1 phosphorylation is necessary for its activation on Senp1 promoter. Upper panel: Western blot with anti-Myc antibodies showing the expression of Yy1 wild type and mutant in the cells applied for luciferase assay. Lower panel: Luciferase activity was measured in Neuro2A cells co-transfected with Senp1 promoter fused to the luciferase reporter and Myc alone, Myc-Yy1-WT, or Myc-Yy1-S184, 247A, respectively. The luciferase activity from cells transfected Myc alone was set at 1. (e) Effects of various phosphatase inhibitors on KCl induced repression of Senp1 mRNA. Cortical neurons were pretreated with 100 nM Okadaic acid (OA) for 4 hrs or 50 μg/ml Cyclosporin A (CsA) for 22 hrs followed with 2 hr treatment with 60 mM KCl. Senp1 expression was detected by qRT-PCR. Graphs indicate three independent biological replicates. Error bars represent one standard deviation from the mean. *(p < 0.05). p value was determined using two-tailed unpaired t test.