Figure 3.

PIASx represses MEF2-dependent transcription in cerebellar granule neurons. A, PIASx RNAi induces specific knockdown of PIASx in cells. HEK293T cells were cotransfected with FLAG-tagged PIASx or PIAS1 expression plasmids, together with the control RNAi plasmid (U6/bs), PIASx RNAi plasmids encoding hpRNAs targeting distinct regions of PIASx mRNA (U6/piasx1 and U6/piasx2, respectively), or a control RNAi plasmid encoding scrambled hairpin RNAs (U6/scri), and FLAG-14-3-3 expression plasmid as a transfection control. After 72 h, cells were lysed and subjected to immunoblotting for FLAG-tagged proteins. Expression of PIASx hpRNAs, but not the scrambled hpRNAs, specifically reduced PIASx expression but not PIAS1 or 14-3-3 expression. B, PIASx represses the Nur77 promoter in primary cerebellar granule neurons. Granule neurons (P6 plus 4 DIV) were cotransfected with a luciferase reporter gene controlled by the Nur77 promoter (pNur77-luc), the PIASx RNAi or control RNAi plasmids, and pRL-TK as a control for transfection efficiency. After 72 h, cells were transferred to medium containing 5 mm KCl, and firefly and renilla luciferase activities were determined 24 h later. Expression of both piasx1 and piasx2 hpRNAs, but not the control scrambled hpRNA or empty RNAi vector, significantly enhanced Nur77 promoter activity (*p < 0.001; ANOVA; n = 4). C, MEF2 binding is necessary for PIASx-repression of the Nur77 promoter in neurons. Granule neurons (P6 plus 4 DIV) were transfected with wild-type (WT) or MEF2 binding site mutant (MREmut) pNur77 luc, the PIASx or control RNAi plasmids, and pRL-TK, and analyzed as in B. Data are normalized as percentage of activation by PIASx hpRNAs relative to the control RNAi plasmid. PIASx hpRNAs significantly enhanced the activity of WT but not MREmut pNur77-luc reporter gene (*p < 0.002; ANOVA; n = 3).