Fig 2: Structures of RORa, YAP, and Tip60 and their possible relationship. a Scheme showing the interaction among YAP, Tip60, and RORa. GST-RORa (a.a. 424–523), used for the pull-down assay in Fig. 5d, is also shown. b Mutants generated for interaction assays. c, d In left panels, immunoprecipitation analyses with primary cerebellar neurons transiently expressing FLAG-YAP2 (full-length YAP) or FLAG-YAPdeltaC show the interaction of endogenous RORa with YAP or YAPdeltaC. In middle panels, YAP2 mutants of the first and/or second WW domain were also examined for interaction with endogenous RORa. In right panels, wild-type RORa and mutant RORa at the PPLY motif were tested for interaction with FLAG-YAP2/YAPdeltaC. The lower graphs show quantitative analyses of the relative intensities of output bands, corrected based on the corresponding input band (n = 3). Double asterisks: p < 0.01 in one-way ANOVA with post hoc Tukey’s HSD test

Fig 3: YAP/YAPdeltaC are co-factors of RORa. a Luciferase assays with COS-7 cells showing that RORa but not mutant RORa (RORa Y507A) lacking the consensus motif for the WW domain of YAP, acts with YAP or YAPdeltaC to transactivate luciferase gene expression via the RORa-responsive element. Single transfection of YAP or YAPdeltaC expression vector yielded insufficient transactivation. Double asterisks indicate statistical significance (p < 0.01, N = 5) in one-way ANOVA with post hoc Tukey’s HSD test. b Protein expression levels of RORa, YAP, and YAPdeltaC in the luciferase assays shown in Fig. 5a, determined by western blot. Because YAP is expressed endogenously in COS-7 cells, the amount of YAP was quantified (N = 5). c Left: effect of YAP knockdown and/or Tip60 knockdown on RORa-mediated transcription, evaluated by luciferase assays in 293T cells expressing RORa. Asterisks indicate statistical significance (p < 0.05, N = 8) in one-way ANOVA with post hoc Tukey’s HSD test. Right: levels of RORa, YAP, YAPdeltaC, and Tip60 are shown in lower panels. Double asterisks indicate statistical significance (p < 0.01, N = 5) in one-way ANOVA with post hoc Tukey’s HSD test. d Pull-down assay showing that YAP and Tip60 compete for interaction with RORa. A GST fusion protein of a part of RORa (a.a. 424–523) was used in this assay, as described in Methods. e Effect of Atxn1 knockdown on RORa-mediated transcription, evaluated by luciferase assays in 293T cells expressing RORa. Expression levels of RORa and Atxn1 are shown in right panel. Asterisks indicate statistical significance (p < 0.05, N = 6) in one-way ANOVA with post hoc Tukey’s HSD test

Fig 4: YAP/YAPdeltaC restores RORa target gene expression in vitro. a In primary cerebellar neurons, luciferase assays were performed 2 days after transfection of reporter and effector plasmids (left upper graph). Expression levels of RORa, Atxn1-86Q, YAP, YAPdeltaC, Tip60, GAPDH, a-tubulin, and ß-actin at the time of the luciferase assay are shown in the lower panels. Atxn1-86Q suppressed RORa-mediated transcription immediately without decreasing the level of RORa or YAP. Transcriptional suppression was rescued by co-expression of YAP or YAPdeltaC (right graphs). Double asterisks indicate statistical significance (p < 0.01, N = 7) in one-way ANOVA with post-hoc Tukey’s HSD test. b Dose-dependent inhibitory effect of Atxn1-86Q (left panels) and dose-dependent rescue effect of YAP2 (right panels) on RORa-mediated transcription. Double asterisks indicate statistical significance (p < 0.01, N = 5) in one-way ANOVA with post hoc Tukey’s HSD test. Lower panels show the amounts of related proteins 2 days after transfection, as determined by western blot. c Protein levels of YAP, YAPdeltaC, Atxn1, GAPDH, a-tubulin, and ß-actin were sequentially monitored until 7 days after transfection of Atxn1-33Q (left panels) or Atxn1-86Q (middle panels) to mouse primary neuron. YAP/YAPdeltaC were decreased from day 4, while RORa was decreased at day 7. The RORa level was not changed at 2 days, when RORa-mediated transcription was suppressed in luciferase assay. MG-132 treatment (0.3 µM, right panels) increased the level of YAP/YAPdeltaC and RORa decreased by Atxn1-86Q. Quantitative analyses are shown in the lower graphs. Double asterisks indicate statistical significance (p < 0.01, N = 5) in Dunnett’s test. d Cell death was not markedly increased at day 7 after transfection of Atxn1-86Q. e Cell viability was examined at days 2, 4, and 7 after transfection by trypan blue dye exclusion test among non-transfected, Atxn1-33Q-transfected, Atxn1-86Q-transfected, and Atxn1-transfected + MG-132 primary cerebellar neurons. Black area represents mean value of % dye-positive neurons (N = 5)

Fig 5: IQGAP1-F inhibits PCAF but activates KAT5 HAT activity. The effect of IQGAP1-F on the HAT activity of two homologues of CBP, PCAF (a) and KAT5 (b) was tested in Western blots developed by anti-AcK (left panel) and anti-His-tag (right panel) antibodies. Acetylation reactions were run in the absence (marked C) and presence of increasing amounts (x10, 100; relative to CBP concentration) of IQGAP1-F (40 kDa). A significant decrease in H4 acetylation by PCAF was observed in presence of 10- or 100-fold excess of IQGAP1-F, however, the activity of KAT5 enzyme was enhanced in presence of IQGAP1-F, in a concentration-dependent manner.