Figure 4: Coordinated functions of Jmjd3 and Set1/MLL regulate Shh-activated gene transcription.

(a) Both the enzymatic and non-enzymatic activities of Jmjd3 are required for its function in Shh-induced gene activation. NIH3T3 cells infected with Jmjd3 shRNA or vector control were transfected with plasmids co-expressing GFP-Gli1 and indicated Jmjd3 proteins or vector controls. Levels of endogenous Gli1 and Ptch1 were determined by RT–qPCR (means±s.d., n=3). (b) Expression of HA-tagged Jmjd3 proteins and exogenous GFP-Gli1 levels in transfected cells as detected by western blot. (c) Binding of Jmjd3 and mutants to Gli1 regulatory region was determined by HA ChIP-qPCR. ‘NS’ indicates not significant (means±s.d., n=3). (d) Set1/MLL complexes are important for Shh-induced target gene activation. RNAi-mediated inhibition of expression of individual Set1/MLL subunits Ash2L, DPY30, RbBP5 and WDR5 significantly impaired Gli1 expression in the presence of Shh as measured by RT–qPCR (means±s.d., n=3). (e) Shh-induced binding of WDR5 to Shh target genes requires the presence of Jmjd3. Shown are ChIP-qPCR analyses with antibodies against endogenous WDR5 and H3K4me3 in NIH3T3 cells expressing shRNA targeting Jmjd3 or scrambled shRNA in the basal or Shh-treated conditions. CD4 was the negative control (means±s.d., n=3). (f) Shh enhances the interactions between Jmjd3 and Set1/MLL subunits. NIH3T3 cells co-transfected with plasmids expressing FLAG-Jmjd3 and HA-Ash2L were treated with or without Shh. An anti-FLAG antibody was used for immunoprecipitation, and antibodies against FLAG or HA were used for western blot. Significance was determined using t-test or ANOVA with post hoc t-test; ** indicates P<0.01 and * indicates P<0.05. The comparisons are all against the control samples.