Figure 2: Shh induces a Gli-dependent local epigenetic switch from PRC2 to Jmjd3.

(a) ChIP-qPCR indicates the dynamic binding of endogenous SUZ12 to the Gli1 regulatory region in wild-type and Jmjd3^−/− MEF cells on Shh induction. (means±s.d., n=3). (b) Increased Jmjd3 binding to Gli1 gene on Shh treatment was shown by ChIP-qPCR (means±s.d., n=3). (c) Shh treatment did not reduce levels of PRC2 subunits as shown by EZH2 and SUZ12 western blot. (d) Jmjd3 expression level was not significantly changed in MEFs by Shh treatment as analysed by RT–qPCR (means±s.d., n=3). (e) Jmjd3 co-immunoprecipitates with Gli2. Lysates of NIH3T3 cells transiently transfected with plasmids expressing HA-Jmjd3 and Myc-Gli2 were immunoprecipitated with anti-HA antibodies and blotted with antibodies against HA and Myc. (f) Jmjd3 co-immunoprecipitates with endogenous Gli1. Cell lysates of Shh-treated NIH3T3 cells expressing HA-Jmjd3 were immunoprecipitated with anti-HA antibodies and blotted with antibodies against HA and Gli1. (g) RNAi-induced inhibition of Gli1 and Gli2 in Shh-treated MEFs led to (h) decreased Jmjd3 binding and (i) increased H3K27me3 levels in the Gli1 regulatory region (means±s.d., n=3). (j) Deletion of Jmjd3 in MEFs led to impaired endogenous Shh target gene (Gli1, Ptch1, Hhip) expression induced by lentiviral expressed HA-Gli1 as shown by RT–qPCR. Expressions of endogenous Gli1 and exogenous HA-Gli1 were measured using primers specific for the 5′-UTR and the HA tag region, respectively. Significance was determined using t-test or ANOVA with post hoc t-test (means±s.d., n=3). ** indicates P<0.01 and * indicates P<0.05.