Figure 3. Set7 improved the stability and DNA binding ability respectably through K436 and K595 methylation.

(A and B) Western blot (A) and qPCR (B) of Gli3 in the condition of Set7 knockdown or overexpression in NIH-3T3 cells. n.s. p>0.05 versus siCtrl (left) or PCDNA (right) respectively. (C and D) Western blot of Gli3^WT, Gli3^K436R or Glli3^K595R in the condition of Set7 knockdown or overexpression in NIH-3T3 cells. (E and F) Western blot (E) and statistic (F) of Gli3^WT, Gli3^K436R or Gli3^K595R in NIH-3T3 cells treated with cycloheximide (CHX) for the indicated times. (G) Schematic representation of three Gli3 binding sites on the promoter regions of Gli1. (H) qPCR of Set7 in Gli3^WT-flag stable cell lines after Set7 knockdown in (I). **p<0.01, ***p<0.00 versus siCtrl. (I) ChIP-qPCR analysis using Ctrl IgG (grey) or anti-flag antibody (black) in NIH-3T3 cells transfected with Ctrl or Set7 siRNAs. ChIP signal levels are represented as fold change of input chromatin. (J) Western blot of flag tagged Gli3^WT and mutants in the stable cell lines used in (K). (K) ChIP-qPCR analysis using anti-flag antibody in NIH-3T3 cells expressing Gli3^WT-flag, Gli3^K436R-flag or Gli3^K595R-flag. ChIP signal levels are represented as fold change of input chromatin. *p<0.05, **p<0.01, ***p<0.001, n.s. p>0.05 versus anti flag siCtrl (I) or Gli3^WT-flag (K). Data are represented as mean ± SEM (n=3). All the qPCR results are normalized to GAPDH. In all the experiments, cells are treated with Shh. Ctrl, Control.

DOI: http://dx.doi.org/10.7554/eLife.15690.013

Figure 3—figure supplement 1. Set7 does not influence the cilium translocation of Gli3.

(A and B) Immunofluorescent staining of Ac-tubulin (blue) and Gli3 (red) in the absence (A-A’’) or presence (B-B’’) of Shh. (C and D) Statistic results of Gli3 accumulation at cilia tips in NIH-3T3 cells with Set7 knockdown (C) or Set7/Set7^H297A over-expression (D). In all experiments of (C and D), cells were treated with Shh. Data are represented as mean ± SEM (n=3). Ctrl, Control.

DOI: http://dx.doi.org/10.7554/eLife.15690.014

Figure 3—figure supplement 2. Set7 improves the DNA binding ability of Gli3 on Gli1 promoter.

(A) ChIP-qPCR analysis at Gli1 loci (Figure 3G) using control IgG or anti-Gli3 antibody in NIH-3T3 cells transfected with control or Set7 siRNAs. ChIP signal levels are represented as fold change of input chromatin. **p<0.01, ***p<0.001, versus anti Gli3 siCtrl. (B) qPCR of Set7 in NIH-3T3 cells with Set7 knockdown. RNAi efficiency was shown. Data are represented as mean ± SEM (n=3). **p<0.01 versus siCtrl. Ctrl, Control. All the qPCR results are normalized to GAPDH.

DOI: http://dx.doi.org/10.7554/eLife.15690.015

Figure 3—figure supplement 3. Set7 improves the DNA binding ability of Gli3 on the promoter regions of Hhip and Ptch1 ChIP-qPCR assays at Hhip.

(A) and Ptch1 (B) loci using control IgG or anti-flag antibody in NIH-3T3 Gli3-flag stable cells transfected with control or Set7 siRNA. ChIP signal levels are represented as fold change of input chromatin. *p<0.05, **p<0.01, ***p<0.001, versus anti flag siCtrl.

DOI: http://dx.doi.org/10.7554/eLife.15690.016