Figure 4: BRMS1L reduces acetylated H3K9 level of FZD10 promoter and suppresses SP1-mediated FZD10 transcription.

(a) T47D cells with stable transfection of pGL3-FZD10 promoter were transfected with BRMS1L-siRNAs or GFP-siRNA and assayed for luciferase activity. **P≪0.01 as compared with GFP-siRNA. (b) qRT–PCR for FZD10 mRNA expression normalized to GAPDH in T47D and BT-474 cells treated with 200 ng ml⁻¹ trichostatin A (TSA) for 24 h. **P≪0.01, ^##P≪0.01 as compared with dimethylsulphoxide (DMSO). (c) ChIP analysis for BRMS1L binding to FZD10 promoter in MDA-MB-231 cells transfected with Myc-BRMS1L or negative control (NC). Enrichment of promoter region was normalized by input. ***P≪0.001 as compared with NC. (d,e) ChIP analysis for HDAC1 binding to FZD10 promoter in T47D cells transfected with BRMS1L-siRNAs or GFP-siRNA (d) and in MDA-MB-231 cells transfected with pcDNA3 vector carrying BRMS1L (BRMS1L) or negative control (NC) (e). Enrichment of promoter region was normalized by input. **P≪0.01 as compared with GFP-siRNA or NC. (f,g) ChIP analysis for H3K9Ac binding to FZD10 promoter in T47D cells transfected with BRMS1L-siRNAs or GFP-siRNA (f) and in MDA-MB-231 cells transfected with pcDNA3 vector carrying BRMS1L (BRMS1L) or negative control (NC; (g). Enrichment of promoter region was normalized by input. **P≪0.01 as compared with GFP-siRNA or NC. (h) Luciferase reporter assays for the truncated or mutant FZD10 promoter constructs transfected in MDA-MB-231 cells. **P≪0.01. (i) FZD10 mRNA normalized to GAPDH in MDA-MB-231 cells that were mock transfected (mock), or transfected with GFP-siRNA or two SP1-siRNAs. **P≪0.01 as compared with GFP-siRNA. (j) FZD10 mRNA normalized to GAPDH in T47D cells that were transfected with pcDNA3 vector carrying SP1 (SP1) or negative control (NC). **P≪0.01 as compared with NC. (k,l) ChIP analysis for SP1 binding to FZD10 promoter in MDA-MB-231 cells (k) and T47D cells (l). Enrichment of promoter region was normalized by input. **P≪0.01 as compared with anti-IgG. (m,n) ChIP analysis for SP1 binding to the FZD10 promoter in MDA-MB-231 cells transfected with pcDNA3 vector carrying BRMS1L (BRMS1L) or negative control (NC; m) and in T47D cells transfected with BRMS1L-siRNAs or GFP-siRNA (n). Enrichment of promoter region was normalized by input. **P≪0.01 as compared with NC or GFP-siRNA. BRL, BRMS1L. Bars correspond to mean±s.d. These data are representative of three independent experiments. Student’s t-test was used for the comparison of two independent groups.