Fig. 5. ZFHX3 and ERβ bind to MYC promoter to coordinately repress its transcription.

a DPN treatment decreased MYC mRNA level in C4-2B cells. Hormone-deprived medium was used for DPN treatment. n = 4. b Inhibition of ERβ function by its antagonist PHTPP eliminated the inhibitory effect of DPN on MYC mRNA expression in C4-2B cells. DPN and PHTPP were at 0.5 µM. n = 4. c Knockout of ZFHX3 increased MYC mRNA level. Wt, control clone; KO3 and KO8, two ZFHX3-null clones of C4-2B cells. n = 4. d, e DPN decreased the activity of MYC promoter. Expression plasmids of pGL3 vector control (pGL3-basic), pGL3 with MYC full-length promoters (a, pGL3-MYC with bases −2455 to 309) or pGL3 with two smaller MYC promoter fragments (d, pGL3-MYC-1 with bases −2024 to −1193 and pGL3-MYC-2 with bases −1200 to −200), and the pRL-TR reporter were transfected into C4-2B cells in phenol red-free medium supplemented with 2% CS-FBS. Twenty-four hours later, DPN treatments (0.1 µM, 48 h) were applied, and relative luciferase activities were then determined. n = 4. f Schematic of the MYC promoter region from base −1200 to base −200 relative to the P2 transcriptional initiation site (TIS), with locations of all four TISs, the first 3 exons, and primers used to amplify promoter regions A–C. Arrows under the promoter indicate primer locations. g Detection of ZFHX3- and ERβ-bound MYC promoter DNA in parental C4-2B cells using ChIP and regular PCR. h, i Binding of ERβ to MYC promoter region A in the presence (Wt) and absence (KO8) of ZFHX3 (h), with or without DPN treatment (i), using ChIP and regular PCR (upper) or real-time PCR (lower) in Wt and KO8 clones of C4-2B cells. The n of h, i is 3. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. ZFHX3, zinc-finger homeobox 3; ERβ, estrogen receptor beta; mRNA, messenger RNA; PHTPP, 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol; Wt, wild type; CS-FBS, calf serum-fetal bovine serum; DPN, diarylpropionitrile, ChIP, chromatin immunoprecipitation