Figure 6: PHF20 is required for the deposition of histone H4K16ac on ERα target genes.

(a) PHF20 is required for the E2-induced accumulation of histone H4K16ac on the promoters of GREB1 and PR. qPCR analysis of histone H4K16ac ChIP in control and PHF20 knockdown cells 15 and 45 min after E2 treatment. The H4K16ac ChIP was calculated as a ratio relative to total H3 ChIP. (b) PHF20 is required for ERα chromatin recruitment. qPCR analysis of ERα ChIP in the cells as in a. ERα ChIP is shown as a ratio relative to input. (c) G9a is required for the E2-induced accumulation of histone H4K16ac on the promoters of GREB1 and PR. qPCR analysis of histone H4K16ac ChIP in control and G9a knockdown cells 15 and 45 min after E2 treatment. (d) G9a is required for ERα chromatin recruitment. qPCR analysis of ERα ChIP in the cells as in c. In a–d, all error bars indicate the mean ±s.e.m. of three experiments. *P<0.05, ^**P<0.01 (Student’s t-test). (e) Working model of G9a-mediated ERα methylation in hormonal response. Left panel: G9a methylates ERα at K235 in the nucleus in response to E2 stimulation, and ERαK235me2 is recognized by the Tudor domain of PHF20, which recruits the MOF complex to acetylate histone H4K16, thereby promoting the expression of ERα target genes GREB1 and PR. Middle panel: depletion of G9a abolishes ERαK235 methylation and the methylation-dependent ERα–PHF20 interaction, thereby reducing the recruitment of PHF20/MOF and ERα to the EREs of ERα target genes GREB1 and PR, consequently reducing their expression. Right panel: depletion of PHF20 reduces the H4K16ac levels on chromatin and the recruitment of ERα to EREs via feedback regulation, thereby reducing the expression of ERα target genes GREB1 and PR.