Figure 4.

Increased uH2A association with genes downregulated in a cell culture model of HD and Ring2 knockdown-mediated reversal of transcriptional repression. A, Increased uH2A association with Vdr, Tcf7, Inhbb, and Dhrs4 gene promoters in STHdh^111/111 cells compared with STHdh^7/7 cells (n = 3 independent experiments; *p < 0.05). B, Western blot showing a reduced Ring2 protein level in STHdh^111/111 cells transfected with 100 nm Ring2 siRNA (Ring2) for 24–72 h. Mock or 100 nm nontargeting control siRNA (NTC)-transfected cells do not show a significant change in Ring2 protein level. For an internal control, the blot was stripped and reprobed for β-actin. Ring2 bands were normalized to β-actin bands (Ring2/β-actin), and densitometry of Ring2/β-actin is shown below (n = 3 independent experiments; *p < 0.05, Ring2 vs NTC). C, Western blot showing reduction in the uH2A level in STHdh^111/111 cells transfected with 100 nm Ring2 siRNA for 24–48 h. Densitometry of uH2A/H2A is shown below (n = 4 independent experiments; *p < 0.05, Ring2 vs NTC). D, Quantitative real-time PCR showing increased mRNA levels of VDR, TCF7, and Inhibin b in STHdh^111/111 cells transfected with 100 nm Ring2 siRNA for 24 h, which is equivalent to those in STHdh^7/7 cells. mRNA levels of each gene normalized to that of β-actin gene are indicated as VDR/β-actin, TCF7/β-actin, and Inhibin b/β-actin. Mock or NTC-transfected cells do not show any changes in mRNA levels of those genes (n = 4 independent experiments; *p < 0.05, Ring2 vs NTC). Error bars indicate SEM. IP, Immunoprecipitation.