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. 2010 Apr 27;38(16):5366–5383. doi: 10.1093/nar/gkq285

Figure 5.

Figure 5.

Reduction of CREB results in changes of chromatin accessibility and histone acetylation. (A) RNAi for CREB reduced Hulc promoter activity in Hep3B and HL-7702 cells. CREB or negative control siRNAs were co-transfected with Luc-HCP into cells. Luciferase assays were performed 24 h after transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. (B) Endogenous HULC mRNA was quantified by real-time PCR and normalized to β-actin RNA. Chromatin from Hep3B and HL-7702 cells treated with control or CREB specific siRNA was harvested and precipitated with anti-diacetyl-H3 (C), anti-tetra-acetyl-H4 (D) and anti-pol II (E) antibodies. After DNA recovery, the precipitates were evaluated by real-time PCR for the level of enrichment over negative control antibody. (F) ChIP and re-ChIP experiments performed with anti-phosph-CREB, anti-P300 and anti-Brg I antibodies on Hep3B and HL-7702 cells. (G) Chromatin from Hep3B cells treated with control or CREB specific siRNA was harvested and precipitated with anti-P300 antibody. All the results (A–E and G) are the means of three independent experiments ± SD. Results of cells transfected siRNA against CREB are normalized to those of cells treated by negative control siRNA. The asterisk indicates statistical significance at the P < 0.05 level using t-test.