Figure 8

CBP enhances the repression of CLOCK/BMAL1-mediated transcription by HDAC3. (A) Northern blot analysis of HDAC3 mRNA expression in mouse brain, COS-1 cells and NIH3T3 cells. 30 μg (mouse brain and COS-1 cells) or 15 μg (NIH3T3 cells) of total RNA were loaded and blotted. (B) COS-1 cells were transiently transfected with pPeriod-1-Luc (10 ng), either with or without pcDNA3CLOCK (10 ng) and pcDNA3BMAL1 (10 ng), in the presence of increasing amounts (0, 20, 50, 100 ng) of pcDNA3HDAC3, as indicated. Empty vector (pcDNA3) was used to standardize the total amount of transfected DNA (210 ng). An asterisk indicates p < 0.05 compared with lane 5 (Student's t test). (C) pPeriod1-Luc cells were transiently cotransfected, with or without pcDNA3CLOCK (10 ng) plus pcDNA3BMAL1 (10 ng), with or without pcDNA3HDAC3 (20 ng), in combination with increasing amounts (0, 10, 30 ng) of pcDNA3CBP, as indicated. Empty vector (pcDNA3) was used to standardize the total amount of transfected DNA (210 ng). An asterisk indicates p < 0.05 (Student's t test). (D) COS-1 cells were transiently transfected with pmyc-HDAC3 (1 μg), in the presence or absence of pcDNA3CBP (7 μg). (E, F) COS-1 cells were transiently transfected with pEYFP-CLOCK (7 μg), pEYFP-BMAL1 (7 μg) or pmyc-HDAC3 (1 μg). Empty vector (pEYFP or pCMV-myc) was used to standardize the total amount of transfected DNA (8 μg). (G) COS-1 cells were transiently transfected with pcDNA3CBP (6 μg), in the presence or absence of pmyc-CLOCK (1 μg) or pmyc-HDAC3 (1 μg). Empty vector (pCMV-myc) was used to standardize the total amount of transfected DNA (8 μg). Immunoprecipitations (IP) was performed using anti-rabbit IgG or anti-CBP antibodies, and then Western blot analysis was performed using anti-myc, anti-CBP or anti-GFP antibodies, as indicated. The input lanes represent 10% of total cell lysates in the binding reaction. (B, C) Luciferase activity was expressed as a ratio of CLOCK/BMAL1-mediated reporter activity.