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Article Title: Histone deacetylase inhibitors stimulate mitochondrial HMG-CoA synthase gene expression via a promoter proximal Sp1 site

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Figure Lengend Snippet: Mitochondrial HMG-CoA synthase, a target gene for HDAC inhibitors: TSA induces mitochondrial HMG-CoA synthase mRNA expression through mitochondrial HMG-CoA synthase promoter acetylation in the chromatin complex. (A) HT-29 and Caco-2 cells were treated with 0.3 µM TSA (+) or DMSO (–) for 24 h. The mitochondrial HMG-CoA synthase mRNA levels were analyzed by northern blot. Levels of 18S rRNA are also shown to correct for gel loading variation. (B) ChIPs were performed using chromatin isolated from HT-29 and CaCo-2 cells either treated with TSA (+) for 24 h or untreated (–). The chromatin preparations were immunoprecipitated with antibodies against acetylated histone H4 (α-AcH4) and analyzed by PCR to detect the HMG-CoA synthase promoter associated to acetylated histones. Aliquots of chromatin were also analyzed before immunoprecipitation (Input). Relative histone acetylation levels (Fold of acetylation) were determined by quantitation of PCR product using BIOPROFIL® image analysis software, BIO-1D (Vilber Lourmat) and by correction with the input data and represented as the ratio of relative acetylation in the presence versus the absence of TSA treatment. (C) ChIP assay of HDAC1 bound to endogenous mitochondrial HMG-CoA synthase promoter. CaCo-2 chromatin, isolated as described above, was immunoprecipitated with increasing amounts of anti-HDAC1 or preimmune serum (IgG). PCRs shown in (B) and (C) were performed with 5 µl of DNA and 30 cycles of amplification were used.

Article Snippet: Relative histone acetylation levels (Fold of acetylation) were determined by quantitation of PCR product using BIOPROFIL® image analysis software, BIO-1D (Vilber Lourmat) and by correction with the input data and represented as the ratio of relative acetylation in the presence versus the absence of TSA treatment. ( C ) ChIP assay of HDAC1 bound to endogenous mitochondrial HMG-CoA synthase promoter.

Techniques: Expressing, Northern Blot, Isolation, Immunoprecipitation, Quantitation Assay, Software, Amplification