Journal: Alcoholism, clinical and experimental research
Article Title: Binge ethanol-induced HDAC3 down-regulates Cpt1α expression leading to hepatic steatosis and injury.
doi: 10.1111/acer.12172
Figure Lengend Snippet: Fig. 5. Evaluation of the role of histone deacetylase 3 (HDAC3) in the transcriptional regulation of Cpt1a gene expression. (A) Schematic representa- tion of a mouse Cpt1a promoter. Locations of chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (qPCR) primer pairs for anal- ysis of HDAC3, N-CoR binding, and histone acetylation in the vicinity of the mouse Cpt1a promoter. The coordinate locations shown are with respect to the transcription start site (TSS) in REFSEQ NM_013495.2. (B) Increased HDAC3 recruitment to the Cpt1a-TRE promoter region in the livers of binge ethanol (EtOH)-treated mice compared to control and Trichostatin A (TSA)-treated animals. (C) N-CoR binding to the Cpt1a-TRE promoter region was unaffected by either EtOH or TSA treatment. (D) Histone acetylation at the Cpt1a (-1) region was decreased in the livers of binge EtOH-treated animals and increased in response to TSA treatment. Chromatin was immunoprecipitated with anti-HDAC3, anti-N-CoR, anti-histone H3 acetylation (H3Ac), and ChIP qPCR was performed to confirm the protein interaction with the target promoters. The “input” lanes represent the results of PCR using diluted frac- tions of nonimmunoprecipitated chromatin as templates. Densitometry analysis was performed to evaluate specific binding of each protein relative to input. HDAC3 and N-CoR recruitment to the Cpt1a promoter was analyzed using Cpt1a-TRE site. Histone acetylation was analyzed at the Cpt1a (-1) site. For semi-qPCR of ChIP assay 29, PCR cycles were used for input, HDAC3, N-CoR, and H3Ac.
Article Snippet: The liver chromatin immunoprecipitation (ChIP) assay was performed following the Enzymatic ChIP protocol (Cell Signaling, Danvers, MA) with slight modifications using minced tissue for formaldehyde cross-linking.
Techniques: Histone Deacetylase Assay, Gene Expression, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Control, Immunoprecipitation, ChIP-qPCR
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