Chromatin immunoprecipitation (ChIP) and re-ChIP assay

ChIP assays were performed as previously described [18]. Hepatocytes were fixed with 1% formaldehyde for 15 min and stopped by adding glycine to a final concentration of 125 mM. Then cells were washed, harvested, lysed, and sonicated to shear chromatin to DNA fragments of 0.5–1 kb. Lysates were centrifuged, and an aliquot (20 μl) of supernatant was saved as input DNA. Supernatants were then immunoprecipitated with indicated antibodies or an IgG as a negative control. Finally, DNA was purified and concentrated using the QIAquick PCR purification kit (Qiagen, CA, USA). Purified DNA was analyzed by conventional and realtime PCR with specific primers (Supplementary Table 6) for the ApoA1 gene promoter. An equal volume of non-precipitated (input) genomic DNA was amplified as positive control. In re-ChIP assays, chromatin was firstly immunoprecipitated with an anti-PPARα antibody, then eluted with 100 μl of elution buffer with 10 mM DTT at 37 °C for 30 min, diluted (25-fold) with dilution buffer (20 mM Tris–HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 1% Triton X-100), and finally reimmunoprecipitated with IgG or an antibody against PARP1, PAR, Sirt1, PGC1α, or RXR (Santa Cruz). Real-time PCR was performed using 1 μg of template DNA with primers specific for Cpt1α, Cyp4a11, or Acox1 (Supplementary Table 6). The input chromosomal DNA and ChIP DNA with non-specific IgG were subjected to the same PCR amplification. PCR products were separated on an ethidium bromide-stained 2% agarose gel.