FIG. 1.

In vivo binding of USF and SREBP to the FAS promoter during fasting and refeeding. (A) Sonicated cross-linked-chromatin samples from livers of mice that were fasted or refed were diluted 1:2, 1:10, 1:100, and 1:500. The diluted samples were subjected to PCR amplification using specific primers complementary to the FAS-CAT transgene. The PCR product was 230 bp in length. Results are representative of those from input chromatin samples recovered from four independent immunoprecipitation experiments. (B) The sonicated cross-linked-chromatin samples from fasted and refed mice were immunoprecipitated with 4 μg of antibody against USF and 30 μg of antibody against SREBP. Precipitation carried out without antibody (No Ab) was used as a negative control. As an additional control, the supernatant from the “no antibody” sample, representing the total input chromatin (Input), was collected, processed as the immunoprecipitates, and included in the PCR. The immunoprecipitates were analyzed by simultaneous PCRs using primers complementary to the endogenous FAS-proximal promoter region and to a region within exon 32 of the FAS gene. The sizes of the PCR products are indicated. Sequences from the proximal CAD promoter were amplified from fasted or refed mice in a separate experiment, and the size of the PCR product is 350 bp. (C) Cross-linked chromatin samples from −444 FAS-CAT mice fasted or refed were immunoprecipitated with anti-USF and anti-SREBP antibodies, and the DNA was analyzed by PCR using the appropriate primers as described for panel B. The sizes of the PCR generated fragments were 269 bp for endogenous FAS, 230 bp for FAS-CAT, and 242 bp for LDL receptor (LDLR). Controls include a precipitation lacking antibody (no Ab), lacking chromatin (mock), and immunoprecipitation with an unrelated antibody (unrelated).