FIG. 6.

Fep1 binds to the abc3⁺ promoter in vivo in an iron-dependent manner. (A) ChIP analysis of the abc3⁺ promoter in fep1Δ php4Δ cells harboring an integrated untagged or TAP-tagged fep1⁺ allele. The cells were precultured in the presence of 100 μM Dip, allowed to grow to an A₆₀₀ of ∼1.0, washed and then incubated (90 min) in the presence of 250 μM Dip or 250 μM FeCl₃ (Fe). Chromatin was immunoprecipitated with anti-mouse IgG antibodies, and a specific region of the abc3⁺ promoter was analyzed by PCR to determine Fep1 occupancy. The top band represents the abc3⁺-specific signal, whereas the lower band is an internal background control derived from a nontranscribed region (intergenic region). (B) Quantitation of the PCR products obtained from anti-IgG immunoprecipitated (IP) chromatin. The results are representative of three independent experiments. The signals are expressed as the relative binding (%) and were calculated as percentages of the largest amount of chromatin measured. Input, input chromatin; IP, immunoprecipitated chromatin.