Mca1 constitutively binds the <i>mfc1</i>⁺ promoter in vivo.

<p><i>A</i>, <i>pat1-114</i>/<i>pat1-114 mca1∆</i>/<i>mca1∆</i> cells expressing Mca1-TAP were synchronously induced into meiosis in the presence of TTM (150 μM) or copper (Cu, 25 μM). The upper panel shows Western blots of Mca1-TAP and α-tubulin protein levels at different time points after meiotic induction. Positions of the molecular weight standards (M) are indicated to the left. <i>B</i>, Schematic representation of <i>mfc1</i>^<i>+</i> and <i>cuf2</i>^<i>+</i> promoter regions. Nucleotide numbers refer to the position relative to the A of the initiator codon of each gene (<i>mfc1</i>^<i>+</i> or <i>cuf2</i>^<i>+</i>). Numbers in parentheses indicate promoter regions that were used for qPCR analysis in ChIP assays. Black boxes depict wild-type TCGGCG sequences known to serve as a binding site for Mca1. <i>C–D</i>, <i>pat1-114</i>/<i>pat1-114 mca1∆</i>/<i>mca1∆</i> cells expressing Mca1-TAP or untagged Mca1 were synchronously induced to undergo meiosis. Following induction of meiosis, chromatin was immunoprecipitated using Sepharose-bound anti-mouse IgG antibodies at the indicated time points. Specific regions of <i>mfc1</i>^<i>+</i> and <i>cuf2</i>^<i>+</i> promoters were analyzed by qPCR to determine Mca1-TAP occupancy in the presence of TTM or copper. Binding of Mca1-TAP to promoters was calculated as the enrichment of specific <i>mfc1</i>^<i>+</i> and <i>cuf2</i>^<i>+</i> promoter regions relative to a 18S ribosomal DNA coding region. The <i>cuf2</i>^<i>+</i> promoter served as a negative control. ChIP data were calculated as values of the largest amount of chromatin measured (fold enrichment). Results are shown as the averages ± S.D. of a minimum of three independent experiments.