Figure 4.

Chp1 displays a similar cen1 association pattern as Swi6. (A) Multiplex PCR was performed to detect association of Chp1–6xMyc with centromeric chromatin. Primers were designed to two sites (imr/otr junction and a region of cnt1), which give amplification specifically from cen1 sequences, and to the euchromatic fbp1⁺ gene locus to act as a control for nonspecific association. Chp1–6xMyc and Swi6 immunoprecipitates both showed enrichment of the imr/otr product relative to fbp1 and showed no enrichment for the central core sequence (cnt1). In contrast, Mis6–3xHA immunoprecipitates showed enrichment for cnt1 and not for imr/otr. (B) Chp1–6xMyc interactions across cen1 were mapped by specific PCR from immunoprecipitates of strains with different cen1–ura4 insertions using various primers from cen1 and one primer anchored in the ura4⁺ gene. Enrichment of centromeric ura4⁺ by immunoprecipitation is reflected by increased intensity of the smaller PCR products, which vary in size from different strains, depending on the location of the centromeric primers, relative to the large PCR product of constant size that reflects association with the euchromatic ura4–DS/E locus. (C) Using this assay, Chp1–6xMyc and Swi6 associate with the flanking repeats but not the central core of cen1. Relative ip values are an average of 2 (Swi6), and 3 (Chp1–myc) experiments. (D) Chp1–6xMyc immunoprecipitation at site 13 is dependent on Clr4 and Rik1 but not Swi6.