The Kinetochore Is an Enhancer of Pericentric Cohesin Binding (original) (raw)
Figure 3
Mcd1p Binding Profiles in Centromere-Proximal and -Distal Regions
Cells containing Mcd1-6HAp were first staged in G1 using αF, and then released from G1 into medium containing nocodazole to arrest the cells in mitosis. For the centromere excision experiments (B–D), the cultures were divided in half after G1 arrest, and one half of each culture was treated with galactose for 2 h to induce centromere excision (see Materials and Methods). Both the induced (acentric) and uninduced (centric) control cultures were then released from the G1 arrest into fresh medium and rearrested in mitosis. Once arrested in mitosis, cells were fixed in formaldehyde and then processed for ChIP using antiserum against epitope-tagged Mcd1p (Mcd1-6HAp) as an indicator of the cohesin complex. DNA isolated from the ChIPs and diluted input DNA not subject to immunoprecipitation were then subjected to PCR analysis using oligonucleotide primer pairs that amplify approximately 300-bp fragments within the indicated regions. Quantitation of DNA in the Mcd1p ChIPs, expressed as a percentage of the input DNA, is plotted as a function of the locations of the midpoints of those DNA fragments based on the SGD coordinates. Centromere position is indicated by an oval (not drawn to scale). (A) The Mcd1p association profile for the CHRI pericentric region in strain 1377A1-4B is shown. Mcd1p binding adjacent to CEN1 is difficult to assess fully because of the presence of a moderately repetitive Ty element in the region from approximately 160 to 166 kb, indicated with the dashed line. Similarly, the Mcd1p binding profiles in the pericentric regions of CHRIII (B) and CHRXIV (C) are shown in the presence (black squares) and absence (gray circles) of CEN3 and CEN14 using strains PMY185 and PMY206, respectively. (D) The Mcd1p binding profiles for a centromere-distal region of CHRIII are shown for comparison in the presence (black squares) and absence (gray circles) of CEN3.