The Centromeric Sister Chromatid Cohesion Site Directs Mcd1p Binding to Adjacent Sequences  Paul C Megee, Cathy Mistrot, Vincent Guacci, Douglas Koshland  Molecular Cell  Volume 4, Issue 3, Pages 445-450 (September 1999) DOI: 10.1016/S1097-2765(00)80347-0

Figure 1 Mcd1-6HAp Association with Endogenous Chromosomes Schematics represent portions of endogenous chromosomes (III, IV, VII, and XVI) with the relevant PCR products (bars). PCR products were generated with oligonucleotides that amplify ∼300 bp DNA fragments (see Experimental Procedures). Ovals indicate positions of the centromeres, and dotted lines represent the remainder of the chromosomes not drawn to scale. Total corresponds to input chromatin before immunoprecipitation. (A) ChIP of CEN3 DNA in strains with (+) or without (−) HA epitope–tagged Mcd1p. The tagged protein is the sole source of Mcd1p and provides wild-type Mcd1p function. (B) ChIP of ARS1 and ADE3 DNA from endogenous chromosomes in G1 and M cells. Cells were staged in G1 or M using mating pheromone and nocodazole, respectively. Lane numbers correspond to the indicated PCR products. (C) Coimmunoprecipitated DNA in Mif2p and Mcd1-6HAp ChIPs as a percentage of input chromatin is shown for centromeric and noncentromeric loci in G1 or M cells. Numbered ARS1 and ADE3 PCR products correspond to those in (B). Inset shows relative pixel intensities for PCRs of input DNA from G1 (squares) and M stage (circles) cells, demonstrating that our PCR conditions respond linearly to the levels of input DNA used for these and all subsequent quantitations of ChIP samples. (D) CEN3-proximal DNA in Mif2p and Mcd1-6HAp CHIPs. The presence of a small amount of CEN-flanking DNA in the Mif2p ChIP is attributed to variable lengths of sheared chromatin, which include occasional long chromatin pieces with both the CEN DNA and flanking sequences. (E) Quantification of CEN3 or CEN16 and proximal sequences as a percentage of total DNA in Mif2p and Mcd1-6HAp ChIPs. Molecular Cell 1999 4, 445-450DOI: (10.1016/S1097-2765(00)80347-0)

Figure 2 Ectopically Placed Centromere Directs Mcd1p Binding to Adjacent Sequences (A) Schematic of the minichromosome centromeric region. The centromere (oval) is flanked by site-specific recombination target sites (triangles, see Figure 3). (B) Mcd1-6HAp ChIP of centromere-proximal DNA from minichromosome-bearing cells staged in G1 or M. Lane numbers correspond to PCR products in (A). (C) Quantification of ARS1 and ADE3 DNA as a percentage of input DNA in Mcd1-6HAp coimmunoprecipitates from M-staged cells with and without the minichromosome. Molecular Cell 1999 4, 445-450DOI: (10.1016/S1097-2765(00)80347-0)

Figure 3 Establishment and Maintenance of Mcd1-6HAp Binding Adjacent to the Centromere Cohesion Site The minichromosome centromere was excised during G1 (A) or M (B) using a site-specific recombination event under the control of an inducible recombinase (see Figure 2A) (Megee and Koshland 1999). It is unlikely that the loss of Mcd1-6HAp association in centromere-proximal regions is the consequence of recombination per se because sister minichromatid cohesion is unaffected by a recombination event that inverts the centromere (Megee and Koshland 1999). Only PCR products from ChIP samples with excised centromeres are shown. Quantification of these results and the corresponding centric (uninduced) controls are shown. Lane and column numbers correspond to PCR products in Figure 2A. Molecular Cell 1999 4, 445-450DOI: (10.1016/S1097-2765(00)80347-0)

Figure 4 Mcd1p-6HAp Association with the Minichromosome Is Biased for A+T-Rich DNA (A) A+T base composition within a sliding 100 bp window was plotted using MacVector software (Oxford Molecular) for 2 kb regions flanking the centromere (ovals) on endogenous chromosomes III and XVI and for pPCM69. (B) The minichromosome is drawn to scale. Red bars indicate PCR products that are specific for the minichromosome. Specificity is derived from minichromosome sequence-specific oligonucleotides in PCR or is inferred because corresponding sequences on endogenous chromosomal loci showed minimal Mcd1p association (see Figure 1). Black bars represent PCR products amplified from either chromosome or minichromosome sequences. (C) Cells harboring the minichromosome were arrested in M and processed for ChIP. The coimmunoprecipitated DNA from two independent experiments (circles or squares) was analyzed using the 47 primer pairs that generate the PCR products shown in (B). Percentages of PCR products bound in Mcd1-6HAp ChIPs are plotted relative to the minichromosome nucleotide using midpoints of amplified PCR products. (D) Plot of minichromosome A+T base composition within a sliding 100 bp window. Molecular Cell 1999 4, 445-450DOI: (10.1016/S1097-2765(00)80347-0)