Localization of Cse4p to STB by ChIP. ChIPs were performed with antibodies to myc-tagged Cse4p, and the precipitated DNA was amplified using primers specific to CEN3, STB, or ORI. In the assays shown in A, in addition to STB, other regions of the 2-μm circle genome were probed (horizontal bars in the schematic representation of the plasmid). The PCR products corresponding to STB and ORI span coordinates 2,969–3,244 and 3,413–3,708, respectively, of the 2-μm circle genome. In the two panels that refer to ORI, the bottom one contained twice the amount of input template relative to the top one. The sizes of the PCR products for REP1, REP2, FLP, and CEN3 are 287, 246, 259, and 222 bp, respectively. The label WCE (whole cell extract) refers to input DNA controls; ChIP indicates experimental samples; and the label beads signifies negative controls.

Depletion of bulk Cse4p after protein synthesis inhibition does not affect Cse4p localized to centromeres or STB. A burst of Cse4p synthesis was induced for a period of 2 h, and translation was arrested by the addition of cycloheximide at time = 0. The cellular pool of Cse4p was assayed by Western blotting, and Cse4p-STB and Cse4p-CEN3 associations were monitored by ChIP. The sizes of the PCR products for STB and CEN3 are 275 and 222 bp, respectively. The loading control for Western blotting was α-tubulin (Tub1p).

Effects of cse4-107 mutation on plasmid segregation, cohesin assembly, and binding of Rep proteins at STB and plasmid localization in chromosome spreads. (A) Segregation of the fluorescence-tagged STB reporter plasmid was followed in wild-type [cir⁺] and [cir⁰] strains and in the cse4 mutant [cir⁺] strain. Cultures grown to mid-log phase at 26°C were split into halves and were incubated for a period of 3 h at 26 or 37°C before scoring plasmid segregation. The data were obtained as the means from 150–200 cells for each assay. (B and C) Cohesin recruitment at STB was assayed by ChIP using antibodies to the HA epitope carried by the Mcd1 protein. In the ChIP analyses for monitoring Rep1p and Rep2p at STB, antibodies to the native proteins were used. The size of the PCR product for STB is 275 bp. (D) The STB reporter plasmid pSV1 was localized in chromosome spreads (50–100 spreads were scored in each assay) as described in Fig. 1. (E) Rsc2 and Rsc8 proteins were localized at STB by ChIP using antibodies to the myc epitope tag harbored by them.

Localization of Cse4p, Rep proteins, and STB reporter plasmid in chromosome spreads. Chromosome spreads were prepared from wild-type [cir⁺], ndc10-1 [cir⁺], and [cir⁰] strains. Rep1p or Rep2p (not depicted) was probed by antibodies to the native protein, and Cse4p was probed by antibodies to the myc epitope harbored by it. The reporter plasmid harboring Lac operator DNA (pSV1; Mehta et al., 2002) was revealed by antibodies to the bound Lac repressor. Secondary antibodies were FITC-conjugated anti–mouse and Texas red–conjugated anti–rabbit antisera. The percentage of spreads (with at least 50 spreads in each assay) that corresponds to a displayed pattern is indicated.

Cse4p association with STB probed by salt extractability of the former and DdeI sensitivity of the latter. (A) Spheroplasts were treated with indicated salt concentrations before performing ChIP using Cse4p-myc–directed antibodies. The sizes of the PCR products for STB and CEN3 are 275 and 222 bp, respectively. (B) A nuclear pellet preparation was extracted with buffers of increasing ionic strength and separated into pellet (P) and supernatant (S) fractions by centrifugation. Western blots were probed using antibodies to histone H3 and to the myc epitope harbored by Cse4p. Under the electrophoresis conditions used, the myc-tagged Cse4p shows an anomalous mobility above the 50-kD marker (see Fig. 4). (C and D) Nuclei were subjected to partial DdeI digestion followed by DNA isolation and complete PstI plus XbaI digestion. DNA was extracted, fractionated by electrophoresis, and probed with the indicated probes derived from distal STB. WT, wild type; MT, mutant.

Association of Cse4p with centromeres and STB as a function of cell cycle. Immunoprecipitations were performed with antibodies to myc-tagged Cse4p except in row 2 of B, where native antibodies to Rep1p were used. (A–D) Cells arrested in G1 (A and B) or in telophase (C and D) were released at time = 0, and the presence of Cse4p at STB and CEN3 was assayed by ChIP. The progression of the cell cycle was followed by the cell morphology/budding index and by FACS analysis. The mitotic spindle was visualized by indirect immunofluorescence using antibodies to α-tubulin and Texas red–conjugated secondary antibodies. The short interval ChIP data shown in the top rows of A–D contained a twofold higher input of template DNA during PCR. (E) Cells were arrested in telophase as in C and D but were released into medium containing α factor. The sizes of the PCR products for STB and CEN3 are 275 and 222 bp, respectively. WCE, whole cell extract.

Cse4p-containing chromatin at STB is a prerequisite for equal segregation of the 2-μm plasmid. (A) Cycling of Rep proteins and Cse4p incorporation at STB during G1→S transition, which is likely followed by RSC2-mediated remodeling of STB chromatin, are postulated to be key events in establishing the plasmid partitioning complex. After plasmid segregation, Cse4p is released from STB during late telophase at spindle disassembly. Cse4p-containing nucleosomes and histone H3-containing nucleosomes are distinguished by coloring them yellow and green, respectively. The associations of Rep proteins with these two chromatin states are thought to be functionally distinct and, thus, are depicted differently. We do not know whether the release of Cse4p from an STB nucleosome is accompanied by its replacement with histone H3. (B) Current and previous results can be accommodated by a model in which a Rep1p–Rep2p-containing prepartitioning complex (green nodule) associated with microtubules mediates the capture of the plasmid cluster (drawn as a ring). Establishment of the mature partitioning complex (red nodule) at STB occurs concomitant with DNA replication, and further downstream events follow as depicted in A. For comparison, a chromosome (blue) attached to the spindle via the kinetochore (yellow) is also shown.