Centromere identity is specified by a single centromeric nucleosome in budding yeast

Abstract

Chromosome segregation ensures that DNA is equally divided between daughter cells during each round of cell division. The centromere (CEN) is the specific locus on each chromosome that directs formation of the kinetochore, the multiprotein complex that interacts with the spindle microtubules to promote proper chromosomal alignment and segregation during mitosis. CENs are organized into a specialized chromatin structure due to the incorporation of an essential CEN-specific histone H3 variant (CenH3) in the centromeric nucleosomes of all eukaryotes. Consistent with its essential role at the CEN, the loss or up-regulation of CenH3 results in mitotic defects. Despite the requirement for CenH3 in CEN function, it is unclear how CenH3 nucleosomes structurally organize centromeric DNA to promote formation of the kinetochore. To address this issue, we developed a modified chromatin immunoprecipitation approach to analyze the number and position of CenH3 nucleosomes at the budding yeast CEN. Using this technique, we show that yeast CENs have a single CenH3 nucleosome positioned over the CEN-determining elements. Therefore, a single CenH3 nucleosome forms the minimal unit of centromeric chromatin necessary for kinetochore assembly and proper chromosome segregation.

Fig. 1.

ChIP with single-nucleosome resolution. (A) Ethidium bromide staining of the input material for ChIP. Chromatin was treated with MNase, and the DNA was isolated and subjected to electrophoresis on a 1.5% agarose gel in the presence of ethidium bromide. Partial MNase digestion generates a nucleosomal DNA ladder with visible mono-, di-, tri-, and tetranucleosomal fragments. (B) CEN DNA is present in extracted MNase-treated chromatin used for ChIP. Southern blot analysis was performed on the isolated input DNA (IN) in A by using a probe specific for the CEN4 nucleosome. M, radiolabeled 100-bp ladder marker. (C) Southern blot analysis on Cse4-associated chromatin preserves size and positional information. Chromatin is digested with MNase (scissors), and Cse4-associated chromatin is immunoprecipitated with antibody. If Cse4 localizes only to the CEN, a mononucleosome-sized fragment is detected only with a probe corresponding to the CEN, whereas probes distal to the CEN detect only increasingly larger fragments (scenario A). In contrast, if Cse4 localizes to both the CEN and the flanking nucleosomes, mononucleosome-sized fragments would be detected with the distal probes (scenario B). C4, Cse4; H3, canonical histone H3.

Fig. 2.

A single Cse4 nucleosome forms over the CEN. MNase-treated chromatin prepared from strain SBY5146 (FLAG-CSE4) was immunoprecipitated with anti-FLAG agarose beads. Cse4-associated DNA was isolated and separated on a 1.5% agarose gel. Southern blotting was performed with probes specific to either the CEN3 or a CEN3-flanking nucleosome. M, radiolabeled 100-bp ladder marker; IP, the DNA bound to Cse4.

Fig. 3.

Detection of a Cse4 mononucleosome at the CEN depends on the inclusion of anti-Cse4 antibody. MNase-treated chromatin prepared from strain SBY3 was immunoprecipitated with protein G Dynabeads in the presence (w/ Ab) and absence (w/o Ab) of anti-Cse4 antibody. Cse4-associated DNA was isolated and separated on a 1.5% agarose gel. Southern blotting was performed with a probe specific to the CEN3. M, radiolabeled 100-bp ladder marker; IN, input DNA; IP, the DNA bound to Cse4.

Fig. 4.

Budding yeast CENs are composed of a single Cse4 nucleosome, regardless of chromosome size. (A) MNase-treated chromatin prepared from strain SBY5146 (FLAG-CSE4) was immunoprecipitated with anti-FLAG agarose beads. Cse4-associated DNA was isolated and separated on 1.5% agarose gel. Southern blotting was performed with probes specific to either the CEN4 or a CEN4-flanking nucleosome. (B) MNase-treated chromatin prepared from strain SBY5146 (FLAG-CSE4) was immunoprecipitated with anti-FLAG agarose beads. Cse4-associated DNA was isolated and separated on 1.5% agarose gel. Southern blotting was performed with 600-bp probes specific to sequences ≈3 kb to the left (−3 kb) or right (+3 kb) of CEN6. M, radiolabeled 100-bp ladder marker; IN, input; IP, the DNA bound to Cse4.