Cell-Cycle-Coupled Structural Oscillation of Centromeric Nucleosomes in Yeast

Abstract

Summary The centromere is a specialized chromosomal structure that regulates chromosome segregation. Centromeres are marked by a histone H3 variant. In budding yeast, the histone H3 variant Cse4 is present in a single centromeric nucleosome. Experimental evidence supports several different models for the structure of centromeric nucleosomes. To investigate Cse4 copy number in live yeast we developed a new method coupling fluorescence correlation spectroscopy and calibrated imaging. We find that centromeric nucleosomes have one copy of Cse4 during most of the cell cycle, whereas two copies are detected at anaphase. The proposal of an anaphase coupled structural change is supported by Cse4-Cse4 interactions, incorporation of Cse4, and the absence of Scm3 in anaphase. Nucleosome reconstitution and ChIP suggests both Cse4 structures contain H2A/H2B. The increase in Cse4 intensity and deposition at anaphase are also observed in Candida albicans. Our experimental evidence supports a cell cycle coupled oscillation of centromeric nucleosome structure in yeast.

Integrative structure and functional anatomy of a nuclear pore complex

“…2A): (1) use of synthetic concatemers of tryptic peptides or QconCATs 51 to define the relative stoichiometry of each component by quantitative MS in affinity-captured NPCs; (2) In vivo calibrated imaging analysis of GFP-tagged Nups 52 , to quantify the absolute copy number per NPC of Nups selected to represent each major module of the NPC; and (3) Charge Detection MS to measure the total mass of affinity-captured NPCs 53 . For the calculation of the integrative NPC structure, the final copy numbers were rounded to fit the known NPC C 8 -symmetry and these values are indicated in Supplementary Table 2A.…”

“…Calibrated imaging data was acquired as described in a previous publication 52 . Using the avalanche photodiode (APD) imaging module of a Zeiss confocor 3, confocal z-stacks of live yeast were acquired with a 40× 1.2 NA Plan-Apochromat water objective.…”

“…FCS was used to convert the amplitude of the Gaussian fit of the Nup spot number of molecules of GFP. Briefly, using a strain expressing only cytosolic GFP, fluorescence correlation spectroscopy determined the average number of molecules in the focal volume, as described previously 52 . Then, the amplitude of the signal of the Nup spot was compared to the intensity of cytosolic GFP, taken with the same imaging setup.…”

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…Fluorescence recovery after photobleaching (FRAP) experiments showed it is deposited during late G1 or early S phase (Pearson et al 2004). Although it was reported that Cse4 is also deposited during anaphase (Shivaraju et al 2012), the marker used for anaphase may not distinguish between late anaphase and G1. Consistent with this, other groups have not observed anaphase incorporation (Pearson et al 2004;Coffman et al 2011;Lawrimore et al 2011).…”

The Centromere: Epigenetic Control of Chromosome Segregation during Mitosis

“…One emerging alternative model, which may reconcile the interpretation of octameric and hemisomal CENP-A nucleosomes, is that CENP-A nucleosome composition may change during progression through the cell cycle. Indeed, two recent studies in two different organisms proposed cell-cycle-dependent transitions between two CENP-A molecules per nucleosome (i.e., an octamer) to one CENP-A molecule per nucleosome (Bui et al 2012;Shivaraju et al 2012). These studies interpreted the changes as an octamer to hemisome transition.…”