Histone dynamics differ in the pericentromere and chromosome arm during metaphase
We determined the dynamics of histones H2B and H4 in the pericentromere and the chromosome arm during metaphase in Saccharomyces cerevisiae
by measuring the half-life (t½
) of fluorescence recovery after photobleaching (FRAP). Strains containing histone tagged with green fluorescent protein (GFP) and spindle pole bodies tagged with red fluorescent protein (RFP; see in Materials and Methods
) allowed us to demarcate the pericentromere from chromosomal arms in a living cell. The pericentromere lies between the spindle pole bodies (Yeh et al., 2008
), and the chromosome arms are distal to the spindle ().
Histones within the pericentromere exhibit faster turnover rates than in the chromosome arms. Histone H2B has a t½
of 62 s in the pericentromere during metaphase, compared with 87 s in the arm ( and and Supplemental Table S1). Histone H4 has a t½
of 76 s in the pericentromere, compared with 121 s in the chromosome arm ( and and Supplemental Table S2). H2B is more dynamic than H4 in both regions of the chromosome. For both H2B and H4, the t½
values of the chromosome arm are significantly slower than those of the pericentromere (Student's t
test, p < 0.05). The final percentage recoveries of histone protein were found to be similar for both regions and histones measured, indicating similar levels of mobile protein ( and Supplemental Tables S1 and S2). The individual dynamics of H2B and H4 both within the pericentromere and the arms are consistent with the observations that each histone pair, H2A/H2B and H3/H4, is independently regulated (Jackson, 1987
; Smith and Stillman, 1991
; Ladoux et al., 2000
; Akey and Luger, 2003
; Jin et al., 2005
; Thiriet and Hayes, 2005
FIGURE 2: Histones in the pericentromere are more dynamic than those of the chromosome arm. (A) Graph of average half-life in seconds measured by FRAP for histones H2B and H4 in the pericentromere and chromosome arm. Asterisks indicate statistically significant (more ...)
Histone dynamics in the pericentromere are reduced on loss of spindle-based tension
To determine whether histone dynamics in the pericentromere were influenced by spindle-based tension, we treated cells with the microtubule-depolymerizing drug nocodazole (noc) and examined histone half-life. In nocodazole-arrested cells, the spindle pole bodies collapse into a single diffraction-limited spot and the pericentromeric chromatin remains adjacent to the spindle pole bodies. On photobleaching, we observed two populations of histone recovery. There was a significant reduction in the number of cells with measurable H4 recovery dynamics in the pericentromere, with the chromosome arm largely unaffected (pericentromere, 92% untreated wild type (WT) vs. 55% noc treated; arm, 97% untreated WT vs. 85% noc treated; Fisher's exact test, p < 0.05; ). H2B also showed a decrease in cells exhibiting measurable dynamics (pericentromere, 92% untreated WT vs. 74% noc treated; arm, 100% untreated WT vs. 83% noc treated), but these were not found to be statistically significant (Fisher's exact test, p < 0.05; ).
FIGURE 3: Loss of spindle tension or chromatin-remodeling activity results in reduced histone dynamics primarily at the pericentromere. (A) Graph showing percentage of samples showing measurable recovery after photobleaching. Samples whose final percentage recovery (more ...)
When histone recovery was measurable, H2B half-life was significantly slowed in the pericentromere (62 s untreated WT vs. 121 s noc treated; Student's t test, p < 0.05; and Supplemental Table S1). There was no significant change in H2B recovery in the chromosome arm upon nocodazole treatment or any significant changes in final percentage recovery ( and ). Therefore, upon reduction of spindle tension by nocodazole treatment, the dynamics of pericentromeric H2B and H4 are reduced.
An alternative method to reduce pericentric tension was used by introducing a conditional allele of the kinetochore protein Nuf2 (Gal-NUF2
). On galactose (gal) media the cells express NUF2
and are able to assemble the kinetochore, whereas on glucose (glu) media NUF2
expression is repressed compromising kinetochore function (Bouck and Bloom, 2005
). Loss of Nuf2p resulted in reduced histone dynamics in the pericentromere but not the chromosome arm for both H2B and H4 (H2B: pericentromere, 62 s WT vs. 94 s Gal-NUF2
on glu; arm, 87 s WT vs. 86 s Gal-NUF2
on glu; H4: pericentromere, 76 s WT vs. 97 s Gal-NUF2
on glu; arm, 121 s WT vs. 135 s Gal-NUF2
on glu; Student's t
test, p < 0.05; and Supplemental Tables S1 and S2). The final percentage recovery of H2B was not significantly affected, whereas the final percentage recovery of H4 in the pericentromere was significantly reduced (64% WT vs. 30% Gal-NUF2
on glu; ), indicating a reduced level of mobile histones. Thus, like nocodazole treatment, the loss of spindle tension via reduction of kinetochore function results in significantly reduced histone dynamics in the pericentromere and not in the chromosome arm.
Increased histone dynamics are the result of increased histone removal
At least two properties of histone dynamics can contribute to the observed behavior in the pericentromere: either the histones are removed from DNA more frequently in the pericentromere, or histones are replaced more quickly, leaving binding sites in the arm unbound longer. To address these possible explanations, we examined the dynamics of H2B tagged with a photoactivatable GFP (paGFP) fluorophore (Vorvis et al., 2008
The highly dynamic nature of proteins can be visualized using photoactivation (). As a control, we examined the dispersion characteristics of photoactivated Erg6p, a membrane protein involved in ergosterol biosynthesis (Gaber et al., 1989
; Vorvis et al., 2008
). Erg6p exhibited dispersion in all of the examined cells, indicative of a high level of dynamics ( and Supplemental Table S3). Dispersion was measured by quantifying the loss of signal intensity in a 2.6 × 2.6 μm area over time (20 × 20 pixels; Materials and Methods
). Photoactivation of H2B in the pericentromere and chromosome arm reveals that the percentage of cells showing histone dispersion is not significantly different in the pericentromere and the chromosome arm (79 vs. 83%, respectively; Fisher's exact test, p < 0.05; ). The removal dynamics of histone protein are not different in the pericentromere and chromosome arm. Therefore the increased histone dynamics observed by FRAP in the pericentromere under tension are likely the result of active processes replacing lost histones more rapidly.
FIGURE 4: Loss of spindle tension results in reduced dispersal of photoactivated histone H2B. (A) Representative images showing nuclear region before photoactivation (preactivation), postphotoactivation, halfway through time lapse (+3 min), and at end of time lapse (more ...)
Loss of spindle tension leads to a significant decrease in the percentage of cells displaying dispersion of photoactivated H2B in the pericentromere but not the chromosome arm (pericentromere, 79% untreated WT vs. 33% noc treated; arm, 83% untreated WT vs. 46% noc treated; Fisher's exact test, p < 0.05; ). The reduced histone dispersion in the pericentromere in collapsed spindles points to reduced histone removal from the DNA in the absence of tension. The FRAP and photoactivation data indicate an active histone replacement time under tension and an increased histone dwell time (slower off rate) in the pericentromere upon loss of spindle tension.
Loss of Sth1p/Nps1p or Isw2p leads to reduced histone turnover in the pericentromere
To address whether chromatin remodelers are involved in nucleosome exchange at the pericentromere, we measured histone dynamics in mutations in RSC (STH1
) and ISW2.
In the absence of RSC activity (nps1-105
temperature-sensitive allele), cells arrest in metaphase with defects in kinetochore assembly and segregation (Tsuchiya et al., 1998
; Hsu et al., 2003
). In the nps1-105
mutant at permissive temperature (24°C), there is a significant decrease in the percentage of cells exhibiting measurable histone-GFP recovery in the pericentromere (H2B, 92% WT vs. 60% nps1-105
; H4, 92% WT vs. 58% nps1-105
; Fisher's exact test, p < 0.05; and Supplemental Tables S1 and S2). Of the cells with measurable histone recovery, the t½
of H2B is significantly slowed as compared with wild type (pericentromere, 62 s WT vs. 116 s nps1-105
; arm: 87 s WT vs. 125 s nps1-105
; Student's t
test, p < 0.05, ). The t½
of H4 is also significantly altered in both the pericentromere and chromosome arm in nps1-105
cells as compared with wild-type (pericentromere, 76 s WT vs. 119 s nps1-105
; arm, 121 s WT vs. 75 s nps1-105
; Student's t
test, p < 0.05; ). The final percentage recovery of histone H4 in nps1-105
cells is significantly reduced from wild type in the pericentromere but unaffected in the chromosome arm (pericentromere, 64% WT vs. 33% nps1-105
; arm, 52% WT vs. 60% nps1-105
; Student's t
test, p < 0.05; ). Histone exchange in the pericentromere is dependent upon a fully functional RSC complex. When the photoactivatable H2B is used, the fraction of cells exhibiting dispersion is unchanged (Fisher's exact test, p < 0.05; ). Thus histones are evicted in nps1-105
, but the mechanisms replacing lost histones are diminished ( and ).
RSC chromatin remodeling during metaphase primarily affects the histone dynamics in the pericentromere, with histone dynamics in the chromosome arm affected to a lesser degree. The nucleus-wide alteration of histone dynamics is consistent with the essential nature of STH1/NPS1. However, histones in the pericentromere more often display no measurable recovery (), indicating a regional specificity for RSC chromatin-remodeling activity.
The requirement for antagonistic chromatin remodeling has been demonstrated at promoter regions to control expression levels (Tomar et al., 2009
; Erkina et al., 2010
). We reasoned that histone occupancy at the pericentromere might also reflect balanced chromatin remodeling. ISW2
has been found to counter the histone removal activity of SWI/SNF chromatin remodeling (Tomar et al., 2009
In the absence of ISW2 activity (isw2Δ), the t½ of both histones H2B and H4 is significantly slower in the pericentromere but not the chromosome arm as compared with wild-type cells. H2B t½ slows from 62 s in wild type to 103 in isw2Δ cells, and H4 t½ slows from 76 s in wild-type cells to 119 s in isw2Δ cells (Student's t test, p < 0.05; and Supplemental Tables S1 and S2). Similarly, the final percentage recovery is significantly lower in the pericentromere but not the chromosome arm for both histones H2B and H4 (Student's t test, p < 0.05; ). Wild-type H2B percentage recovery in the pericentromere is 58% and is reduced to 40% in isw2Δ cells. H4 percentage recovery in the pericentromere is 64% in wild-type cells and is reduced to 37% in isw2Δ cells. Consistent with the nonessential nature of ISW2, there is no significant difference in the percentage of cells showing measurable histone recovery between wild-type and isw2Δ cells (Fisher's exact test, p < 0.05; ). As in the nps1-105 cells, there was no significant change in percentage of cells exhibiting dispersion after photoactivation in isw2Δ cells as compared with wild type (Fisher's exact test, p < 0.05; ). These data suggest that the primary role for ISW2 is maintenance of nucleosome occupancy under tension by reloading histones rather than eviction, as there is no decrease in percentage of cells exhibiting measurable recovery ().
Chromatin packaging contributes to kinetochore organization
In yeast, the 16 kinetochores are clustered into a close-to-diffraction–limited spot. To address whether histone occupancy is important for this organization, we examined the structure of the inner (Ame1p-GFP or Ndc10p-GFP) and outer (Spc24p-GFP or Nuf2p-GFP) kinetochores ( and ). From this analysis, we observed significant disruption of the kinetochores in conditions that perturb chromatin packaging.
FIGURE 5: Disruption of the underlying chromatin platform results in disruption of the kinetochore. (A) Diagram of kinetochore location in relation to pericentromeric chromatin, as denoted by green dotted line. (B) Representative images of both normal and disrupted (more ...)
We first examined kinetochore structure upon the depletion of histone H3 and found that the inner, but not the outer (as in Bouck and Bloom, 2007
), kinetochore is disrupted ( and Supplemental Table S4). Cells expressing the sole copy of H3 under the galactose promoter exhibit disruption of the inner kinetochore (Ndc10p-GFP) in 5% of cells. On reduction of histone H3—resulting in an approximately twofold reduction in nucleosome concentration—55% of the cells show disruption of the inner kinetochore, a significant increase (Fisher's exact test, p < 0.05; ). Decreasing histone density specifically affects the inner kinetochore organization, leaving the microtubule-binding components (Nuf2p-GFP) structurally intact. The significant disruption of the inner kinetochores observed in H3-repressed cells is not simply the disaggregation of the 16 individual kinetochores, because the outer kinetochore components remain properly organized. Thus the underlying pericentromeric chromatin contributes to the structure of the inner kinetochore and the correct linkage with the microtubule-binding outer kinetochore.
Loss of RSC function (nps1-105 at restrictive temperature, 37°C) results in significant disruption of both the inner and outer kinetochores. The inner (Ame1p-GFP) and outer (Spc24p-GFP) kinetochores of nps1-105 cells are disrupted 37 and 24%, respectively, as compared with 6 and 7% in wild-type cells (Fisher's exact test, p < 0.05; and Supplemental Table S4). The increase in disruption is more dramatic in the inner kinetochore (6% WT vs. 37% nps1-105), supporting the hypothesis that disruption of the underlying chromatin results in disrupted kinetochore organization. The disruption of the outer kinetochore (Spc24p-GFP) in nps1-105 cells may suggest a role for RSC in kinetochore organization or stability. We did not observe increased kinetochore disruption in isw2Δ cells (Fisher's exact test, p < 0.05; ). Thus nucleosome density and mobility within pericentromeric chromatin is essential in maintaining kinetochore structure.