Nucleosome architecture throughout the cell cycle

Abstract

Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity.

Figure 1. Nucleosome organization around TSSs and ORIs at G1 phase.

(a) Heatmap of MNase-seq coverage (a) around TSSs (b) around ORIs (aligned taking strand directionality into account). Darker colors represent higher nucleosome occupancy. Rows are sorted according NFR-width and grouped by adjacent nucleosome classification (see methods for details). (c) Distribution of −1/ + 1 nucleosome dyads distance at TSSs and ORIs. While TSS-NFRs show a bi-modal distribution (peaks at 170 bp and 270 bp), ORI-NFRs are in general wider with a dyad-to-dyad distance of 300 bp (d) Mean nucleosome positioning scores (nucleR scores) of + 1/−1 nucleosomes at TSSs and ORIs. Value 1 indicates the highest coverage and phasing of nucleosomes, while value 0 points to disorganized and lowest-covered structure. Error bars, when visible, indicate the 95% confidence interval (CI); dashed horizontal line represents the global mean. (e) Mean fuzziness score and NFR width ratio between maximum and minimum values observed in triplicates. Higher values indicate more variability among G1 replicas. Error bars indicate the 95% CI (f) Comparison of the mean nucleosome coverage between the genes with constitutively low-expression and constitutively high-expression. Error bars indicate the 95% CI. (g) NFR distribution of the genes with constitutively low-expression and constitutively high-expression. (h) nucleR aggregated score for −1/ + 1 nucleosomes of early- and late-firing replication origins at each the cell cycle stage. Symbol indicates the mean nucleR score and error bars the 95% CI.

Figure 2. Chromatin sensitivity to MNase digestion along cell cycle.

(a) Chromatin collected at indicated time points after alpha factor release was formaldehyde cross-linked and digested with increasing amounts of MNase (0.005, 0.01 and 0.025 U) for 25′ at 37 °C, as indicated by the triangles above the lanes. De-crosslinked nucleosomal DNA was separated on 2% agarose gel to compare the digestion pattern along cell cycle. (b) Ratio of mono- to di- and mono- to tri-nucleosomes, calculated using IMAGEJ, of MNase digested cell cycle samples from two biological replicas. Error bars signal the 95% confidence interval. (c) The mean positioning and coverage scores, based on nucleR score, of −1 and + 1 nucleosomes are shown along cell cycle. Error bars (only visible by the end of the horizontal crossbar) signal the 95% confidence interval.

Figure 3. The transition of the nucleosome clusters of the plastic genes along cell cycle.

Directional graphs show the number of genes that keep or change the nucleosome architecture around the TSSs (see Methods for clustering details and description) at four transitions along the cell cycle (G1-S, S-G2, G2-M, M-G1).

Figure 4. Functional implications of chromatin dynamics along cell cycle.

(a) Distribution of stable, plastic and mobile genes. (b) Ratio between maximum and minimum mRNA expression levels observed during cell cycle for stable, plastic and mobile genes. Error bars indicate 95% CI. (c) Absolute expression difference between mobile genes in adjacent stages. Error bars indicate 95% CI. (d) Differences in positioning scores of −1/ + 1 nucleosomes relative to the TSSs for genes that decrease (left) and increase (right) their expression in adjacent stages (ie, if A = G1 stage then B = S stage, if A = G2 then B = M, stage). Only genes with an expression fold change of at least 1.2x between considered stages were selected. GO enrichment for (e) stable, (f) mobile genes. Shown elements represent top 10 terms according enrichment p-value (FDR corrected, minimum significance allowed 0.01). Similar terms have been reduced using ReviGO (see Methods). (g) Average nucleosome coverage around TSSs of mobile genes with higher expression in G1 compared to M and S stages. (h) GO enrichment of mobile genes with higher expression in G1 compared to M and S stages

Figure 5. Nucleosome coverage at TSSs and expression levels along CC for genes

(a) STE12, (b) MFA1, (c) SST2, (d) SIC1, (e) CLB1 and (f) CLB6. In each panel, a MNase-seq coverage plot is shown for the G1 (red), S (blue), G2 (green) and M (yellow) phases. Gene expression levels are indicated as log2 values of the hybridization ratios from the Affymetrix GeneChip Yeast Genome 2.0 arrays. Expression levels for the four stages of the corresponding genes are shown with the same color code. Note that horizontal axis in mRNA plot is not aligned anyhow with the upper plot. Genes in the first row (a–c) are related to alpha-factor response and sensibilitzation. Bottom three (d–f) are genes involved in other cell cycle functions.