Presentation_1_Phenotypic selection during laboratory evolution of yeast populations leads to a genome-wide sustainable chromatin compaction shift.PDF

In a previous study, we have shown how microbial evolution has resulted in a persistent reduction in expression after repeatedly selecting for the lowest PGAL1-YFP-expressing cells. Applying the ATAC-seq assay on samples collected from this 28-day evolution experiment, here we show how genome-wide chromatin compaction changes during evolution under selection pressure. We found that the chromatin compaction was altered not only on GAL network genes directly impacted by the selection pressure, showing an example of selection-induced non-genetic memory, but also at the whole-genome level. The GAL network genes experienced chromatin compaction accompanying the reduction in PGAL1-YFP reporter expression. Strikingly, the fraction of global genes with differentially compacted chromatin states accounted for about a quarter of the total genome. Moreover, some of the ATAC-seq peaks followed well-defined temporal dynamics. Comparing peak intensity changes on consecutive days, we found most of the differential compaction to occur between days 0 and 3 when the selection pressure was first applied, and between days 7 and 10 when the pressure was lifted. Among the gene sets enriched for the differential compaction events, some had increased chromatin availability once selection pressure was applied and decreased availability after the pressure was lifted (or vice versa). These results intriguingly show that, despite the lack of targeted selection, transcriptional availability of a large fraction of the genome changes in a very diverse manner during evolution, and these changes can occur in a relatively short number of generations.</p

Table_1_Phenotypic selection during laboratory evolution of yeast populations leads to a genome-wide sustainable chromatin compaction shift.xlsx

In a previous study, we have shown how microbial evolution has resulted in a persistent reduction in expression after repeatedly selecting for the lowest PGAL1-YFP-expressing cells. Applying the ATAC-seq assay on samples collected from this 28-day evolution experiment, here we show how genome-wide chromatin compaction changes during evolution under selection pressure. We found that the chromatin compaction was altered not only on GAL network genes directly impacted by the selection pressure, showing an example of selection-induced non-genetic memory, but also at the whole-genome level. The GAL network genes experienced chromatin compaction accompanying the reduction in PGAL1-YFP reporter expression. Strikingly, the fraction of global genes with differentially compacted chromatin states accounted for about a quarter of the total genome. Moreover, some of the ATAC-seq peaks followed well-defined temporal dynamics. Comparing peak intensity changes on consecutive days, we found most of the differential compaction to occur between days 0 and 3 when the selection pressure was first applied, and between days 7 and 10 when the pressure was lifted. Among the gene sets enriched for the differential compaction events, some had increased chromatin availability once selection pressure was applied and decreased availability after the pressure was lifted (or vice versa). These results intriguingly show that, despite the lack of targeted selection, transcriptional availability of a large fraction of the genome changes in a very diverse manner during evolution, and these changes can occur in a relatively short number of generations.</p

Table_2_Phenotypic selection during laboratory evolution of yeast populations leads to a genome-wide sustainable chromatin compaction shift.xlsx

In a previous study, we have shown how microbial evolution has resulted in a persistent reduction in expression after repeatedly selecting for the lowest PGAL1-YFP-expressing cells. Applying the ATAC-seq assay on samples collected from this 28-day evolution experiment, here we show how genome-wide chromatin compaction changes during evolution under selection pressure. We found that the chromatin compaction was altered not only on GAL network genes directly impacted by the selection pressure, showing an example of selection-induced non-genetic memory, but also at the whole-genome level. The GAL network genes experienced chromatin compaction accompanying the reduction in PGAL1-YFP reporter expression. Strikingly, the fraction of global genes with differentially compacted chromatin states accounted for about a quarter of the total genome. Moreover, some of the ATAC-seq peaks followed well-defined temporal dynamics. Comparing peak intensity changes on consecutive days, we found most of the differential compaction to occur between days 0 and 3 when the selection pressure was first applied, and between days 7 and 10 when the pressure was lifted. Among the gene sets enriched for the differential compaction events, some had increased chromatin availability once selection pressure was applied and decreased availability after the pressure was lifted (or vice versa). These results intriguingly show that, despite the lack of targeted selection, transcriptional availability of a large fraction of the genome changes in a very diverse manner during evolution, and these changes can occur in a relatively short number of generations.</p

Data_Sheet_1_Phenotypic selection during laboratory evolution of yeast populations leads to a genome-wide sustainable chromatin compaction shift.XLSX

In a previous study, we have shown how microbial evolution has resulted in a persistent reduction in expression after repeatedly selecting for the lowest PGAL1-YFP-expressing cells. Applying the ATAC-seq assay on samples collected from this 28-day evolution experiment, here we show how genome-wide chromatin compaction changes during evolution under selection pressure. We found that the chromatin compaction was altered not only on GAL network genes directly impacted by the selection pressure, showing an example of selection-induced non-genetic memory, but also at the whole-genome level. The GAL network genes experienced chromatin compaction accompanying the reduction in PGAL1-YFP reporter expression. Strikingly, the fraction of global genes with differentially compacted chromatin states accounted for about a quarter of the total genome. Moreover, some of the ATAC-seq peaks followed well-defined temporal dynamics. Comparing peak intensity changes on consecutive days, we found most of the differential compaction to occur between days 0 and 3 when the selection pressure was first applied, and between days 7 and 10 when the pressure was lifted. Among the gene sets enriched for the differential compaction events, some had increased chromatin availability once selection pressure was applied and decreased availability after the pressure was lifted (or vice versa). These results intriguingly show that, despite the lack of targeted selection, transcriptional availability of a large fraction of the genome changes in a very diverse manner during evolution, and these changes can occur in a relatively short number of generations.</p

Data_Sheet_2_Phenotypic selection during laboratory evolution of yeast populations leads to a genome-wide sustainable chromatin compaction shift.XLSX

In a previous study, we have shown how microbial evolution has resulted in a persistent reduction in expression after repeatedly selecting for the lowest PGAL1-YFP-expressing cells. Applying the ATAC-seq assay on samples collected from this 28-day evolution experiment, here we show how genome-wide chromatin compaction changes during evolution under selection pressure. We found that the chromatin compaction was altered not only on GAL network genes directly impacted by the selection pressure, showing an example of selection-induced non-genetic memory, but also at the whole-genome level. The GAL network genes experienced chromatin compaction accompanying the reduction in PGAL1-YFP reporter expression. Strikingly, the fraction of global genes with differentially compacted chromatin states accounted for about a quarter of the total genome. Moreover, some of the ATAC-seq peaks followed well-defined temporal dynamics. Comparing peak intensity changes on consecutive days, we found most of the differential compaction to occur between days 0 and 3 when the selection pressure was first applied, and between days 7 and 10 when the pressure was lifted. Among the gene sets enriched for the differential compaction events, some had increased chromatin availability once selection pressure was applied and decreased availability after the pressure was lifted (or vice versa). These results intriguingly show that, despite the lack of targeted selection, transcriptional availability of a large fraction of the genome changes in a very diverse manner during evolution, and these changes can occur in a relatively short number of generations.</p

Degradation of cyclin Cln3 by exceeding CENs: Mad3 physical and functional interactions with SCF.

(A) Analysis of Cln3 stability by promoter shut-off experiments in the presence (orange circles) or absence (gray circles) of two YCp–CENGALp vectors in wild-type cells grown under permissive conditions. After tetracycline addition, cells were collected at the indicated times, and obtained Cln3–6FLAG levels are plotted relative to an unspecific cross-reacting band (asterisk) used as loading control. (B) Analysis of Cln3 stability in Mad3-deficient cells as in (A). (C) Analysis of mCitrine–Cln3–11A stability by time-lapse microscopy in the presence (orange circles) or absence (gray circles) of three YCp vectors. Nuclear levels of mCitrine–Cln3–11A in cells were determined at the indicated times after cycloheximide addition, and mean values (N = 100) are plotted. (D) Analysis of mCitrine–Cln3–11A accumulation in the nucleus in the presence (orange circles) or absence (gray circles) of three YCp vectors. Nuclear levels of mCitrine–Cln3–11A were determined in G1 daughter cells with 50–60 μm3 of volume. Individual data (N = 90) and median values are plotted. (E) Cell extracts (input) and GST PDs of cdc4ts grr1 cells expressing Cln3–13myc and GST fusions to Cdc4ΔFbox, Mad3, or Mad3ΔGLEBS were analyzed by immunoblotting with either αmyc (top panels) or αGST (bottom panel) antibodies. (F) Cell extracts (input) and GST PDs of cells expressing Mad3–3HA or Mad3 ΔGLEBS–3HA and either GST or GST–Cdc4 were analyzed by immunoblotting with either αHA (top panels) or αGST (bottom panel) antibodies. (G) Cells with the indicated genotypes carrying three YCp vectors were analyzed as in Fig 1B to determine cell size at budding as a function of copy number. Individual budding volumes (small dots) were binned, and mean values (large circles, N = 50) and a regression line are plotted. Correlation analysis and pairwise comparisons were performed with nonparametric tests as described in Materials and methods. Underlying data can be found in S1 Data. GST, glutathione S-transferase; PD, pulldown; YCp, yeast centromeric plasmid.</p

Centromeric signaling proteins boost G1 cyclin degradation and modulate cell size in budding yeast

<div><p>Cell size scales with ploidy in a great range of eukaryotes, but the underlying mechanisms remain unknown. Using various orthogonal single-cell approaches, we show that cell size increases linearly with centromere (CEN) copy number in budding yeast. This effect is due to a G1 delay mediated by increased degradation of Cln3, the most upstream G1 cyclin acting at Start, and specific centromeric signaling proteins, namely Mad3 and Bub3. Mad3 binds both Cln3 and Cdc4, the adaptor component of the Skp1/Cul1/F-box (SCF) complex that targets Cln3 for degradation, these interactions being essential for the CEN-dosage dependent effects on cell size. Our results reveal a pathway that modulates cell size as a function of CEN number, and we speculate that, in cooperation with other CEN-independent mechanisms, it could assist the cell to attain efficient mass/ploidy ratios.</p></div

Exceeding CENs require centromeric Mad3/Bub3 signaling proteins to modulate cell size.

(A) Cells with the indicated genotypes carrying three YCp vectors were analyzed as in Fig 1B to determine cell size at budding as a function of copy number. Individual budding volumes (small dots) were binned, and mean values (large circles, N = 50) and a regression line are plotted. (B) Newborn daughter cells with the indicated genotypes were analyzed to determine cell size at budding. Mad3 overexpression (oMAD3) was attained by inducing a GAL1p–MAD3 construct with 1 mM estradiol in newborn cells expressing the Gal4–hER–VP16 transactivator. Individual data (N > 300) and median values are plotted. Correlation analysis and pairwise comparisons were performed with nonparametric tests as described in Materials and methods. Underlying data can be found in S1 Data. CEN, centromere; YCp, yeast centromeric plasmid.</p

CEN signaling proteins cooperate with SCF–Cdc4 to enhance degradation of the yeast G1 cyclin and modulate cell size in budding yeast.

When present in excess, centromeres accelerate Cln3 degradation in the nucleus with the essential participation of Mad3, a centromeric signaling protein that interacts with Cdc4 and requires SCF function to modulate cell size as a function of centromere number. CEN, centromere.</p

Exceeding CENs modulate cell size in a Cln3-dependent manner.

<p>(<b>A</b>) Cells with the indicated genotypes carrying three YCp vectors (3YCp) or none (ctrl) were analyzed to determine cell size at budding. Individual data (<i>N</i> > 400), and median values are plotted. (<b>B</b>) Cells with the indicated genotypes carrying three YCp vectors were analyzed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005388#pbio.2005388.g001" target="_blank">Fig 1B</a> to determine cell size at budding as a function of copy number. Individual budding volumes (small dots) were binned, and mean values (large circles, <i>N</i> = 50) and a regression line are plotted. Correlation analysis and pairwise comparisons were performed with nonparametric tests as described in Materials and methods. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005388#pbio.2005388.s008" target="_blank">S1 Data</a>. CEN, centromere; YCp, yeast centromeric plasmid.</p