Daughter-specific transcription factors regulate cell size control in budding yeast.

Strain and Plasmid Construction

Standard methods were used throughout. All strains are W303-congenic. All integrated constructs were characterized by qPCR. Mutations of the Ace2/Swi5 and Ash1 binding sites on the CLN3 promoter were verified by sequencing.

Time-Lapse Microscopy

Preparation of cells for microscopy and time-lapse microscopy were performed as previously described [5] , [6] . Growth of microcolonies was observed with fluorescence time-lapse microscopy at 30°C using a Leica DMIRE2 inverted microscope with a Ludl motorized XY stage. Images were acquired every 3 min for cells grown in glucose and every 6 min for cells grown in glycerol/ethanol with a Hamamatsu Orca-ER camera. Custom Visual Basic software integrated with ImagePro Plus was used to automate image acquisition and microscope control.

Image Analysis

Automated image segmentation and fluorescence quantification of yeast grown under time-lapse conditions and semi-automated assignment of microcolony pedigrees were performed as previously described [6] . The nuclear residence of Whi5-GFP was scored by visual inspection of composite phase contrast-fluorescent movies. Cell size was measured as the total cell fluorescence from DsRed protein, expressed from the constitutively active ACT1pr , as previously described [5] . Cell size at every time point was extrapolated from a linear fit of the ln(M) as a function of time for cells grown in glucose and from a smoothing spline fit for cells grown in glycerol/ethanol. Individual cell growth in glycerol/ethanol appears to be intermediate between a linear and an exponential model (unpublished data); this deviation from exponentiality has very little effect on this analysis.

Data Analysis

Time-lapse fluorescence microscopy, microarray data, and sequencing data were analyzed with custom software written in MATLAB software (see Text S1 for details on the analysis of the microarray data) [5] . For cluster analysis, the log 2 of the arrays data or of the subtracted arrays data were hierarchically clustered by the agglomerative algorithm [41] . Data were visually presented using JavaTreeView. For sequencing data, the area associated to every wild-type or mutated nucleotide was evaluated manually by using the MATLAB software.

Cell Cycle Synchronization

YEP medium was used for all cell cycle synchronization experiments, supplemented with the appropriate carbon source as indicated below.

Cell cycle synchronization by the cdc20

GALL-CDC20 block release was achieved by growing cells to early log phase in YEP+galactose (3%), then filtering and growing them in YEP+glucose (2%) for 3 h to arrest cells in metaphase. Cells were released from the block by filtering back into YEP+galactose (3%). GALL is a truncated version of the GAL1 promoter that shows inducible but significantly lower expression than the full-length GAL1 promoter [37] . Microarrays Microarrays were performed as previously described [71] but using microarrays carrying PCR fragments from open reading frames of S. cerevisiae . Each array had each PCR fragment independently spotted four to eight times, leading to a high redundancy of data and small errors in expression ratios. RNA extraction, cDNA synthesis and labeling, and hybridization and scanning were carried out by the Stony Brook spotted microarray facility, as described previously [71] . ChIPs Standard methods were used for ChIP experiments. Early log phase cells were fixed for 15 min in 1% formaldehyde at room temperature. Immunoprecipitations were performed with IgG Sepharose beads. Immunoprecipitated DNA was amplified by PCR.

Supporting Information Dataset S1 Microarrays data of wild-type, ace2 , swi5 , and ace2 swi5 synchronized cell populations. (1.48 MB TXT) Click here for additional data file. Dataset S2 Microarrays data of wild-type, ace2 ash1 , ACE2* ASH1* , ace2 , ACE2* , ash1 , ASH1* synchronized cell populations. (2.42 MB TXT) Click here for additional data file. Figure S1 Hierarchical clustering analysis of genes regulated by Ace2 and Swi5. (9.90 MB TIF) Click here for additional data file. Figure S2 Ash1 is a modulator of Swi5-dependent expression. Average expression for Ace2/Swi5 and Swi5 targets (45 genes) in response to Ash1 (data were obtained by subtracting the ASH1* dataset from ash1 dataset). This graph shows that Ash1 weakly represses the expression of many Ace2/Swi5 and Swi5 targets in daughter cells. (0.87 MB TIF) Click here for additional data file. Figure S3 Activation of SBF and MBF is delayed by Ace2 and Ash1. Average expression of 20 SBF/MBF targets in (A) ace2 and ACE2* , (B) ash1 and ASH1* , (C) ace2 ash1 and ACE2* ASH1* cells. Distribution of cell size at birth after release from the cdc20 arrest for (D) ace2 ash1 and (E) ACE2* ASH1* cells. (1.48 MB TIF) Click here for additional data file. Figure S4 Deletion of ACE2 and ASH1 result in similar T 1 only in mothers and daughter of similar size. Histogram of the difference in T 1 for mother-daughter pairs in wild-type (A, B), ace2 (C, D), ash1 (E, F), and ace2 ash1 (G, H) cells. T 1 is longer in daughters for almost all mother-daughter pairs, indicating that symmetrical regulation of Start is restricted to mothers and daughters of similar size upon deletion of ACE2 and ASH1 . (1.98 MB TIF) Click here for additional data file. Figure S5 Symmetrical distribution of Ace2 and Ash1 result in similar T 1 only in mothers and daughter of similar size. Histogram of the difference in T 1 for mother-daughter pairs in wild-type (A, B), ACE2* (C, D), ASH1* (E, F), and ACE2* ASH1* (G, H) cells. T 1 is longer in daughters for almost all mother-daughter pairs, indicating that symmetrical regulation of Start is restricted to mothers and daughters of similar size. (2.07 MB TIF) Click here for additional data file. Figure S6 Deletion or symmetrical regulation of CLN3 result in similar T 1 only in mothers and daughter of similar size. Histogram of the difference in T 1 for mother-daughter pairs in wild-type (A, B), cln3 (C, D), 6xCDC28pr-CLN3 (E), 4xCDC28pr-CLN3 (F), and ADH1pr-CLN3 (G) cells. T 1 is longer in daughters for almost all mother-daughter pairs, indicating that symmetrical regulation of Start is restricted to mothers and daughters of similar size. (1.98 MB TIF) Click here for additional data file. Figure S7 Start control is similar in mothers and “pseudo-mothers.” Plot of αT 1 versus ln(M birth ) for the average “mother-like” (red dots and error bars, see Figure 3 ) compared to mothers and “pseudo-mothers” (black dots). (1.45 MB TIF) Click here for additional data file. Figure S8 Start control is similar in daughters and “pseudo-daughters.” Plot of αT 1 versus ln(M birth ) for the average “daughter-like” (blue dots and error bars, see Figure 3 ) compared to daughters and “pseudo-daughters” (black dots). (1.53 MB TIF) Click here for additional data file. Figure S9 Correlation between αT 1 and ln(M birth ) for cells grown in glucose in mutants lacking the Ace2/Swi5 and/or Ash1 sites on the CLN3 promoter. (A) wild-type, (B) Ace2/Swi5 sites mutated, (C) Ash1 sites mutated, (D) Ace2/Swi5 and Ash1 sites mutated. Red dots, mothers; blue dots, daughters. (1.21 MB TIF) Click here for additional data file. Figure S10 Ace2 nuclear residence is independent of cell size. Correlation between αT A2 , that is, the time of Ace2 nuclear residence scaled with growth rate α, and ln(M birth ) for wild-type daughter cells grown in glucose. Red line: least square fit, slope ≈−0.2. (0.46 MB TIF) Click here for additional data file. Figure S11 Phylogenetic analysis of Ace2/Swi5 putative binding sites on the CLN3 promoter. Ace2/Swi5 consensus-binding site identified by PhyloGibbs as over-represented motif in the promoter of the Ace2 and Swi5 targets. Conserved Ace2/Swi5 putative binding sites identified by PhyloGibbs. (0.72 MB TIF) Click here for additional data file. Table S1 Strains list. (0.10 MB PDF) Click here for additional data file. Table S2 Plasmids list. (0.06 MB PDF) Click here for additional data file. Table S3 Analysis of Ace2 and Ash1 shared targets. (0.06 MB PDF) Click here for additional data file. Table S4 Average daughter delay in new-born cells of the same size. (0.07 MB PDF) Click here for additional data file. Table S5 Size-independent noise is similar in daughters and pseudo-daughters. (0.06 MB PDF) Click here for additional data file. Table S6 Mutation of Ace2/Swi5 and Ash1 putative sites results in reduced binding of these factors to the CLN3 promoter. (0.07 MB PDF) Click here for additional data file. Text S1 Supplementary materials and methods and supplementary results. (0.13 MB PDF) Click here for additional data file.