Journal: Molecular Biology of the Cell

Article Title: Layers of regulation of cell-cycle gene expression in the budding yeast Saccharomyces cerevisiae

doi: 10.1091/mbc.E18-04-0255

Figure Lengend Snippet: Proteome dynamics of cell-cycle regulators follow the transcriptome with delay during the cell cycle in S. cerevisiae . Wild-type budding yeast cells were grown in 2% YEPD rich media, synchronized by alpha-factor mating pheromone, released into YEPD, and monitored over ∼2 cell cycles. Samples were collected every 5 min for RNA sequencing ( Kelliher et al. , 2016 ) (A) or every 7 min for total protein extraction (B, C). Population synchrony was monitored by counting at least 200 cells per time point for the presence or absence of a bud (bottom). A total of 31 high-confidence proteins (44 total peptides) are shown, with multiple high-confidence peptides per protein for Cdc28, Swi4, Cln2, Tos4, Hcm1, Plm2, Nrm1, Ndd1, Fhl1, Swi5, Ace2, and Sic1. Genes and proteins were ordered on the y -axis by peak time of mRNA expression (A). Transcript and protein levels are depicted as z -score changes relative to expression mean in the respective data sets, where values represent the number of standard deviations away from the mean. Each column represents a lifeline point on a common cell-cycle timeline determined by the CLOCCS algorithm (Supplemental File 1). Individual line plots from are shown for core cell-cycle TFs (Supplemental Figure 1), non-strongly-cycling regulators (Supplemental Figure 2), and periodic regulators (Supplemental Figure 3). Eleven regulators (Gat1, Cdc28, Swi4, Fhl1, Fkh1, Fkh2, Msn2, Swi6, Mbp1, Ixr1, and Mcm1) were somewhat constitutively expressed, and 20 regulators (Phd1, Pcl2, Msn4, Yox1, Clb5, Pcl1, Cln2, Tos4, Hcm1, Plm2, Nrm1, Cin8, Pds1, Ndd1, Swi5, Ace2, Sfg1, Cdc20, Ash1, and Sic1) appeared to be cycling.

Article Snippet: Alternatively, the nuclear localization of Swi4, Swi6, and/or Mbp1 could be altered without changing the total protein abundance, which has been demonstrated for Swi6 nuclear export following Clb6/CDK phosphorylation ( Geymonat et al. , 2004 ).

Techniques: RNA Sequencing, Protein Extraction, Expressing

Journal: Molecular Biology of the Cell

Article Title: Layers of regulation of cell-cycle gene expression in the budding yeast Saccharomyces cerevisiae

doi: 10.1091/mbc.E18-04-0255

Figure Lengend Snippet: An integrated cell-cycle network includes windows of targeted E3 ubiquitin ligase activity. Cell-cycle ordering is maintained by cyclin/CDK activity, an interconnected network of transcription factors, and E3 ubiquitin ligase activity. Approximate windows of peak degradation machinery are shown based on wild-type data . Periodic TFs (activators in green, repressors in red) are placed on the cell-cycle timeline approximately by peak mRNA expression. In blue, G1 (Cln) and B-type (Clb) cyclin/CDKs, APC/C, Cdc14, and Sic1 regulate each other and TFs in the network. Edges from TFs represent evidence for transcriptional regulation: ChIP-chip data for TF binding and/or genetic evidence for regulation type (compiled in Orlando et al. , 2008 ; McGoff et al. , 2016 ; Cho et al. , 2017a ). Edges between regulatory proteins and TFs represent protein-level modifications (e.g., phosphorylation or ubiquitination). Pointed arrows indicate activation, and blunted arrows mark repression or protein degradation. Proteins outlined in black were detected with high confidence in our study (Supplemental Table 2), and gray indicates low-confidence peptides. Dashed outlines mark periodic cell-cycle regulators, and solid boxes represent more stable TF expression. Paralogues from the whole genome duplication include Yhp1 and Yox1, Plm2 and Tos4, Fkh1 and Fkh2, and Ace2 and Swi5. Complexes of TFs include SBF (Swi4 and Swi6), MBF (Mbp1 and Swi6), and SFF (Mcm1, Fkh1-2, and Ndd1).

Article Snippet: Alternatively, the nuclear localization of Swi4, Swi6, and/or Mbp1 could be altered without changing the total protein abundance, which has been demonstrated for Swi6 nuclear export following Clb6/CDK phosphorylation ( Geymonat et al. , 2004 ).

Techniques: Ubiquitin Proteomics, Activity Assay, Expressing, ChIP-chip, Binding Assay, Phospho-proteomics, Activation Assay