Bck2 activates Mcm1-driven <i>lacZ</i> reporter constructs.

<p>(A) Diagrams of plasmid reporter constructs used to assess the effect of <i>BCK2</i> deletion or overexpression. Constructs containing either multiple synthetic Mcm1-binding sites upstream of the <i>lacZ</i> gene (4 x P-site, pCLM771) or endogenous promoters that contain ECB elements (<i>CLN3</i> pBD1790, <i>SWI4</i> pBD1577, <i>CDC6</i> pBD1637, <i>CDC47</i> pBD1951) are shown. Black boxes represent distinct Mcm1-binding sites such as Mcm1-binding P-site elements or ECB elements, whereas white boxes represent MCB elements or GRE elements. (B) WT (grey bars) or <i>bck2</i>Δ (black bars) yeast transformants carrying P-<i>lacZ</i>, <i>CLN3</i>-<i>lacZ</i>, <i>SWI4</i>-<i>lacZ</i>, <i>CDC6</i>-<i>lacZ</i>, <i>CDC47</i>-<i>lacZ</i>, and <i>ACT1</i>-<i>lacZ</i> were assessed for <i>lacZ</i> expression level. Asynchronous cells were grown to mid-log phase in selective medium and subjected to quantitative β-galactosidase assays to measure <i>lacZ</i> expression. Y-axis values are expressed in Miller units. Error bars reflect values obtained from 3 independent transformants in separate experiments.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Truncation analysis of the <i>BCK2</i> gene.

<p>Fragments of the <i>BCK2</i> gene (black bar) were amplified from genomic DNA using primer positions shown and designated as “amino acid+F (Forward), or R (reverse)”. PCR products were cloned into a yeast two-hybrid vector to create <i>BCK2</i> fragments fused to the N-terminal <i>GAL4</i> DBD (DNA Binding Domain). High density growth spots (in either the <i>ADE2</i> transcription activation assay or the complementation assay) were called “+”, “++”, or “+++” depending on extent of growth. A complete absence of growth was called “−”. Numbers in the β-Gal column represent averaged quantities (per fusion protein) in Miller Units (U).</p

Bck2 localizes to the promoters of M/G₁, G₁/S and G₂/M genes.

<p>(A) Bck2 localization to M/G₁ genes depends on ECBs. WT strain (BY2125; W303) or a strain containing mutated ECB elements in the <i>CLN3</i> and <i>SWI4</i> promoters (BY2680; <i>cln3</i>(ecb)<i>swi4</i>(ecb)) was transformed with a pGAL-<i>BCK2</i>-FLAG plasmid and grown separately in raffinose- (non-inducing conditions) or galactose-containing medium (inducing conditions) to mid-log phase. Cultures were harvested and anti-FLAG ChIPs were analyzed for <i>CLN2</i>, <i>CLN3</i> and <i>SWI4</i> promoter DNA by Q-PCR. The Y-axis measures enrichment of promoter DNA for the target gene indicated relative to enrichment of non-promoter DNA from an untranscribed region of chromosome II. (B) Bck2 localization to the <i>CLN2</i> promoter is reduced when SCBs or Mcm1-binding sites are mutated. Vector and pGAL-BCK2-FLAG were transformed into WT (GC46), and strains with 3 SCBs mutated (yLB76-scb*) and 2 Mcm1-binding sites mutated (yLB76-mcm1*), cells were grown in inducing conditions, and Bck2-Flag localization to the <i>CLN2</i> promoter was analyzed by Q-PCR. (C) Bck2 localizes to the <i>CLB2</i> promoter. WT cells containing vector or pGAL-BCK2-FLAG were grown in inducing conditions and Bck2-Flag localization to the <i>CLN3</i> and <i>CLB2</i> promoter was analyzed by ChIP.</p

Bck2 requires intact ECB elements for transcriptional activation.

<p>(A) WT (grey bars) or <i>bck2</i>Δ (black bars) yeast transformants harboring a <i>CLN3</i>-<i>lacZ</i> or <i>CLN3</i>_ecb-<i>lacZ</i> reporter plasmid were grown to mid-log phase in selective medium and subjected to quantitative β-galactosidase assays. (B) WT yeast strains containing a <i>CLN3</i>-<i>lacZ</i>, <i>CLN3</i>ecb-<i>lacZ</i> or <i>ACT1</i>-<i>lacZ</i> reporter plasmid were co-transformed with pGAL-<i>BCK2</i>-FLAG (black bars) or vector (grey bars) and grown to mid-log phase in selective medium containing galactose (inducing conditions) and subjected to quantitative β-galactosidase assays to measure <i>lacZ</i> expression. Y-axis values are expressed in Miller units. Error bars reflect values obtained from 3 independent transformants in separate experiments. (C) A WT (BY2125) strain and a strain lacking functional ECB elements in the <i>CLN3</i> and <i>SWI4</i> promoters (BY2680; <i>cln3</i>(ecb)<i>swi4</i>(ecb)) were transformed with pGAL-<i>BCK2</i>-FLAG (hatched bars in WT; black bars in mutant) or vector (white bars in WT; grey bars in mutant). Transformants were grown to saturation in plasmid selective medium and subcultured in YPGal to mid-log phase before harvesting for quantification of mRNA levels by Q-PCR analysis using <i>ACT1</i> mRNA levels as a normalizing control. Relative enrichment of <i>CLN3</i>, <i>SWI4</i>, <i>CLN2</i>, <i>ALG9</i> and <i>CLB2</i> mRNA normalized against <i>ACT1</i> mRNA is shown in the left panel. Relative enrichment of <i>BCK2</i> mRNA from the same samples normalized against <i>ACT1</i> mRNA is shown in the right panel.</p

Effect of <i>BCK2</i> deletion on <i>CLN2</i>, <i>ALG9</i>, <i>CLN3</i>, <i>SWI4</i>, <i>BCK2</i>, and <i>CLB2</i> mRNA accumulation during the cell cycle.

<p>Y8890 (<i>cdc20</i>-<i>3</i> WT, dark blue line with diamonds) and BY4897 cultures (<i>cdc20</i>-<i>3 bck2</i>Δ, dark pink line with squares) were grown to log-phase, arrested at M/G₁ by incubating for 3.5 hours at 37°C (block), then released into the cell cycle by re-incubation at 21°C. Samples were harvested every 15 minutes and mRNA levels quantified by Q-PCR using <i>ACT1</i> mRNA levels as a normalizing control. In WT cells, the peak of <i>CLN2</i> transcription marks the G₁/S transition, the peak of <i>CLN3</i> and <i>SWI4</i> marks M/G₁, and the peak of <i>CLB2</i> marks G₂/M.</p

Bck2-interacting proteins identified in a genome-wide yeast two-hybrid screen.

<p>Yeast transformants carrying <i>ADH1</i>-<i>GAL4</i> DBD (vector; <i>LEU2</i>) or <i>ADH1</i>-<i>GAL4</i> DBD-<i>BCK2</i> Fragment 11 (Bck2) in a two-hybrid bait strain (Y8930) were mated to yeast transformants of a two-hybrid prey strain (Y8800) bearing specific gene ORFs fused to the N-terminal <i>GAL4</i> AD (activation domain; <i>TRP1</i>, i.e. <i>ADH1</i>-<i>GAL4</i> AD-ORF plasmid). Diploids were selected by streaking on double plasmid selection medium (SD – Leu – Trp). Strains were grown to equivalent optical density, and spotted in serial 10-fold dilutions on double plasmid selection medium (SD – Leu – Trp) or medium where growth is proportional to transcription of the <i>ADE2</i> gene (SD – Leu – Trp - Ade). Plates were incubated for 48 h at 30°C.</p

Summary of Bck2-dependent regulation of cell cycle gene expression.

<p>In pre-START cells Bck2 binds to Mcm1 and promotes expression of M/G₁ genes such as <i>SWI4</i> and <i>CLN3</i>, which encode important constituents of the transcriptional ‘switch’ for START. Cln3 then contributes to activation of Swi4, a component of SBF, which induces expression of G₁/S genes such as <i>CLN2</i>. Bck2 also induces <i>CLN2</i> expression directly through physical association with Swi4 and with Mcm1. In G₂/M Bck2 promotes expression of <i>CLB2</i>, possibly through interaction with Mcm1. We speculate that Bck2 may be responding to environmental signals such as nutrients in its role of activating cell cycle gene expression (see text for details). Bold lines indicate interactions, both protein-protein (in blue) and protein-DNA (in green) described in this work.</p

Bck2 may compete with Yox1 for interaction with Mcm1.

<p>(A) <i>YOX1</i>-TAP, <i>YHP1</i>-TAP and <i>MCM1</i>-TAP strains carrying a pGAL-<i>BCK2</i>-FLAG plasmid (black bars) or vector (grey bars), were individually grown in plasmid selective medium containing galactose (inducing conditions) to mid-log phase. Cultures were harvested and anti-TAP ChIPs were performed using IgG sepharose resin and analyzed for <i>CLN3</i> promoter DNA using Q-PCR. Error bars reflect values obtained after multiple Q-PCR runs from the same experiment. (B) A WT strain (Y7092) was co-transformed with a GAL-<i>YOX1</i> (<i>URA3</i>) plasmid and one of Vector, GAL-<i>CLN3</i>, or GAL-<i>BCK2</i> (<i>LEU2</i>) plasmid. Transformant cultures of equivalent optical density were spotted in serial 5-fold dilutions on selective medium that was either non-inducing (glucose) or inducing (galactose), and incubated for 72 h at 30°C. (C) A yeast two-hybrid bait strain (Y8930) carrying either plasmid <i>ADH1</i>-<i>GAL4</i> DB (vector) or plasmid <i>ADH1</i>-<i>GAL4</i> DB-<i>BCK2</i> Fragment 11 (Bck2) were mated to yeast two-hybrid prey strain (Y8800) carrying either <i>ADH1</i>-<i>GAL4</i> AD-<i>MCM1</i>^WT or <i>ADH1</i>-<i>GAL4</i>-<i>MCM1</i>^V69E to create diploid yeast strains. Diploids were spotted in serial 10-fold dilutions on double plasmid selection medium, or medium where growth is proportional to transcription of the <i>ADE2</i> gene, and incubated for 48–72 h at 30°C.</p

<i>Saccharomyces cerevisiae</i> strains used in this study.

<p>Strains are isogenic with BY4741, except for BY2125 and BY2680, and GC46, yLB76-mcm1*, and yLB76-scb*, which are isogenic to W303, and the Y2H strains Y8930 and Y8800. Where strains are not listed in this table, strains from the yeast gene-deletion set <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003507#pgen.1003507-Winzeler1" target="_blank">[77]</a> were used.</p