Bck2 acts through the MADS box protein Mcm1 to activate cell-cycle-regulated genes in budding yeast

Abstract

The Bck2 protein is a potent genetic regulator of cell-cycle-dependent gene expression in budding yeast. To date, most experiments have focused on assessing a potential role for Bck2 in activation of the G1/S-specific transcription factors SBF (Swi4, Swi6) and MBF (Mbp1, Swi6), yet the mechanism of gene activation by Bck2 has remained obscure. We performed a yeast two-hybrid screen using a truncated version of Bck2 and discovered six novel Bck2-binding partners including Mcm1, an essential protein that binds to and activates M/G1 promoters through Early Cell cycle Box (ECB) elements as well as to G2/M promoters. At M/G1 promoters Mcm1 is inhibited by association with two repressors, Yox1 or Yhp1, and gene activation ensues once repression is relieved by an unknown activating signal. Here, we show that Bck2 interacts physically with Mcm1 to activate genes during G1 phase. We used chromatin immunoprecipitation (ChIP) experiments to show that Bck2 localizes to the promoters of M/G1-specific genes, in a manner dependent on functional ECB elements, as well as to the promoters of G1/S and G2/M genes. The Bck2-Mcm1 interaction requires valine 69 on Mcm1, a residue known to be required for interaction with Yox1. Overexpression of BCK2 decreases Yox1 localization to the early G1-specific CLN3 promoter and rescues the lethality caused by overexpression of YOX1. Our data suggest that Yox1 and Bck2 may compete for access to the Mcm1-ECB scaffold to ensure appropriate activation of the initial suite of genes required for cell cycle commitment.

Figure 1. Truncation analysis of the BCK2 gene.

Fragments of the BCK2 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 BCK2 fragments fused to the N-terminal GAL4 DBD (DNA Binding Domain). High density growth spots (in either the ADE2 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).

Figure 2. Bck2-interacting proteins identified in a genome-wide yeast two-hybrid screen.

Yeast transformants carrying ADH1-GAL4 DBD (vector; LEU2) or ADH1-GAL4 DBD-BCK2 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 GAL4 AD (activation domain; TRP1, i.e. ADH1-GAL4 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 ADE2 gene (SD – Leu – Trp - Ade). Plates were incubated for 48 h at 30°C.

Figure 3. Bck2 activates Mcm1-driven lacZ reporter constructs.

(A) Diagrams of plasmid reporter constructs used to assess the effect of BCK2 deletion or overexpression. Constructs containing either multiple synthetic Mcm1-binding sites upstream of the lacZ gene (4 x P-site, pCLM771) or endogenous promoters that contain ECB elements (CLN3 pBD1790, SWI4 pBD1577, CDC6 pBD1637, CDC47 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 bck2Δ (black bars) yeast transformants carrying P-lacZ, CLN3-lacZ, SWI4-lacZ, CDC6-lacZ, CDC47-lacZ, and ACT1-lacZ were assessed for lacZ expression level. Asynchronous cells were grown to mid-log phase in selective medium and subjected to quantitative β-galactosidase assays to measure lacZ expression. Y-axis values are expressed in Miller units. Error bars reflect values obtained from 3 independent transformants in separate experiments.

Figure 4. Effect of BCK2 deletion on CLN2, ALG9, CLN3, SWI4, BCK2, and CLB2 mRNA accumulation during the cell cycle.

Y8890 (cdc20-3 WT, dark blue line with diamonds) and BY4897 cultures (cdc20-3 bck2Δ, 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 ACT1 mRNA levels as a normalizing control. In WT cells, the peak of CLN2 transcription marks the G₁/S transition, the peak of CLN3 and SWI4 marks M/G₁, and the peak of CLB2 marks G₂/M.

Figure 5. Bck2 requires intact ECB elements for transcriptional activation.

(A) WT (grey bars) or bck2Δ (black bars) yeast transformants harboring a CLN3-lacZ or CLN3 _ecb-lacZ reporter plasmid were grown to mid-log phase in selective medium and subjected to quantitative β-galactosidase assays. (B) WT yeast strains containing a CLN3-lacZ, CLN3ecb-lacZ or ACT1-lacZ reporter plasmid were co-transformed with pGAL-BCK2-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 lacZ 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 CLN3 and SWI4 promoters (BY2680; cln3(ecb)swi4(ecb)) were transformed with pGAL-BCK2-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 ACT1 mRNA levels as a normalizing control. Relative enrichment of CLN3, SWI4, CLN2, ALG9 and CLB2 mRNA normalized against ACT1 mRNA is shown in the left panel. Relative enrichment of BCK2 mRNA from the same samples normalized against ACT1 mRNA is shown in the right panel.

Figure 6. Bck2 localizes to the promoters of M/G₁, G₁/S and G₂/M genes.

(A) Bck2 localization to M/G₁ genes depends on ECBs. WT strain (BY2125; W303) or a strain containing mutated ECB elements in the CLN3 and SWI4 promoters (BY2680; cln3(ecb)swi4(ecb)) was transformed with a pGAL-BCK2-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 CLN2, CLN3 and SWI4 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 CLN2 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 CLN2 promoter was analyzed by Q-PCR. (C) Bck2 localizes to the CLB2 promoter. WT cells containing vector or pGAL-BCK2-FLAG were grown in inducing conditions and Bck2-Flag localization to the CLN3 and CLB2 promoter was analyzed by ChIP.

Figure 7. Bck2 may compete with Yox1 for interaction with Mcm1.

(A) YOX1-TAP, YHP1-TAP and MCM1-TAP strains carrying a pGAL-BCK2-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 CLN3 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-YOX1 (URA3) plasmid and one of Vector, GAL-CLN3, or GAL-BCK2 (LEU2) 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 ADH1-GAL4 DB (vector) or plasmid ADH1-GAL4 DB-BCK2 Fragment 11 (Bck2) were mated to yeast two-hybrid prey strain (Y8800) carrying either ADH1-GAL4 AD-MCM1 ^WT or ADH1-GAL4-MCM1 ^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 ADE2 gene, and incubated for 48–72 h at 30°C.

Figure 8. Summary of Bck2-dependent regulation of cell cycle gene expression.

In pre-START cells Bck2 binds to Mcm1 and promotes expression of M/G₁ genes such as SWI4 and CLN3, 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 CLN2. Bck2 also induces CLN2 expression directly through physical association with Swi4 and with Mcm1. In G₂/M Bck2 promotes expression of CLB2, 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.