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

Bastajian N, Friesen H, Andrews BJ - PLoS Genet. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Donnelly Centre and the Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

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.

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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.
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pgen-1003507-g003: 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.

Mentions: Given the clear roles for Mcm1 in cell-cycle-dependent gene expression, we chose to focus our follow-up analysis on the Mcm1-Bck2 interaction. In addition to the Y2H interaction between Bck2 and Mcm1, several observations from earlier studies implicate Bck2 in the activation of Mcm1 target genes in M/G1 phase: (1) mRNA from the M/G1 gene SWI4 accumulates much more slowly in bck2Δ than WT cells in synchronized cultures, whereas SWI4 is upregulated in cells that overexpress BCK2[12]; (2) high-copy BCK2 stimulates the expression of the Mcm1-dependent reporter gene, P-lacZ[47]; (3) overexpression of BCK2 causes increased transcription of CLN3 and SWI4 by microarray analysis [48]. However, up to now, no direct connection between BCK2 and M/G1 genes has been established. To evaluate the significance of the Mcm1-Bck2 physical interaction in vivo, we first assessed the effect of BCK2 deletion on expression of lacZ reporter genes whose expression was dependent on either multiple Mcm1-binding sites (4 x P-sites) [47] or the upstream activating sequences of four Mcm1-regulated genes expressed in M/G1 phase – CLN3, CDC6, CDC47, SWI4[49] (Figure 3A). In these plasmid reporter assays, deletion of BCK2 had no effect on expression of a control ACT1-lacZ reporter gene. However, we saw a pronounced reduction in expression of the CLN3-lacZ, CDC6-lacZ, CDC47-lacZ, SWI4-lacZ, and P-lacZ reporter genes in the bck2Δ strain (Figure 3B). These results suggest a role for BCK2 in M/G1 gene expression. To verify the results of the reporter gene assays, we next examined endogenous levels of Mcm1 target gene expression in a bck2Δ strain (Figure 4). Since Mcm1 controls CLN3 and SWI4 transcript accumulation at a very early point in G1, we synchronized cultures in mitosis with a cdc20-3 temperature-sensitive allele and released them into the subsequent cell cycle. Cells in this experiment were slow growing and enriched in large-budded cells that precluded FACS analysis of cell cycle synchrony (data not shown). However, CLN2 transcription was highly periodic in both WT and bck2Δ cells, indicating that these cultures were synchronously released from the mitotic block. Consistent with previous reports, CLN2 transcript was reduced in the bck2Δ strain [11], [12] while levels of a control transcript (ALG9) were unaffected. Strikingly, the accumulation of CLN3 and SWI4 mRNAs was significantly reduced in bck2Δ cells, and peak expression was also delayed, at least for the CLN3 transcript. In wild-type cells, CLB2 mRNA peaked after G1 transcripts as expected. However, in bck2Δ cells CLB2 transcripts were delayed and only began to accumulate near the end of the time-course, after the peak of CLB2 expression seen in wild-type cells. Thus, BCK2 is required for the appropriate expression of the M/G1 genes CLN3 and SWI4, the G1/S gene CLN2, and the G2/M gene CLB2.


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

Bastajian N, Friesen H, Andrews BJ - PLoS Genet. (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3649975&req=5

pgen-1003507-g003: 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.
Mentions: Given the clear roles for Mcm1 in cell-cycle-dependent gene expression, we chose to focus our follow-up analysis on the Mcm1-Bck2 interaction. In addition to the Y2H interaction between Bck2 and Mcm1, several observations from earlier studies implicate Bck2 in the activation of Mcm1 target genes in M/G1 phase: (1) mRNA from the M/G1 gene SWI4 accumulates much more slowly in bck2Δ than WT cells in synchronized cultures, whereas SWI4 is upregulated in cells that overexpress BCK2[12]; (2) high-copy BCK2 stimulates the expression of the Mcm1-dependent reporter gene, P-lacZ[47]; (3) overexpression of BCK2 causes increased transcription of CLN3 and SWI4 by microarray analysis [48]. However, up to now, no direct connection between BCK2 and M/G1 genes has been established. To evaluate the significance of the Mcm1-Bck2 physical interaction in vivo, we first assessed the effect of BCK2 deletion on expression of lacZ reporter genes whose expression was dependent on either multiple Mcm1-binding sites (4 x P-sites) [47] or the upstream activating sequences of four Mcm1-regulated genes expressed in M/G1 phase – CLN3, CDC6, CDC47, SWI4[49] (Figure 3A). In these plasmid reporter assays, deletion of BCK2 had no effect on expression of a control ACT1-lacZ reporter gene. However, we saw a pronounced reduction in expression of the CLN3-lacZ, CDC6-lacZ, CDC47-lacZ, SWI4-lacZ, and P-lacZ reporter genes in the bck2Δ strain (Figure 3B). These results suggest a role for BCK2 in M/G1 gene expression. To verify the results of the reporter gene assays, we next examined endogenous levels of Mcm1 target gene expression in a bck2Δ strain (Figure 4). Since Mcm1 controls CLN3 and SWI4 transcript accumulation at a very early point in G1, we synchronized cultures in mitosis with a cdc20-3 temperature-sensitive allele and released them into the subsequent cell cycle. Cells in this experiment were slow growing and enriched in large-budded cells that precluded FACS analysis of cell cycle synchrony (data not shown). However, CLN2 transcription was highly periodic in both WT and bck2Δ cells, indicating that these cultures were synchronously released from the mitotic block. Consistent with previous reports, CLN2 transcript was reduced in the bck2Δ strain [11], [12] while levels of a control transcript (ALG9) were unaffected. Strikingly, the accumulation of CLN3 and SWI4 mRNAs was significantly reduced in bck2Δ cells, and peak expression was also delayed, at least for the CLN3 transcript. In wild-type cells, CLB2 mRNA peaked after G1 transcripts as expected. However, in bck2Δ cells CLB2 transcripts were delayed and only began to accumulate near the end of the time-course, after the peak of CLB2 expression seen in wild-type cells. Thus, BCK2 is required for the appropriate expression of the M/G1 genes CLN3 and SWI4, the G1/S gene CLN2, and the G2/M gene CLB2.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Donnelly Centre and the Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

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.

Show MeSH
Related in: MedlinePlus