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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 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.
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pgen-1003507-g006: 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.

Mentions: Since Bck2 functions through ECB elements (Figure 5) and physically interacts with Mcm1 (Figure 2), we next asked if Bck2 localized to the promoter regions of M/G1-phase genes. We first used chromatin immunoprecipitation (ChIP) with a strain carrying a TAP-tagged allele of Bck2 to assess association of Bck2 with various promoters. We detected a reproducible enrichment of promoter DNA in the Bck2 ChIP, but the signal was very low relative to Swi4 or Mcm1 ChIPs (data not shown). To improve our assay, we repeated the ChIP experiment using a strain in which a FLAG-tagged derivative of Bck2 was conditionally overproduced (Figure 6A). Under inducing conditions (galactose), Bck2-FLAG IPs were enriched in CLN2, CLN3 and SWI4 promoter DNA relative to non-inducing conditions (raffinose) (Figure 6A) or vector control (data not shown). The enhanced enrichment of CLN3 promoter DNA compared to SWI4 promoter DNA in Bck2 IPs likely reflects the presence of more ECB elements in the CLN3 promoter (6 versus 1). Association of Bck2 with the CLN3 and SWI4 promoters was entirely dependent on the presence of ECB elements, while association with the CLN2 promoter was unaffected, consistent with our gene expression analysis (Figure 5). Our findings are supported by a recent study that identified Bck2 as a constituent of DNA-bound complexes containing Mcm1 [52]. We conclude that Bck2 localizes to the promoters of CLN3 and SWI4 in a manner that depends on ECB elements.


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 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.
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Related In: Results  -  Collection

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

pgen-1003507-g006: 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.
Mentions: Since Bck2 functions through ECB elements (Figure 5) and physically interacts with Mcm1 (Figure 2), we next asked if Bck2 localized to the promoter regions of M/G1-phase genes. We first used chromatin immunoprecipitation (ChIP) with a strain carrying a TAP-tagged allele of Bck2 to assess association of Bck2 with various promoters. We detected a reproducible enrichment of promoter DNA in the Bck2 ChIP, but the signal was very low relative to Swi4 or Mcm1 ChIPs (data not shown). To improve our assay, we repeated the ChIP experiment using a strain in which a FLAG-tagged derivative of Bck2 was conditionally overproduced (Figure 6A). Under inducing conditions (galactose), Bck2-FLAG IPs were enriched in CLN2, CLN3 and SWI4 promoter DNA relative to non-inducing conditions (raffinose) (Figure 6A) or vector control (data not shown). The enhanced enrichment of CLN3 promoter DNA compared to SWI4 promoter DNA in Bck2 IPs likely reflects the presence of more ECB elements in the CLN3 promoter (6 versus 1). Association of Bck2 with the CLN3 and SWI4 promoters was entirely dependent on the presence of ECB elements, while association with the CLN2 promoter was unaffected, consistent with our gene expression analysis (Figure 5). Our findings are supported by a recent study that identified Bck2 as a constituent of DNA-bound complexes containing Mcm1 [52]. We conclude that Bck2 localizes to the promoters of CLN3 and SWI4 in a manner that depends on ECB elements.

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