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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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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).
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pgen-1003507-g001: 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).

Mentions: We reasoned that an exploration of Bck2-interacting proteins might illuminate the function of Bck2 in G1 progression. Bck2 is difficult to work with biochemically (for example endogenous Bck2 is undetectable by Western blot [16], [32, and data not shown], so we turned to the yeast two-hybrid (Y2H) system for our protein interaction screens. Bck2 has a potent transcriptional activation domain, and activates reporter gene expression when fused to the Gal4 DNA binding domain (DBD) in a Y2H reporter strain [33] (Figure 1), a property that has precluded identification of Bck2 binding partners using the Y2H screening method. To discover Bck2 derivatives that might be useful for two-hybrid screening, we created 20 truncations of the BCK2 gene and fused them to the GAL4 DBD (Figure 1). We first assessed the ability of each truncation construct to activate transcription of two reporter genes, in which the GAL4 UAS is upstream of either lacZ (Figure S1) or ADE2 (Figure S1B). Bck2 residues 662 to 851 were required for transcriptional activation (fragments 11–20), while the Bck2 N-terminal region had some apparent inhibitory activity (Figure S1A), as suggested by our finding that deletions of the N-terminus resulted in significant increases in reporter gene expression. A construct containing fragment 5, lacking the first 529 amino acids of Bck2, was the most potent Y2H auto-activator in the lacZ reporter assay (Figure S1A).


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003507-g001: 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).
Mentions: We reasoned that an exploration of Bck2-interacting proteins might illuminate the function of Bck2 in G1 progression. Bck2 is difficult to work with biochemically (for example endogenous Bck2 is undetectable by Western blot [16], [32, and data not shown], so we turned to the yeast two-hybrid (Y2H) system for our protein interaction screens. Bck2 has a potent transcriptional activation domain, and activates reporter gene expression when fused to the Gal4 DNA binding domain (DBD) in a Y2H reporter strain [33] (Figure 1), a property that has precluded identification of Bck2 binding partners using the Y2H screening method. To discover Bck2 derivatives that might be useful for two-hybrid screening, we created 20 truncations of the BCK2 gene and fused them to the GAL4 DBD (Figure 1). We first assessed the ability of each truncation construct to activate transcription of two reporter genes, in which the GAL4 UAS is upstream of either lacZ (Figure S1) or ADE2 (Figure S1B). Bck2 residues 662 to 851 were required for transcriptional activation (fragments 11–20), while the Bck2 N-terminal region had some apparent inhibitory activity (Figure S1A), as suggested by our finding that deletions of the N-terminus resulted in significant increases in reporter gene expression. A construct containing fragment 5, lacking the first 529 amino acids of Bck2, was the most potent Y2H auto-activator in the lacZ reporter assay (Figure S1A).

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