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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-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.
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pgen-1003507-g002: 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.

Mentions: We used the ORFeome Y2H screening method [35] to discover potential Bck2-interacting proteins. We identified six proteins that interacted with Bck2: Mcm1, Yap6, Tpd3, Std1, Mth1, and Mot3 (Figure 2 and data not shown). With the exception of Tpd3, all of these proteins are transcriptional regulators that act proximal to DNA. As noted in the introduction, Mcm1 is a member of a class of MADS box transcription factors found in all eukaryotic organisms [36]–[38] and we explore the Mcm1-Bck2 interaction in detail below. Four of the other Bck2 partners we identified in our screen were also DNA binding proteins that had roles in ion homeostasis and nutrient sensing: (1) Yap6 is a basic leucine zipper (bZIP) transcription factor that activates a number of genes involved in sodium and lithium tolerance [39], [40]; (2) Std1 and Mth1 are both controllers of glucose-regulated gene expression [41] and are required for transcriptional repression of HXT (hexose transport) genes [42], [43]; (3) Mot3 is a Zn-finger transcription factor that activates a number of cell wall genes [44], and represses transcription of the DAN/TIR group of genes that encode cell wall mannoproteins during anaerobic growth [44]. The only protein that is not a transcription factor, Tpd3, is the scaffold subunit A of the heterotrimeric protein phosphatase 2A (PP2A) [45], which has roles at several points in the cell cycle and is involved in the TOR pathway for nutrient sensing [46]. We did not identify SWI4 or MBP1 in our Y2H screen, but a direct test revealed a weak interaction between Bck2 and Swi4 (Figure 2). The identification of genes with known roles in transcription and nutrient response is consistent with the apparent functions of Bck2, and validates our two-hybrid screen as a tool for identifying Bck2-interacting proteins.


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-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.
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pgen-1003507-g002: 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.
Mentions: We used the ORFeome Y2H screening method [35] to discover potential Bck2-interacting proteins. We identified six proteins that interacted with Bck2: Mcm1, Yap6, Tpd3, Std1, Mth1, and Mot3 (Figure 2 and data not shown). With the exception of Tpd3, all of these proteins are transcriptional regulators that act proximal to DNA. As noted in the introduction, Mcm1 is a member of a class of MADS box transcription factors found in all eukaryotic organisms [36]–[38] and we explore the Mcm1-Bck2 interaction in detail below. Four of the other Bck2 partners we identified in our screen were also DNA binding proteins that had roles in ion homeostasis and nutrient sensing: (1) Yap6 is a basic leucine zipper (bZIP) transcription factor that activates a number of genes involved in sodium and lithium tolerance [39], [40]; (2) Std1 and Mth1 are both controllers of glucose-regulated gene expression [41] and are required for transcriptional repression of HXT (hexose transport) genes [42], [43]; (3) Mot3 is a Zn-finger transcription factor that activates a number of cell wall genes [44], and represses transcription of the DAN/TIR group of genes that encode cell wall mannoproteins during anaerobic growth [44]. The only protein that is not a transcription factor, Tpd3, is the scaffold subunit A of the heterotrimeric protein phosphatase 2A (PP2A) [45], which has roles at several points in the cell cycle and is involved in the TOR pathway for nutrient sensing [46]. We did not identify SWI4 or MBP1 in our Y2H screen, but a direct test revealed a weak interaction between Bck2 and Swi4 (Figure 2). The identification of genes with known roles in transcription and nutrient response is consistent with the apparent functions of Bck2, and validates our two-hybrid screen as a tool for identifying Bck2-interacting proteins.

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