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. 2005 Mar;169(3):1329-42.
doi: 10.1534/genetics.104.036103. Epub 2005 Jan 16.

Meiosis-specific regulation of the Saccharomyces cerevisiae S-phase cyclin CLB5 is dependent on MluI cell cycle box (MCB) elements in its promoter but is independent of MCB-binding factor activity

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Meiosis-specific regulation of the Saccharomyces cerevisiae S-phase cyclin CLB5 is dependent on MluI cell cycle box (MCB) elements in its promoter but is independent of MCB-binding factor activity

Sheetal A Raithatha et al. Genetics. 2005 Mar.

Abstract

In proliferating S. cerevisiae, genes whose products function in DNA replication are regulated by the MBF transcription factor composed of Mbp1 and Swi6 that binds to consensus MCB sequences in target promoters. We find that during meiotic development a subset of DNA replication genes exemplified by TMP1 and RNR1 are regulated by Mbp1. Deletion of Mbp1 deregulated TMP1 and RNR1 but did not interfere with premeiotic S-phase, meiotic recombination, or spore formation. Surprisingly, deletion of MBP1 had no effect on the expression of CLB5, which is purportedly controlled by MBF. Extensive analysis of the CLB5 promoter revealed that the gene is largely regulated by elements within a 100-bp fragment containing a cluster of MCB sequences. Surprisingly, induction of the CLB5 promoter requires MCB sequences, but not Mbp1, implying that another MCB-binding factor may exist in cells undergoing meiosis. In addition, full activation of CLB5 during meiosis requires Clb5 activity, suggesting that CLB5 may be regulated by a positive feedback mechanism. We further demonstrate that during meiosis MCBs function as effective transcriptional activators independent of MBP1.

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Figures

F<sc>igure</sc> 1.—
Figure 1.—
CLB5 expression during meiosis is independent of MBP1. (A) Homozygous diploid cells with the deletion of the gene or genes indicated at the left were induced to enter meiosis and RNA from samples collected at the indicated time points was analyzed by Northern blot for CLB5 or ACT1 mRNA. (B) RNA from the wild-type strain in A was probed for MBP1 and ACT1 mRNA. (C) Homozygous diploid cells with tagged alleles of the transcription factor indicated at the left were induced to enter meiosis, and protein samples isolated at each time point were probed by Western blot for Mbp1-Myc or Ndt80-HA. The same samples were probed for Cdc28 as a loading control. Swi4HA was isolated by immunoprecipitation from extracts made from equal numbers of cells at each of the indicated time points. The immune complexes were probed by Western blot for the HA epitope. A nonspecific background HA reactive species is indicated by an asterisk.
F<sc>igure</sc> 2.—
Figure 2.—
MBP1 is not required for effective completion of S-phase or homologous recombination but does control some MCB-regulated genes during meiotic development. (A) DNA content of homozygous MBP1 NDT80, mbp1 NDT80, and MBP1 ndt80 cells was monitored by FACS during meiotic progression. (B) Homologous recombination in mbp1 (•) or MBP1 (▪) homozygous diploids was measured by the appearance of Arg+ or His+ recombinants. (C) MBP1 and mbp1 homozygous diploids were induced to enter meiosis and RNA collected at the indicated time points was analyzed by Northern blot for the mRNAs indicated at the left.
F<sc>igure</sc> 3.—
Figure 3.—
A 100-bp region of the CLB5 promoter provides the majority of regulation to CLB5 transcription during meiosis. Schematics of the wild-type and mutant CLB5 promoters are shown at the left. (▪) Putative MCB elements; (□) putative MSE sequence elements. The position of each element relative to the initiating codon of CLB5 is indicated below each box in the wild-type CLB5 promoter. Homozygous wild-type diploid cells carrying the clb5 reporter gene construct indicated at the left were induced to enter meiosis and RNA samples collected at the indicated time points were probed by Northern blot for CLB5, SPS1, and ACT1 mRNA. The reporter gene (clb5) migrates more slowly than the endogenous CLB5 gene (CLB5). (A) Wild-type promoter. (B) Δ179 promoter, which has a deletion from −222 to −398 relative to the start codon. (C) Δ178 promoter, which has a deletion from −298 to −398. (D) Δmse, promoter which has a mutation in the MSE located at position −230. (E) Δmcb5 promoter, which has mutations in the MCB sequences at −389, −354, −337, −315, and −248.
F<sc>igure</sc> 4.—
Figure 4.—
Mutation of MCBs in the CLB5 promoter reduces CLB5 mRNA and associated kinase activity. (A) clb5 clb6 homozygous diploids carrying a CLB5 open reading frame under the regulation of a wild-type or mutant CLB5 promoter (indicated at the left) were induced to enter meiosis and RNA collected at the indicated time points was probed by Northern blot for CLB5 and ACT1 mRNA. (B) clb5 clb6 homozygous diploid cells carrying a CLB5 open reading frame under the regulation of a CLB5, Δ178, Δmcb4, or Δmcb5 promoter were induced to enter meiosis and RNA collected at the indicated time points was probed for CLB5 and ACT1. All the samples were separated on a single gel and the Northern blot signals were quantitated by a phosphorimager; the relative CLB5/ACT1 mRNA are displayed as a histogram. (C) Histone H1 kinase activity associated with Clb5 expressed in clb5 clb6 diploids that carry a CLB5-HA open reading frame regulated by a CLB5, Δmcb4, or Δmcb5 promoter. Samples were taken from clb5 clb6 cells carrying an untagged construct (“No Tag”) from asynchronously growing cells (“Asynch”) or at the indicated time points from cells progressing through meiosis. (D) FACS analysis during synchronous meiotic development of homozygous clb5 clb6 diploids that carry a CLB5-HA open reading frame under the regulation of a CLB5, Δmcb4, or Δmcb5 promoter.
F<sc>igure</sc> 5.—
Figure 5.—
Mutation of the MCBs in the CLB5 promoter affect cell morphology and sporulation efficiency. Cells with the indicated genotype carrying the indicated construct were grown in YEPD; photographs were taken from representative cells in the asynchronously growing populations. (A) Wild-type CLB5 CLB6 diploids. (B) clb5 clb6 diploids transformed with an empty vector. (C) clb5 clb6 diploids transformed with CLB5 regulated by a wild-type promoter. (D) clb5 clb6 diploids transformed with CLB5 regulated by a Δ178 promoter. (E) clb5 clb6 diploids transformed with CLB5 regulated by a Δmcb4 promoter. (F) clb5 clb6 diploids transformed with CLB5 regulated by a Δmcb5 promoter. The cell volume of cultures of the indicated diploid strains growing asynchronously in YEPD medium is indicated below each image. (G) clb5 clb6 homozygous diploids carrying a CLB5 open reading frame under the regulation of a CLB5 promoter or the indicated mutant promoter were induced to enter meiosis. After 24 hr the percentage of cells that had completed the two meiotic divisions (indicated by more than two DAPI-staining chromatin masses) was scored.
F<sc>igure</sc> 6.—
Figure 6.—
MCB sequences are sufficient to drive transcription during meiosis. (A) Homozygous clb5 clb6 MBP1 diploids (open symbols) or clb5 clb6 mbp1 diploids (solid symbols) carrying a CLB5 open reading frame regulated by wild-type MCB sequences CGCG (□, ▪) or a mutant MCB sequence CGAG (○, •) were induced to enter meiosis and RNA collected at the indicated time points was probed by Northern blot for CLB5 and ACT1. The abundance of CLB5 is presented as a ratio of CLB5/ACT1. (B) Homozygous clb5 clb6 MBP1 and clb5 clb6 mbp1 diploids carrying a CLB5 open reading frame regulated by a wild-type MCB (CGCG) or a mutant MCB (CGAG) were induced to enter meiosis and after 24 hr the cultures were microscopically examined for the percentage of cells forming asci.

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