Regulation of Ace2-dependent genes requires components of the PBF complex in Schizosaccharomyces pombe

Abstract

The division cycle of unicellular yeasts is completed with the activation of a cell separation program that results in the dissolution of the septum assembled during cytokinesis between the 2 daughter cells, allowing them to become independent entities. Expression of the eng1(+) and agn1(+) genes, encoding the hydrolytic enzymes responsible for septum degradation, is activated at the end of each cell cycle by the transcription factor Ace2. Periodic ace2(+) expression is regulated by the transcriptional complex PBF (PCB Binding Factor), composed of the forkhead-like proteins Sep1 and Fkh2 and the MADS box-like protein Mbx1. In this report, we show that Ace2-dependent genes contain several combinations of motifs for Ace2 and PBF binding in their promoters. Thus, Ace2, Fkh2 and Sep1 were found to bind in vivo to the eng1(+) promoter. Ace2 binding was coincident with maximum level of eng1(+) expression, whereas Fkh2 binding was maximal when mRNA levels were low, supporting the notion that they play opposing roles. In addition, we found that the expression of eng1(+) and agn1(+) was differentially affected by mutations in PBF components. Interestingly, agn1(+) was a major target of Mbx1, since its ectopic expression resulted in the suppression of Mbx1 deletion phenotypes. Our results reveal a complex regulation system through which the transcription factors Ace2, Fkh2, Sep1 and Mbx1 in combination control the expression of the genes involved in separation at the end of the cell division cycle.

Keywords: cell cycle regulation; cell separation; forkhead; transcription.

Figure 1.

Ace2 regulates the expression of its target genes by periodically binding the CCAGCC sequence. (A) Schematic representation of the position of the 2 copies of the CCAGCC sequence in the eng1⁺ promoter and constructs generated. The different deletions were cloned upstream of the E. coli lacZ gene. The arrow indicates the orientation of the CCAGCC sequence. The graph shows the results of the β-galactosidase activity assay in the wild-type strain (PN1870) carrying the wild-type eng1⁺ promoter (WT; pMAN4), eng1-Δ1 (pMAN5), eng1-Δ2 (pMAN6), eng1-Δ1Δ2 (pMAN7) or empty vector and in the ace2Δ mutant (OL163) transformed with the wild-type eng1⁺ promoter (WT; pMAN4). The data are means (±SD ) of 2 independent experiments and are normalized to the wild-type strain. (B) Chromatin immunoprecipitation of Ace2-HA in a strain carrying the wild-type eng1⁺ promoter (WT; YMAT15) and in a strain carrying the deletion of the putative Ace2-binding sites in the eng1⁺ promoter (eng1-Δ1Δ2; YMAT85). Data are shown relative to the background binding in untagged control cells (OL264). The binding of Ace2 to the his3⁺ promoter was used as a negative control to normalize the data. The columns represent the mean of 2 independent biological repeats, indicated by dots. Each dot is the mean of at least 2 technical replicates. (C) eng1⁺ expression level in the wild-type strain (WT, YMAT15) and in the eng1-Δ1Δ2 (YMAT85) and ace2Δ (YMAT71) mutants determined by quantitative RT-PCR and normalized using his3⁺ expression. The columns represent the mean of 2 independent biological repeats, indicated by dots. Each dot is the mean of at least 2 technical replicates. (D) Expression of eng1⁺ and agn1⁺ in synchronized cells. Data were normalized using his3⁺ expression. The results shown are representative of the results obtained in 2 different experiments. (E) Ace2 binding to the promoters of eng1⁺ and eng1⁺ along the cell cycle. A cdc25–22 ace2-HA strain (YMAT15) was synchronized by arrest-release and samples were taken at the indicated times (minutes) after the release for mRNA purification (D) or chromatin immunoprecipitation (E) using anti-HA antibodies. The anaphase and septation indices are indicated in E. Data are shown relative to the background binding in untagged control cells (OL264). The binding of Ace2-HA to the his3⁺ promoter was used as a negative control to normalize the data. The results shown are representative of the results obtained in 2 different experiments.

Figure 2.

Diverse binding sites are present in the promoters of Ace2-dependent genes. Schematic representation of the promoter region of Ace2-dependent genes. Blue rectangles indicate the position of the CCAGCC sequence; green rectangles correspond to TGTTTA motifs, and red rectangles mark GNAACR sequences. The position of the rectangles indicates their orientation: direct if it is above the line and inverted if it is below. NDR regions in the promoters are indicated by yellow boxes. Genes were ordered according to the abundance of these sites within NDR. The asterisks indicate the sites mutated in this study (TGTTTA to CGGCTA and GNAACR to GNGCCR).

Figure 3.

PBF components bind to Ace2-target promoters in vitro and in vivo. (A) Gel retardation assay with the eng1⁺ promoter. A labeled DNA fragment corresponding to the region marked with a rectangle in the schematic representation to the left was incubated with protein extracts from the wild-type strain (PN1870, WT) and the ace2Δ (OL163), mbx1Δ (GG503), fkh2Δ (GG523), mbx1Δ fkh2Δ (GG552), ace2Δ fkh2Δ (YMAN91), ace2Δ mbx1Δ (YMAN92) and ace2Δ mbx1Δ fkh2Δ (YMAN106) mutants. The probe without protein extract (F) is also shown. (B) Chromatin immunoprecipitation of Ace2-HA, Fkh2-HA and Sep1-myc in ace2-HA (YMAT15), fkh2-HA (YMAT14) or sep1-myc (YMAT16) strains. Data are shown relative to the background binding in untagged control cells (OL264). Binding of the 3 tagged proteins to his3⁺ promoter was used as a negative control to normalize the data. (C) eng1⁺ promoter scanning. Schematic representation of the intergenic region that separates slp1⁺ and eng1⁺, in which the binding sites for Ace2 are indicated with light gray boxes and those for forkhead transcription factors with dark gray. The oligonucleotide pairs used for ChIP experiments are also indicated. Below, quantitative results of the ChIP experiments with Ace2-HA or Fkh2-HA. Data are shown relative to the background binding in untagged control cells (OL264). The binding of Ace2-HA and Fkh2-HA to the his3⁺ promoter was used as a negative control to normalize the data. For B and C, the columns represent the mean of 2 independent biological repeats, indicated by dots. Each dot represents the mean of at least 2 technical replicates.

Figure 4.

Regulation of eng1⁺ and agn1⁺ requires different factors. (A) Expression of ace2⁺ and the Ace2-target genes eng1⁺ and agn1⁺ measured by quantitative RTPCR in the wild-type strain (OL432) and the ace2Δ (YMAN30), sep1Δ (A131), fkh2Δ (GG523), mbx1Δ (GG503) and mbx1Δ fkh2Δ (GG552) mutants. his3⁺ expression was used for normalization. (B) Expression of eng1⁺ and agn1⁺ measured by quantitative RT-PCR in strains P_nmt⁺-ace2⁺ (YMAT59), mbx1Δ P_nmt1-ace2⁺ (YMAT61), sep1Δ P_nmt1-ace2⁺ (YMAT62), and fkh2Δ P_nmt1-ace2⁺ (YMAT60). The graph represents the quantification of the expression of each gene with respect to strain P_nmt1-ace2. act1⁺ expression was used for normalization. In both panels, the columns represent the mean of 2 independent biological repeats, indicated by dots. Each dot represents the mean of at least 2 technical replicates.

Figure 5.

Fkh2 binds to the promoters of Ace2-dependent genes at different moments of the cell cycle. (A) Chromatin immunoprecipitation of Fkh2-HA in the cdc25–22 fkh2-HA strain (YMAT14) (left), and expression of eng1⁺ and agn1⁺ (right) measured by quantitative RT-PCR along the cell cycle and normalized using his3⁺ expression. Synchrony was induced by arrest-release and samples were taken at the indicated times (minutes) after the release for chromatin immunoprecipitation (left) using anti-HA antibodies or for mRNA purification (right). The anaphase and septation index is indicated in the left graph. Data are shown relative to the background binding in untagged control cells (OL264). Binding of Fkh2-HA to the his3⁺ promoter was used as a negative control to normalize the data. (B) Expression of eng1⁺ determined by quantitative RT-PCR in strains nda3-KM311 Pnmt1⁺-ace2⁺ (YMAT17), and nda3-KM311 fkh2Δ Pnmt1⁺-ace2⁺ (YMAT94) along the cell cycle. Cells were arrested in early mitosis by incubation at 18°C and samples were taken at the indicated times (minutes) after release for mRNA purification. Expression data were normalized using his3⁺ expression. (C) Chromatin immunoprecipitation of Fkh2-HA and Sep1-myc in strains carrying mutations in the 3 forkhead binding sites and an adjacent PCB site in the eng1⁺ promoter (Peng1–4m allele) (strains YMAT43 and YMAT70, respectively). Data are shown relative to the binding in wild-type cells (YMAT14 and YMAT69, respectively). The binding of Fkh2-HA and Sep1-myc to the his3⁺ promoter was used as a negative control to normalize the data. The columns represent the mean of 2 independent biological repeats, indicated by dots. Each dot represents the mean of at least 2 technical replicates. (D) Expression of eng1⁺ and agn1⁺ during the cell cycle. Synchrony was induced by arrest-release of cdc25–22 (YMAT14) or cdc25–22 Peng1–4m (YMAT43) mutants, and samples were taken at the indicated times (minutes) after release for RNA extraction. Expression was measured by quantitative RT-PCR and normalized using his3⁺ expression. For the time course experiments (A, B and D), the results shown are representative of the results obtained in 2 different experiments.

Figure 6.

Mbx1 controls agn1⁺ expression. (A) Expression of eng1⁺ and agn1⁺ during the cell cycle. Synchrony was induced by arrest-release of cdc25–22 (OL264) or cdc25–22 mbx1Δ (GG549) mutants, and samples were taken at the indicated times (minutes) after release for RNA extraction. RNA blots were probed with specific probes for eng1⁺ and agn1⁺, using act1⁺ as a loading control. The percentage of septation at each time-point is indicated below, and was determined by counting the percentage of cells with a septum after calcofluor staining. The graph represents the quantification of the expression of each gene with respect to the wild-type (wt, value 1). (B) Overexpression of agn1⁺ complements the separation defects of mbx1Δ mutants. The wild-type (WT; OL432) and the agn1Δ (YSAB156), mbx1Δ (GG503) and mbx1Δ carrying Pnmt1⁺-agn1⁺ (YMAT91) mutants were grown in EMM5S medium without thiamine for 17 hours before staining the cells with aniline blue. Images show fields and details of separating cells for each strain. The graph to the right indicates the percentage of cells with a septum that have a wild-type or a V-shaped phenotype in each strain (n = 350).