Transcriptional regulatory network for sexual differentiation in fission yeast

Abstract

Background: Changes in gene expression are hallmarks of cellular differentiation. Sexual differentiation in fission yeast (Schizosaccharomyces pombe) provides a model system for gene expression programs accompanying and driving cellular specialization. The expression of hundreds of genes is modulated in successive waves during meiosis and sporulation in S. pombe, and several known transcription factors are critical for these processes.

Results: We used DNA microarrays to investigate meiotic gene regulation by examining transcriptomes after genetic perturbations (gene deletion and/or overexpression) of rep1, mei4, atf21 and atf31, which encode known transcription factors controlling sexual differentiation. This analysis reveals target genes at a genome-wide scale and uncovers combinatorial control by Atf21p and Atf31p. We also studied two transcription factors not previously implicated in sexual differentiation whose meiotic induction depended on Mei4p: Rsv2p induces stress-related genes during spore formation, while Rsv1p represses glucose-metabolism genes. Our data further reveal negative feedback interactions: both Rep1p and Mei4p not only activate specific gene expression waves (early and middle genes, respectively) but are also required for repression of genes induced in the previous waves (Ste11p-dependent and early genes, respectively).

Conclusion: These data give insight into regulatory principles controlling the extensive gene expression program driving sexual differentiation and highlight sophisticated interactions and combinatorial control among transcription factors. Besides triggering simultaneous expression of gene waves, transcription factors also repress genes in the previous wave and induce other factors that in turn regulate a subsequent wave. These dependencies ensure an ordered and timely succession of transcriptional waves during cellular differentiation.

Figure 1

Meiotic gene expression program in rep1Δ and mei4Δ mutants. (a) Left: hierarchical cluster analysis with columns representing experimental time points and rows representing early genes. Vegetatively growing cells (V) are synchronized in G1 by nitrogen removal and enter meiosis by temperature shift at time 0. The mRNA levels at each time point of pat1(from [4]) and rep1Δ pat1 time courses relative to the levels in vegetative cells are color-coded as indicated at the bottom with missing data in gray. Rep1p-independent and -dependent genes are indicated at right as drawn-out or dashed blue lines, respectively. Right: average expression profiles of Rep1p-dependent and Rep1p-independent genes in wild-type (pat1-synchronized cells from [4]) and rep1Δ cells. (b) Average expression profiles of the four main gene clusters upregulated during pat1-induced meiosis: nitrogen-starvation response (black), early (blue), middle (red) and late (green), as defined in [4]. Experimental details are as in (a). The y-axis shows the expression level at the corresponding time point relative to expression in vegetative cells. Graphs from left to right: pat1 cells (from [4]) and mei4Δ pat1 cells. (c) Hierarchical cluster analysis with columns representing experimental time points and rows representing genes. The mRNA levels at each time point of pat1 time courses relative to the levels in vegetative cells are color-coded as indicated in (a). The last column (OE) shows the expression level in cells overproducing Mei4p relative to an empty vector control. The four gene clusters are indicated on the left.

Figure 2

Atf21p and Atf31p cooperate to induce expression of meiotic genes. Effects of overproduction of (a) Atf21p, (b) Atf31p, and (c) both Atf21p and Atf31p. The Venn diagrams on the left show the overlaps between genes induced by overproduction of the different transcription factors (OE) and potential meiotic Atf21p target genes (obtained by analysis of atf21Δ cells [4]). The numbers in brackets show the overlap between the two lists expected by chance given the sizes of the gene sets considered and the total number of genes. The overlap between Atf31p-induced genes and meiotic Atf21p targets is not significant, while the overlaps between Atf21p and Atf21p/Atf31p-induced genes and Atf21p targets are both significant (P = 2.3 × 10^-3 and 1.5 × 10^-62, respectively; note, however, that only four Atf21p targets are induced by Atf21p overexpression). The panels on the right show the meiotic expression profiles of genes induced by the overproduction of the corresponding transcription factors (pat1-synchronized cells from [4], experiment and labeling as in Figure 1). The profiles of atf21 and atf31 are highlighted in blue and green, respectively.

Figure 3

Identification of Rsv2p-dependent genes. (a) Expression profile of rsv2 during meiosis and sporulation (from [4], experiment and labeling as in Figure 1). (b) Comparison of gene expression levels between wild-type and rsv2Δ meiotic cells. Genes outside the blue lines differ by more than two-fold in expression levels. rsv2 is indicated by an arrow. (c) Expression profiles of the potential Rsv2p-dependent genes identified in (b) (pat1-synchronized cells from [4]).

Figure 4

Identification of Rsv1p-dependent genes. (a) Expression profile of rsv1 during meiosis and sporulation (experiment and labeling as in Figure 1). (b) Comparison of gene expression levels between wild-type and rsv1Δ meiotic cells. Genes outside the blue lines differ by more than two-fold in expression levels. rsv1 is indicated by an arrow. (c) Expression profiles of the potential Rsv1p-dependent genes identified in (b) (pat1-synchronized cells from [4]).

Figure 5

Transcription factor genes regulated by Mei4p. Expression profiles of genes encoding transcription factors in pat1-synchronized wild-type (blue lines, data from [4]) and mei4Δ (red lines) cells: (a) atf21, (b) atf31, (c) rsv1 and (d) rsv2. Experiments and labeling are as in Figure 1.

Figure 6

Rep1p is required to down-regulate a subset of nitrogen starvation-induced genes. (a) Average expression profiles in wild-type (pat1-synchronized cells from [4]) and rep1Δ cells of two subclusters of genes induced in response to nitrogen starvation: delayed (blue) and transient (red), as defined by [4]. (b) Hierarchical cluster analysis of the two gene clusters shown in (a), with columns representing experimental time points and rows representing genes. The mRNA levels at each time point of pat1 time courses relative to the levels in vegetative cells are color-coded as indicated at the bottom with missing data in gray. Labeling is as in Figure 1. (c) Histogram showing the gene expression levels of rep1Δ relative to wild-type meiotic cells. The two clusters are colored as in (a).

Figure 7

Model for transcriptional regulatory network controlling meiosis and sporulation. Arrows indicate activation and cross bars indicate repression. The colors reflect the different stages of sexual differentiation as in Figure 1. Mei4p controls transcription of its own gene in a positive feedback [14], but no other transcription factors have been identified that trigger the initial induction of mei4. This activation could be achieved at the posttranscriptional level [7]. Refer to the main text for further details on interactions.