Mapping transcriptional-regulatory networks requires the identification of target genes, binding specificities and signalling pathways of transcription factors. However, the characterization of each transcription factor sufficiently for deciphering such networks remains laborious. The recent availability of overexpression and deletion strains for almost all of the transcription factor genes in the fission yeast Schizosaccharomyces pombe provides a valuable resource to better investigate transcription factors using systematic genetics. In the present paper, I review and discuss the utility of these strain collections combined with transcriptome profiling and genome-wide chromatin immunoprecipitation to identify the target genes of transcription factors.
Transcriptional-regulatory networks determine the global gene expression profiles that give rise to specific cellular processes and phenotypes. Central to the network are sequence-specific transcription factors (referred to as transcription factors hereinafter) that control the expression of downstream target genes and are themselves regulated by upstream signalling pathways. A major objective is to fully map these networks of various model organisms in order to obtain a comprehensive systems view of specific developmental programmes and disease states. However, achieving this in any organism remains elusive as it requires the elucidation of a vast number of transcription factor–target gene interactions.
The fission yeast Schizosaccharomyces pombe is an ideal model system for deciphering transcriptional-regulatory networks of an organism. Its transcriptional-regulatory network that governs all fundamental eukaryotic cellular processes is relatively small, consisting of approximately 100 transcription factors controlling the expression of 5200 genes [1–3]. Moreover, S. pombe possesses the functional genomic tools such as microarray expression profiling and ChIP-chip (chromatin immunoprecipitation on chip) that allow for rapid characterization of transcription factors [4–6]. In the present mini-review, I describe our research with S. pombe transcription factors and the use of systematic deletion and overexpression analyses with these microarray-based approaches to identify their direct target genes.
S. pombe transcription factors: DNA-binding domains and biological functions
One of the first tasks in our endeavour to map the transcriptional-regulatory network of S. pombe was to determine the actual number of transcription factor genes in the genome. We searched for gene products that primarily contained DNA-binding domains characteristic of transcription factors and removed those that appeared to function in general transcription and other cellular processes. Our list consisted of 99 transcription factor genes (~1.9% of encoding genes), which is proportionally less than other ascomycete genomes such as Yarrowia lipolytica (2.3%), Saccharomyces cerevisiae (2.6%), Candida albicans (2.9%) and Aspergillus nudilans (3.7%) [3,7]. Since the number of coding genes is also lower in S. pombe compared with these yeasts, fewer transcription factors would have to be analysed to map the entire network of this organism. Among the 99 transcription factors, the most common class of DNA-binding domains is the fungal C6 zinc finger (30), followed by the C2H2 finger (18), bZIP (basic leucine zipper) (6) and HMG box (5). Other DNA-binding domain classes include forkhead/FHA (5), bHLH (4), GATA (4), APSES (4), MADS box/SRF (3), CBF/NF-Y (3), homeobox (2), Myb/SANT (2), HSF-type (2), copper fist (2), CBF/LAG1 (2) and RFX-type (1) . Of these, only the CBF/LAG1 DNA-binding domain is not found in S. cerevisiae . The remaining proteins did not have a defined DNA-binding domain, but were classified as transcription factors by sequence homology. Searching in Pombase revealed that 68 of the 99 transcription factor genes have been assigned some biological role, but the remaining 31 have no known function . For the former, the biological processes include mating/meiosis (17.2%), cell cycle regulation (16.2%), metabolism (11.1%), stress response (8.1%), ion homoeostasis (4.0%) and flocculation (3.0%). However, only a small proportion of target genes have been identified for many of these transcription factors. Therefore much remains to be done in order to complete the mapping of the S. pombe transcriptional-regulatory network.
Transcription factor deletion strains: complications and solutions in identifying target genes
In S. pombe, the proportion of essential genes encoding transcription factors under optimal growth conditions is 9.1%, substantially less than the overall essentiality in its genome (26.1%) [7,9]. To identify target genes, we constructed haploid deletions of the 91 non-essential transcription factor genes for transcriptome profiling. The target genes of transcriptional activators and repressors could be potentially identified by down-regulated and up-regulated transcripts respectively in the deletion strains relative to wild-type. One complication with this approach is that only ten of the 91 transcription factor haploid deletions displayed significant differences in their generation times compared with the wild-type in rich medium . These observations suggest that most transcription factors are probably not required or active under these conditions. Therefore microarray expression profiling of these deletion strains in rich medium would probably not reveal the target genes. Consistent with this hypothesis, we found that microarray expression profiling of deletion strains of 15 uncharacterized transcription factor genes that do not exhibit a growth defect in rich medium showed few differentially expressed genes relative to wild-type (G. Chua, unpublished work). In S. cerevisiae, comparable difficulties to identify target genes have also been encountered with transcriptome studies of transcription factor deletion mutants, as well as ChIP-chip analysis of condition-specific transcription factors in rich medium [6,10–12]. Despite these obstacles, target genes were identified for a few S. pombe transcription factors by microarray expression profiling the deletion strain or loss-of-function conditional alleles in rich medium. These transcription factors functioned primarily in the vegetative cell cycle and included Sep1, Ace2, Yox1 and Cdc10 [13–16]. The absence of phenotypes in single deletions of transcription factor genes could also be attributed to functional redundancy between transcription factor pairs. However, this is unlikely to be a major cause. Synthetic genetic array analysis in S. pombe has examined approximately 25% of all possible transcription factor double mutants under optimal growth conditions, and the frequency of negative genetic interactions is estimated to be 3–6% [17,18].
These results demonstrate the importance of finding the physiological conditions that induce transcription factor activity in order to identify the target genes. This could be potentially accomplished by screening the transcription factor deletion strains for hypersensitivity to various environmental perturbations. The hypersensitivity of the deletion strain would indicate that transcription factor activity is required in the presence of the perturbation. Therefore target genes could be revealed by comparing transcriptomes of the transcription factor deletion strain and wild-type in the presence of the perturbation. This strategy was successful in identifying the target genes of Zip1 and Atf1 by expression microarrays under conditions (cadmium and osmotic stress respectively) that caused hypersensitivity in the deletion strain [19,20]. Although this appears straightforward in principle, the target genes of only a few condition-specific transcription factors have been identified in this way. One limitation is that only a small number of hypersensitivity by transcription factor deletion mutants has been recovered from genome-wide chemical screens so far. Furthermore, extensive chemical genetic profiling in S. cerevisiae has suggested that hypersensitivity of the deletion strain is usually not indicative of gene activity, which could also be the case in S. pombe [21,22].
Several other approaches have been successful in elucidating the inducing conditions of S. pombe transcription factors. The initial discovery of interesting phenotypes in mei4∆ and pho7∆ strains indicated that these transcription factors were active during meiosis and phosphate starvation respectively [23,24]. In addition, the activity of certain transcription factors could be inferred from their periodic mRNA expression obtained from time-course transcriptomes of synchronized mitotic and meiotic wild-type cells . These genome-wide studies identified functions for Yox1 and Atf21/Atf31 in S-phase and late meiosis respectively [13,25,26]. For some transcription factors, their function and condition-specific activity were discovered through sequence homology. For example, Sre1 and Cuf1 are functional homologues of the human SREBP (sterol-regulatory-element-binding protein) and S. cerevisiae Mac1p and Ace2p copper fist transcription factors respectively [27–29]. From their homology with these transcription factors, Sre1 and Cuf1 activity was shown to be dependent on low levels of sterol and copper respectively [27,29]. The inducing conditions of a transcription factor could also be determined if the biological function of the target genes is known. Fep1 was initially discovered in a search for transcription factor candidates that bound to cis-regulatory GATA-like sequences within the promoters of several iron-starvation genes . On the basis of the known biological roles of these target genes, Fep1 was shown to specifically function in the presence of external iron. Transcriptome profiling of several of these transcription factor deletion mutants under the inducing conditions uncovered from these approaches have been effective in identifying their target genes.
Systematic transcription factor overexpression: identifying target genes by phenotypic activation
Our deletion mutant studies revealed that determination of the inducing conditions for most S. pombe transcription factors is necessary to identify their target genes. However, accomplishing this task is not trivial. Overexpression of transcription factors has the potential to promote their activity without the need to elucidate the specific inducing conditions. In S. cerevisiae, 32.6% of transcription factor genes were detrimental to growth when overexpressed under the GAL1/10 promoter . These observations suggested that the growth defect from overexpression could be caused by inappropriate induction of transcription factor activity and aberrant expression of target genes (known as ‘phenotypic activation’) . Indeed, transcriptome profiling these toxic overexpression strains identified known target genes and binding specificities in the majority of well-characterized transcription factors . In contrast, similar experimentation on the non-toxic transcription factor overexpressors did not yield any positive results .
On the basis of the promising results in S. cerevisiae, we conducted systematic overexpression studies of 99 transcription factor genes in S. pombe . Some 64 transcription factor genes caused reduced fitness when overexpressed under the nmt1 promoter . Of these transcription factor genes, 20 were not named and remained uncharacterized. The frequency of toxic transcription factor overexpressors was double that of S. cerevisiae and was probably due to differences in fitness scoring and promoter strength [7,31]. In addition, almost half of S. pombe transcription factors displayed cell elongation when overexpressed, suggesting potential roles in the cell cycle . These included most transcription factors annotated as a regulator of transcription during mitosis (Cdc10, Res1, Res2, Rep1, Fkh2, Ams2 and Mbx1; GO:0045896) [1,7].
Transcriptome and ChIP-chip profiling of several transcription factor overexpression strains were successful in identifying their target genes. We discovered novel cell cycle transcription factors, including Toe1 and Toe3, which positively regulate target genes implicated in pyrimidine salvage and polyamine synthesis respectively . Moreover, profiling several transcription factor-overexpression strains that exhibited constitutive flocculation, as well as reduced fitness identified target genes encoding flocculins and cell wall-remodelling enzymes . These studies also uncovered a flocculation transcriptional-regulatory network that contained various network motifs such as positive autoregulation of mbx2+ and cbf12+, negative autoregulation of rfl1+ and regulation of the flocculin target gene pfl1+/gsf2+ by an inhibitory feedforward loop involving the opposing transcription factors Mbx2 and Rfl1/Gsf1 [33,34].
Why are target genes detected for only certain overexpressed transcription factors by these microarray-based approaches? The most likely reason is that overexpression of these transcription factors induces the regulation of target genes by increasing promoter occupancy. For example, pheromone response and meiotic genes are induced in vegetative cells when ste11+ and mei4+ are overexpressed respectively [26,35,36]. Other transcription factors such as Pap1 are retained in the cytoplasm, but translocate into the nucleus in response to the inducing condition . Pap1 overexpression transcriptionally activates target genes probably by promoting entry into the nucleus through saturation of the nuclear import machinery. In contrast, target genes are not revealed by the single overexpression of atf21+, atf31+ and sre1+. Atf21 and Atf31 are bZIP transcription factors that heterodimerize and only their co-overexpression induces their meiotic target genes . Sre1 is an endoplasmic reticulum membrane-bound transcription factor that undergoes proteolytic processing and activation in response to ergosterol and oxygen deprivation . Therefore overexpression of full-length Sre1 under non-inducing conditions does not result in activation of its target genes, as the transcription factor remains attached to the endoplasmic reticulum membrane. Altogether, these results indicate that the target genes of a considerable number of S. pombe transcription factors can potentially be identified by overexpression analysis with transcriptome and ChIP-chip profiling.
Validation of putative target genes by genetic perturbation
Putative target genes identified from these transcription factor overexpression studies can be validated further by several genetic approaches. If it is presumed that the transcription factor is a positive regulator and the overexpression phenotype is caused by inappropriate induction of its target genes, then we anticipate that the transcription factor overexpression phenotype could be: (i) recapitulated by the ectopic expression of the target gene, and (ii) suppressed by deletion of the target gene. We found that several putative target genes of Toe1–Toe3 could either replicate or suppress the transcription factor overexpression phenotype by ectopic expression or gene deletion respectively . Only the putative target gene SPBC3H7.05c was able to both replicate and suppress the toe2+ overexpression phenotype by its ectopic expression and deletion respectively . These results also suggest that the toxic phenotypes of toe+ overexpression are more likely to be due to abnormal up-regulation of target genes rather than sequestration of the RNA polymerase initiation complex by ectopic expression of the transcription factor (transcriptional squelching) [38–40]. This is because the overexpression phenotypes of these transcription factors can be recapitulated by ectopic expression of target genes that do not have a known role in transcription .
The observation that replication and suppression of the transcription factor-overexpression phenotype did not occur by ectopic expression and deletion of all the putative target genes (presuming they are real) respectively is not surprising. Target genes are likely to exhibit various levels of contribution to transcription factor function. The single ectopic expression of a target gene with a major contribution to transcription factor function would more resemble the transcription factor overexpression phenotype compared with a target gene with a lesser contribution. Moreover, the contribution of the target genes to transcription factor function may be cumulative, and therefore an increased phenotypic resemblance could be seen by their co-overexpression. These occurrences have been observed with the transcriptional activator Mbx2 and its nine flocculin target genes (pfl1+–pfl9+). The single ectopic expression of the pfl+ genes varied in resemblance to the flocculent phenotype of mbx2+ overexpression with pfl1+/gsf2+ producing the most similar size flocs . In addition, enhanced flocculation was observed in several combinations of double pfl+ co-overexpression .
Similarly, the degree of suppression of the transcription factor overexpression phenotype by deletion of the target gene is also likely to depend on the relative contribution of the target gene to transcription factor function. Indeed, the level of loss-of-function suppression by the pfl+ genes of the mbx2+ overexpression flocculent phenotype was correlated with the ability of these genes to induce flocculation when ectopically expressed . Furthermore, complete suppression of the flocculent phenotype in the mbx2+ overexpression strain did not occur for any single pfl+ gene deletion, but was observed in certain double pfl+ deletion combinations . These genetic approaches should also work in principle with transcription factors that have gain-of-function overexpression phenotypes, but exhibit repressive activities. For these transcription factors, the overexpression phenotype would potentially resemble loss-of-function alleles of target genes and be suppressed by their ectopic expression.
The complete set of overexpression and deletion transcription factor strains will allow for novel systematic genetic approaches to advance the deciphering of transcriptional-regulatory networks in S. pombe. Large-scale genetic interaction mapping of these transcription factor strains and the Bioneer deletion collection by synthetic genetic array analysis will enable further identification of upstream regulatory pathways and target genes. For example, digenic interactions of an overexpressed transcription factor gene and a gene deletion have the potential to uncover positive upstream regulators and target genes. Deletion backgrounds that rescue the toxic effects of transcription factor overexpression could represent positive upstream regulators or target genes, whereas deletion backgrounds that sensitize the overexpression of non-toxic transcription factors could represent negative upstream regulators. Furthermore, as the number of transcription factor-target gene interactions identified in S. pombe increases, greater comparative analysis of transcriptional-regulatory networks with other yeasts will be possible, thereby enhancing our knowledge in the evolution of such networks.
The 7th International Fission Yeast Meeting: Pombe 2013: An Independent Meeting/EMBO Conference held at University College London, London, U.K., 24–29 June 2013. Organized and Edited by Jürg Bähler (University College London, U.K.) and Jacqueline Hayles (Cancer Research UK London Research Institute, U.K.).
This work was supported by the Canadian Institutes of Health Research [grant number MOP-89741] and Canada Foundation for Innovation [grant number 16855] to G.C.