Kate Chatfield-Reed

Deciphering the transcriptional-regulatory network of flocculation in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe, the transcriptional-regulatory network that governs flocculation remains poorly understood. Here, we systematically screened an array of transcription factor deletion and overexpression strains for flocculation and performed microarray expression profiling and ChIP–chip analysis to identify the flocculin target genes. We identified five transcription factors that displayed novel roles in the activation or inhibition of flocculation (Rfl1, Adn2, Adn3, Sre2, and Yox1), in addition to the previously-known Mbx2, Cbf11, and Cbf12 regulators. Overexpression of mbx2+ and deletion of rfl1+ resulted in strong flocculation and transcriptional upregulation of gsf2 +/pfl1+ and several other putative flocculin genes (pfl2+–pfl9+). Overexpression of the pfl+ genes singly was sufficient to trigger flocculation, and enhanced flocculation was observed in several combinations of double pfl+ overexpression. Among the pfl1+ genes, only loss of gsf2+ abrogated the flocculent phenotype of all the transcription factor mutants and prevented flocculation when cells were grown in inducing medium containing glycerol and ethanol as the carbon source, thereby indicating that Gsf2 is the dominant flocculin. In contrast, the mild flocculation of adn2+ or adn3+ overexpression was likely mediated by the transcriptional activation of cell wall–remodeling genes including gas2+, psu1+, and SPAC4H3.03c. We also discovered that Mbx2 and Cbf12 displayed transcriptional autoregulation, and Rfl1 repressed gsf2+ expression in an inhibitory feed-forward loop involving mbx2+. These results reveal that flocculation in S. pombe is regulated by a complex network of multiple transcription factors and target genes encoding flocculins and cell wall–remodeling enzymes. Moreover, comparisons between the flocculation transcriptional-regulatory networks of Saccharomyces cerevisiae and S. pombe indicate substantial rewiring of transcription factors and cis-regulatory sequences.

Identification of detoxification pathways in plants that are regulated in response to treatment with organic compounds isolated from oil sands process-affected water.

Bitumen mining in the Athabasca oil sands region of northern Alberta results in the accumulation of large volumes of oil sands process-affected water (OSPW). The acid-extractable organic (AEO) fraction of OSPW contains a variety of compounds, including naphthenic acids, aromatics, and sulfur- and nitrogen-containing compounds that are toxic to aquatic and terrestrial organisms. We have studied the effect of AEO treatment on the transcriptome of root and shoot tissues in seedlings of the model plant, Arabidopsis thaliana. Several genes encoding enzymes involved in the xenobiotic detoxification pathway were upregulated, including cytochrome P450s (CYPs), UDP-dependent glycosyltransferases (UGTs), glutathione-S-transferases (GSTs), and membrane transporters. In addition, gene products involved in oxidative stress, β-oxidation, and glucosinolate degradation were also upregulated, indicating other potential mechanisms of the adaptive response to AEO exposure. These results provide insight into the pathways that plants use to detoxify the organic acid component of OSPW. Moreover, this study advances our understanding of genes that could be exploited to potentially develop phytoremediation and biosensing strategies for AEO contaminants resulting from oil sands mining.

Functional Characterization of Fission Yeast Transcription Factors by Overexpression Analysis

In Schizosaccharomyces pombe, over 90% of transcription factor genes are nonessential. Moreover, the majority do not exhibit significant growth defects under optimal conditions when deleted, complicating their functional characterization and target gene identification. Here, we systematically overexpressed 99 transcription factor genes with the nmt1 promoter and found that 64 transcription factor genes exhibited reduced fitness when ectopically expressed. Cell cycle defects were also often observed. We further investigated three uncharacterized transcription factor genes (toe1+–toe3+) that displayed cell elongation when overexpressed. Ectopic expression of toe1+ resulted in a G1 delay while toe2+ and toe3+ overexpression produced an accumulation of septated cells with abnormalities in septum formation and nuclear segregation, respectively. Transcriptome profiling and ChIP-chip analysis of the transcription factor overexpression strains indicated that Toe1 activates target genes of the pyrimidine-salvage pathway, while Toe3 regulates target genes involved in polyamine synthesis. We also found that ectopic expression of the putative target genes SPBC3H7.05c, and dad5+ and SPAC11D3.06 could recapitulate the cell cycle phenotypes of toe2+ and toe3+ overexpression, respectively. Furthermore, single deletions of the putative target genes urg2+ and SPAC1399.04c, and SPBC3H7.05c, SPACUNK4.15, and rds1+, could suppress the phenotypes of toe1+ and toe2+ overexpression, respectively. This study implicates new transcription factors and metabolism genes in cell cycle regulation and demonstrates the potential of systematic overexpression analysis to elucidate the function and target genes of transcription factors in S. pombe.

Deciphering the Stigmatic Transcriptional Landscape of Compatible and Self-Incompatible Pollinations in Brassica napus Reveals a Rapid Stigma Senescence Response Following Compatible Pollination

Dear Editor, Self-incompatibility (SI) is a genetic mechanism through which flowering plants prevent self-pollination to ensure outcrossing and genetic diversity. In Brassica sp., this mechanism is controlled by the self-incompatibility (S) locus, in which, the stigmatic ‘S-locus receptor kinase (SRK)’ recognizes the ‘S-locus cysteine rich protein (SCR)’ from the self-pollen to elicit an active rejection response. This results in blocking of compatibility factors from being delivered to the site of pollen attachment leading to self-pollen rejection (Chapman and Goring, 2010). In contrast, following recognition of compatible signals from the cross-pollen or compatible pollen (CP), the stigma releases its resources such as water and nutrients to the dry pollen so that the pollen tube can germinate and penetrate the stigmatic cuticle leading to successful fertilization. Thus, an incompatible or self-pollen is fully capable of eliciting a compatible response, but is actively rejected before compatible responses can occur. Following landing of self-pollen or cross-pollen on stigmas of Brassica napus (canola), there is a latent period of 30 min when signals are exchanged between the highly lipophilic pollen coat proteins and the stigmatic components. CP tubes can be observed to emerge between 30 and 90 min after initiation of this interaction. Given that stigmas control the outcome of pollen acceptance or rejection, deciphering the transcriptional changes during this latent period would reveal genes involved in compatible and self-incompatible responses. As expected, when self-incompatible W1 canola stigmas were stained with aniline blue to observe pollen tubes, SI-pollinated stigmas lacked any pollen attachment or pollen tubes at 30 min and 6 h after pollination (Figure 1A, right panel). The weakly attached pollen without any positive interactions is washed away during the staining process. Following compatible pollination, although pollen attachment and pollen tubes could be observed at 6 h, at 30 min after pollination, no pollen attachment could be observed (Figure 1A, right panel). This is due to lack of complete adhesion and pollen tube germination at 30 min after pollination. These observations suggest that analyzing the transcriptome changes 0–30 min following SI and compatible pollinations would likely reveal genes that are triggered by pollen landing on the stigma and could represent genes that are required for promoting SI and compatible responses, respectively. To identify the genes that are differentially regulated by SI and compatible pollinations (Figure 1A, left panel), RNA extracted from self-incompatible W1 stigmas, pollinated with self-pollen or cross-pollen for either 15 or 30 min, were compared against RNA from unpollinated (UP) stigmas through transcriptome profiling, using the Agilent 4 × 44K Brassica Gene Expression Microarrays (G2519F). Following normalization, filtering based on P-values (<0.001) and then by two-fold up-regulation in at least one of the microarray experiments, we identified 621 genes that were differentially regulated. Clustering of these genes (Supplemental Table 1) clearly indicated strong up-regulation of multiple genes across all four treatments. This suggested to us that these were likely expressed pollen genes when SI and compatible pollinated stigmas were compared with UP stigmas that lacked any pollen. Utilizing the high sequence similarity between Arabidopsis and Brassica, we identified the orthologous Arabidopsis genes for the 621 canola genes to facilitate further bioinformatic analyses. Following filtering of the pollen genes from the differentially expressed genes in the microarray experiments (see Supplementary Data), the 621 genes were subdivided into stigma genes (287), pollen genes (181), and stigma–pollen genes (153) (Supplemental Table 2). Since the focus of this study was to identify stigmatic genes specifically regulated by SI and CP, we focused our attention on the 287 stigmatic genes. Clustering the stigmatic genes (Supplemental Table 3) revealed a clear pattern of changes between SI and compatible pollination. Heat maps were generated for the subset of genes that were specifically induced or repressed in SI and compatible pollination (Figure 1B–1E and Supplemental Table 4).

The NuA4 Complex Promotes Translesion Synthesis (TLS) – Mediated DNA Damage Tolerance

Lesions in DNA can block replication fork progression, leading to its collapse and gross chromosomal rearrangements. To circumvent such outcomes, the DNA damage tolerance (DDT) pathway becomes engaged, allowing the replisome to bypass a lesion and complete S phase. Chromatin remodeling complexes have been implicated in the DDT pathways, and here we identify the NuA4 remodeler, which is a histone acetyltransferase, to function on the translesion synthesis (TLS) branch of DDT. Genetic analyses in Saccharomyces cerevisiae showed synergistic sensitivity to MMS when NuA4 alleles, esa1-L254P and yng2Δ, were combined with the error-free bypass mutant ubc13Δ. The loss of viability was less pronounced when NuA4 complex mutants were disrupted in combination with error-prone/TLS factors, such as rev3Δ, suggesting an epistatic relationship between NuA4 and error-prone bypass. Consistent with cellular viability measurements, replication profiles after exposure to MMS indicated that small regions of unreplicated DNA or damage were present to a greater extent in esa1-L254P/ubc13Δ mutants, which persist beyond the completion of bulk replication compared to esa1-L254P/rev3Δ. The critical role of NuA4 in error-prone bypass is functional even after the bulk of replication is complete. Underscoring this observation, when Yng2 expression is restricted specifically to G2/M of the cell cycle, viability and TLS-dependent mutagenesis rates were restored. Lastly, disruption of HTZ1, which is a target of NuA4, also resulted in mutagenic rates of reversion on level with esa1-L254P and yng2Δ mutants, indicating that the histone variant H2A.Z functions in vivo on the TLS branch of DDT.

Conserved and Diverged Functions of the Calcineurin-Activated Prz1 Transcription Factor in Fission Yeast.

Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeast Schizosaccharomyces pombe. Genome-wide studies of the Crz1 and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This article identifies an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2 and tunicamycin treatment, as well as a ∆pmr1 genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of the Saccharomyces cerevisiae ortholog Crz1. These genes were functionally enriched for Crz1-conserved processes such as cell-wall biosynthesis. Overexpression of prz1+ increased resistance to the cell-wall degradation enzyme zymolyase, likely from upregulation of the O-mannosyltransferase encoding gene omh1+. Loss of omh1+ abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss of prz1+ resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the ∆prz1 strain was abrogated by the loss of gsf2+ or cbf12+. This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell-wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes between Crz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional regulatory network.

Using a Chemical Genetic Screen to Enhance Our Understanding of the Antibacterial Properties of Silver

It is essential to understand the mechanisms by which a toxicant is capable of poisoning the bacterial cell. The mechanism of action of many biocides and toxins, including numerous ubiquitous compounds, is not fully understood. For example, despite the widespread clinical and commercial use of silver (Ag), the mechanisms describing how this metal poisons bacterial cells remains incomplete. To advance our understanding surrounding the antimicrobial action of Ag, we performed a chemical genetic screen of a mutant library of Escherichia coli—the Keio collection, in order to identify Ag sensitive or resistant deletion strains. Indeed, our findings corroborate many previously established mechanisms that describe the antibacterial effects of Ag, such as the disruption of iron-sulfur clusters containing proteins and certain cellular redox enzymes. However, the data presented here demonstrates that the activity of Ag within the bacterial cell is more extensive, encompassing genes involved in cell wall maintenance, quinone metabolism and sulfur assimilation. Altogether, this study provides further insight into the antimicrobial mechanism of Ag and the physiological adaption of E. coli to this metal.

A multiplexed transcription activator-like effector system for detecting specific DNA sequences.

Transcription activator-like effectors (TALEs), originating from the Xanthomonas genus of bacteria, bind to specific DNA sequences based on amino acid sequence in the repeat-variable diresidue (RVD) positions of the protein. By altering these RVDs, it has been shown that a TALE protein can be engineered to bind virtually any DNA sequence of interest. The possibility of multiplexing TALEs for the purposes of identifying specific DNA sequences has yet to be explored. Here, we demonstrate a system in which a TALE protein bound to a nitrocellulose strip has been utilized to capture purified DNA, which is then detected using the binding of a second distinct TALE protein conjugated to a protein tag that is then detected by a dot blot. This system provides a signal only when both TALEs bind to their respective sequences, further demonstrating the specificity of the TALE binding.

Identification of novel transcriptional regulators of PKA subunits in Saccharomyces cerevisiae by quantitative promoter-reporter screening

The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits.

Building with the beeoids: guided automatic construction using pheromones

Artist Aganetha Dyck collaborates with bees to produce inventive and unusual art. We demonstrate a swarm system that mimics this collaboration allowing designers to interact with simple agents called beeoids to produce virtual three-dimensional constructions. We show how the system responds to changes in user input and how interactive evolution can be used to change the behaviour of the system.