Genetic-interaction screens uncover novel biological roles and regulators of transcription factors in fission yeast.

In Schizosaccharomyces pombe, systematic analyses of single transcription factor deletion or overexpression strains have made substantial advances in determining the biological roles and target genes of transcription factors, yet these characteristics are still relatively unknown for over a quarter of them. Moreover, the comprehensive list of proteins that regulate transcription factors remains incomplete. To further characterize Schizosaccharomyces pombe transcription factors, we performed synthetic sick/lethality and synthetic dosage lethality screens by synthetic genetic array. Examination of 2,672 transcription factor double deletion strains revealed a sick/lethality interaction frequency of 1.72%. Phenotypic analysis of these sick/lethality strains revealed potential cell cycle roles for several poorly characterized transcription factors, including SPBC56F2.05, SPCC320.03, and SPAC3C7.04. In addition, we examined synthetic dosage lethality interactions between 14 transcription factors and a miniarray of 279 deletion strains, observing a synthetic dosage lethality frequency of 4.99%, which consisted of known and novel transcription factor regulators. The miniarray contained deletions of genes that encode primarily posttranslational-modifying enzymes to identify putative upstream regulators of the transcription factor query strains. We discovered that ubiquitin ligase Ubr1 and its E2/E3-interacting protein, Mub1, degrade the glucose-responsive transcriptional repressor Scr1. Loss of ubr1+ or mub1+ increased Scr1 protein expression, which resulted in enhanced repression of flocculation through Scr1. The synthetic dosage lethality screen also captured interactions between Scr1 and 2 of its known repressors, Sds23 and Amk2, each affecting flocculation through Scr1 by influencing its nuclear localization. Our study demonstrates that sick/lethality and synthetic dosage lethality screens can be effective in uncovering novel functions and regulators of Schizosaccharomyces pombe transcription factors.

African Americans With p16+ and p16- Oropharyngeal Squamous Cell Carcinomas Have Distinctly Poor Treatment Outcomes Independent of Medical Care Access.

PURPOSE: Human papilloma virus-positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC), diagnosed with p16 immunohistochemistry, is associated with favorable prognosis; however, this connection was established using European American (EA)-skewed populations. The impact of p16/human papillomavirus status on outcomes in African American (AA) OPSCC patients remains to be settled. In this study, we determine the association between cancer disparity and p16 status in an OPSCC cohort controlling for time to treatment initiation (TTI), a surrogate for medical care access. MATERIALS AND METHODS: We analyzed data from all patients diagnosed with OPSCC (N = 440) between 2010 and 2017, who received treatment at our academic medical center. Associations between age, disease stage, sex, p16 status, race, TTI, and overall survival (OS) were investigated. RESULTS: TTI was similar between AA and EA OPSCC patients in our p16+ (P = .291) or p16- (P = .715) cohorts. Among p16+ OPSCC patients, the median OS was > 8.65 years for EA patients compared with 5.038 years (95% CI, 2.019 to 5.30; P = .003, log-rank) for AA patients. For p16- patients, the median OS was 5.74 years (95% CI, 3.32 to 6.99) for EA patients and 1.85 years (95% CI, 0.978 to 4.50; P = .03, log-rank) for AA patients. Multivariate Cox regression analysis showed that race was an independent prognostic biomarker and the most impactful co-variate for OS (hazard ratio, 0.40; 95% CI, 0.00 to 0.69; P = .001). CONCLUSION: Our work showed that AAs with p16+ OPSCC have surprisingly poor clinical outcomes and are thus poor candidates for treatment de-escalation regimens. Caution should be exercised when extending clinical guidelines based on EA-majority studies to non-EA populations.

cfDNA detection for HPV+ squamous cell carcinomas.

High-risk human papillomavirus (HPV) is an etiologic factor in a spectrum of squamous cell carcinomas including anal, cervical, and oropharyngeal. HPV cell free DNA (cfDNA) is shed from the primary tumor into systemic circulation and can be detected using several platforms including quantitative PCR, digital droplet PCR, or next generation sequencing. Levels of HPV cfDNA at time of initial presentation is associated with known poor prognostic clinicopathologic variables, such as advanced stage and, locoregional and distant metastases. Moreover, longitudinal sampling revealed that persistent or increasing HPV cfDNA levels are indicative of treatment relapse and, in some studies, HPV cfDNA detection predicted treatment failures prior to routine post-treatment clinical imaging. A liquid biopsy platform using HPV cfDNA offers unique advantages over traditional approaches and may have clinical utility for detection of minimum residual disease, treatment response, and disease progression in patients with HPV+ cancers.

p53 functional states are associated with distinct aldehyde dehydrogenase transcriptomic signatures.

p53 and aldehyde dehydrogenase (ALDH) have been implicated in key tumorigenesis processes including cancer initiating cell (CIC) maintenance; however, the relationship between these two mediators remains poorly defined. In this study, ALDH isoform expression diversity was revealed in CICs with disparate p53 functional states: gain of function, high risk p53 mutation (p53HRmut) and wildtype p53 (p53WT) inactivated by the human papillomavirus 16 (HPV16) E6 oncogene. Interrogation of head and neck squamous cell carcinoma (HNSCC) cell lines and patient tumors showed that HPV16+/p53WT cases have higher ALDH variance score (AVS), a measure of tumor ALDH isoform expression diversity, compared to HPV-/p53HRmut cases (p = 0.03). AVS and several individual ALDH isoforms were associated with prognosis in HPV16+/p53WT HNSCC but not in HPV-/p53HRmut HNSCC. Knockdown of the dominant ALDH isoform in high AVS HNSCC depleted the CIC pool in vitro and in vivo. Our results demonstrate that p53 functional states are associated with distinct ALDH isoform transcriptomic signatures. Moreover, tumor ALDH profiling may provide insight on which ALDH isoform to target in high AVS HNSCC tumors to deplete the CIC population.

HPV33+ HNSCC is associated with poor prognosis and has unique genomic and immunologic landscapes.

OBJECTIVE: To determine the influence of high-risk HPV genotype on outcomes in HNSCC patients. MATERIALS AND METHODS: This is a retrospective analysis of The Cancer Genome Atlas HNSCC cohort. RESULTS: Using multivariate Cox regression analysis, we revealed that HPV33+ HNSCC patients have inferior overall survival compared to HPV16+ HNSCC patients independent of anatomical site (HR 3.59, 95% CI 1.58-8.12; p = 0.002). A host anti-viral immune response, apolipoprotein B mRNA editing enzyme, and catalytic polypeptide-like mutational signature, was under represented and, aneuploidy and 3p loss were more frequent in HPV33+ tumors. A deconvolution RNA-Seq algorithm to infer immune cell fractions revealed that CD8+ cytotoxic T-cell infiltration was reduced in HPV33+ compared to HPV16+ tumors (1.3% vs. 2.7%, p = 0.007). TGFB1, a negative modulator of T-cell infiltration and function, showed expression and pathway enrichment in HPV33+ tumors. CONCLUSIONS: Our work reveals that HPV genotype, in particular HPV33, has a powerful impact on HNSCC patient survival. We argue that p16 immunohistochemistry as a surrogate biomarker for HPV+ status will lead to sub-optimal risk stratification and advocate HPV genotype testing as standard of care.

Using a Chemical Genetic Screen to Enhance Our Understanding of the Antimicrobial Properties of Gallium against Escherichia coli.

The diagnostic and therapeutic agent gallium offers multiple clinical and commercial uses including the treatment of cancer and the localization of tumors, among others. Further, this metal has been proven to be an effective antimicrobial agent against a number of microbes. Despite the latter, the fundamental mechanisms of gallium action have yet to be fully identified and understood. To further the development of this antimicrobial, it is imperative that we understand the mechanisms by which gallium interacts with cells. As a result, we screened the Escherichia coli Keio mutant collection as a means of identifying the genes that are implicated in prolonged gallium toxicity or resistance and mapped their biological processes to their respective cellular system. We discovered that the deletion of genes functioning in response to oxidative stress, DNA or iron⁻sulfur cluster repair, and nucleotide biosynthesis were sensitive to gallium, while Ga resistance comprised of genes involved in iron/siderophore import, amino acid biosynthesis and cell envelope maintenance. Altogether, our explanations of these findings offer further insight into the mechanisms of gallium toxicity and resistance in E. coli.

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.

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.

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 theO-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.

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, ß-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.

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.

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.

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 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.

Conserved rules govern genetic interaction degree across species.

BACKGROUND: Synthetic genetic interactions have recently been mapped on a genome scale in the budding yeast Saccharomyces cerevisiae, providing a functional view of the central processes of eukaryotic life. Currently, comprehensive genetic interaction networks have not been determined for other species, and we therefore sought to model conserved aspects of genetic interaction networks in order to enable the transfer of knowledge between species. RESULTS: Using a combination of physiological and evolutionary properties of genes, we built models that successfully predicted the genetic interaction degree of S. cerevisiae genes. Importantly, a model trained on S. cerevisiae gene features and degree also accurately predicted interaction degree in the fission yeast Schizosaccharomyces pombe, suggesting that many of the predictive relationships discovered in S. cerevisiae also hold in this evolutionarily distant yeast. In both species, high single mutant fitness defect, protein disorder, pleiotropy, protein-protein interaction network degree, and low expression variation were significantly predictive of genetic interaction degree. A comparison of the predicted genetic interaction degrees of S. pombe genes to the degrees of S. cerevisiae orthologs revealed functional rewiring of specific biological processes that distinguish these two species. Finally, predicted differences in genetic interaction degree were independently supported by differences in co-expression relationships of the two species. CONCLUSIONS: Our findings show that there are common relationships between gene properties and genetic interaction network topology in two evolutionarily distant species. This conservation allows use of the extensively mapped S. cerevisiae genetic interaction network as an orthology-independent reference to guide the study of more complex species.