Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Functional Characterization of Fission Yeast Transcription Factors by Overexpression Analysis

ABSTRACT: 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. Screening the overexpression array revealed 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+) which 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, the phenotypes of toe1+and toe2+ overexpression could be suppressed by deletion of the putative target genes urg2+ and SPAC1399.04c, and SPBC3H7.05c, SPACUNK4.15 and rds1+, 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. We generated 3 overexpression microarrays and 2 deletion microarray with dye swaps, 3 deletion microarrays with drug treatments with dye swaps, 1 deletion single replicate microarray experiment, and 3 single replicate chIP-chip experiments. The effect of the mutant strains were all compared to wild type or empyty vector strains except for two experiments one that looked at the toe1 mutant with and without chlorpromazine and another that looked at wild type with and without chlorpromazine.

ORGANISM(S): Schizosaccharomyces pombe

SUBMITTER: Gordon Chua

PROVIDER: E-GEOD-46811 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description: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 paper identified 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 âpmr1 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 âprz1 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. We generated 3 overexpression microarrays and 2 deletion microarrays with dye swaps, and 2 deletion microarrays with drug treatments with dye swaps, each with a biological replicate performed as a dye swap. The effect of the mutant strains were all compared to wild type or empty vector strains, except for one experiment where the prz1 deletion mutant was compared to the pmr1 deletion mutant.

Project description: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 Rfl1, 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. We generated 2 overexpression microarrays and 1 deletion microarray with dye swaps, 2 overexpression single replicate microarray experiments, 3 deletion single replicate microarray experiments, and 3 single replicate ChIP-chip experiments. The effect of the mutant strains were all compared to wild type or empty vector strains.

Project description:Visceral fat (VF) and subcutaneous fat (SF) are developmentally different tissues with different gene expression. Islet-1 (ISL1), a LIM-homeobox transcription factor with important developmental and regulatory function in islet, neural, and cardiac tissue, is virtually absent in SF but substantially expressed in the stromovascular [preadipocyte containing] fraction of VF; expression correlates negatively with adiposity in rodents and man. ISL1 expression is transiently increased in 3T3-L1 preadipocytes during early differentiation, suggesting a functional role. To examine the role of ISL1 in adipogenesis, we tested whether retroviral overexpression of ISL1 in 3T3-L1 preadipocytes affected their ability to differentiate into mature adipocytes. Terminal differentiation was assessed by Oil Red O [lipid droplet] staining and by immunoblot detection of adipocyte marker proteins, including aP2 and GLUT4. ISL1 significantly inhibited lipid droplet formation, reduced lipid accumulation (about 80% inhibition, p<0.05), and substantially inhibited aP2 and GLUT4 expression. ISL1 did not inhibit expression of C/EBPb and C/EBPd after induction of differentiation, but reduced PPARg and C/EBPa by >50% at both mRNA and protein level. In addition, the PPARg agonist, rosiglitazone, substantially rescued ISL1 inhibited adipogenesis in the absence of exogenous PPARg, and fully rescued in the presence of exogenous PPARg. In summary, ISL1 overexpression inhibited fat droplet formation, lipid accumulation, and adipocyte-specific gene expression; there was accompanying inhibition of C/EBPa, PPARg and downstream gene expression. We conclude that ISL1 overexpression inhibited adipocyte differentiation by inhibition of PPARg regulated gene expression. As abdominal obesity strongly correlates with insulin resistance, and cardiovascular risk, ISL1 up-regulation may impact abdominal obesity and its concomitant metabolic derangements. Total cellular RNA was isolated from 3T3-L1 cells expressing Flag-ISL1 or not at 48 h following treatment with differentiation cocktail. Individual RNA from biological triplicates was used for microarray analysis.

Project description:Heterochromatin plays a key role in gene repression, maintaining genome integrity and chromosome segregation. Fission yeast, Schizosaccharomyces pombe, utilizes conserved components to direct heterochromatin formation using siRNA generated by RNA interference (RNAi) to guide a histone H3 lysine 9 methyltransferase to cognate chromatin. To identify compounds that inhibit heterochromatin formation, an in vivo phenotypic screen for loss of silencing was performed. A tester strain harbouring a silent dominant selectable kanMX reporter gene within fission yeast centromeric heterochromatin was used to screen a diverse library of chemicals. HMS-I1 and HMS-I2 were identified as compounds that reproducibly increased G418 resistance due to loss of kanMX silencing, and decreased the level of repressive H3K9 methylation on centromeric repeats. The pattern of changes induced by HMS-I1 and HMS-I2 were consistent with inhibition of the histone deacetylases (HDACs) Clr3 and/or Sir2. Chemical-genetic interactions and expression profiling indicated that both HMS-I1 and HMS-I2 affect the activity of the Clr3-containing Snf2/HDAC repressor complex (SHREC). Exposure to HMS-I1 was also found to alleviate silencing of reporter genes in an Arabidopsis transgenic plant line and a mouse cell line. HMS-I2 also disrupted reporter gene silencing in Arabidopsis. In vitro assays indicate that HMS-I1 impairs the activity of human HDAC6 and HDAC10. As HMS-I1 and HMS-I2 bear no resemblance to known inhibitors of chromatin-based activities they represent potentially novel and valuable reagents for experimental and therapeutic purposes. Our findings highlight the use of in vivo chemical screening conducted in fission yeast to identify compounds that disrupt heterochromatin across plant, fungi and animal kingdoms. 16 RNA samples: 2 replicates of WT untreated (vehicle DMSO) (KE1-A, KE2-A), HMS-I1 treated (KE1-B, KE2-B), HMS-I2 treated (KE1-C, KE2-C), and 2 additional replicates of WT (KE05_wt_1, KE10_wt_2), 4 replicates of clr2M-bM-^HM-^F (KE01_2118_1, KE06_2118_2, KE03_0080_1, KE08_0080_2) and mit1M-bM-^HM-^F (KE02_1295_1, KE07_1295_2, KE04_1278_1, KE09_1278_2).

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Functional Characterization of Fission Yeast Transcription Factors by Overexpression Analysis

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