Genetic interaction analysis of RSA1 deletion strains (GIM screen)
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ABSTRACT:
PROVIDER: PRJNA485996 | ENA |
REPOSITORIES: ENA
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Project description:A S. cerevisiae strain with deletion of RSA1 gene was combined with two collections of yeast mutants: one in which non-essential genes were deleted and one in which an long 3' UTR extension has been added to the mRNA of essential genes (GIM method, Decourty et al., 2008). Combined double-mutant deletion cells growth was quantified using barcodes that are specific for each gene mutation. A reference population was obtained by mixing the results of 15 GIM screens DNA prior to barcode amplification and labeling (Decourty et al., in preparation). The identification of genetic interactions between deletion of RSA1 and deletion of RTT106, a histone chaperone, indicate a potential role of Rtt106 in snoRNP formation.
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Project description:This is a partial data set that corresponds to 16 GIM screen results. A larger data set, using a different microarray platform is included in a separate submission. The results correspond to relative growth defect estimates for combinations of a given mutant with more than 5000 other yeast (Saccharomyces cerevisiae) mutants.
2019-06-30 | E-MTAB-7191 | biostudies-arrayexpress
Project description:A S. cerevisiae strain a gene of interest mutated or deleted was combined with two collections of yeast mutants: one in which non-essential genes were deleted and one in which an long 3' UTR extension has been added to the mRNA of essential genes (GIM method, Decourty et al., 2008). Combined double-mutant deletion cells growth was quantified using barcodes that are specific for each gene mutation. A reference population was obtained by mixing the results of 15 GIM screens DNA prior to barcode amplification and labeling (Decourty et al., in preparation). The identification of genetic interactions between genes involved in different cellular pathways indicate new functions for previously characterized genes and new links between
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Project description:Dynamic acetylation of metabolic proteins has emerged as a ubiquitous post-translational modification of human metabolic proteins. However, the corresponding modifying enzymes and the functions of the modification await exploration. Using a genome-wide synthetic lethality screen, we constructed a genetic interaction network of human histone deacetylases (HDACs) and discovered many metabolic substrates of these enzymes. We further confirmed that the adenosine monophosphate-activated protein kinase (AMPK) catalytic subunit is acetylated and deacetylated by EP300 and HDAC1, respectively. Deacetylation of AMPK catalytic subunit enhances physical interaction with the upstream kinase LKB1, and leads to AMPK phosphorylation and activation. These findings highlight the importance of genetic interaction profiling to identify specific substrates of individual HDACs and elucidate how cells use protein (de)acetylation to coordinate nutrient availability and cellular energy status. To study the functional specificity of individual HDAC, we developed a genome-wide genetic interaction profiling technology in cultured human cells by RNAi using pooled TRC (The RNAi Consortium) 75k human shRNA library and complexity deconvolution by high-density microarray with a half-hairpin barcode design. The HCT116 colon cancer cell line was chosen as the screen platform because of its quasinormal diploid karyotype. The performance of the customized microarray we designed was first evaluated by the receiver operating characteristic (ROC) curve showing high sensitivity (89%), specificity (94%) and area under curve (0.95), as well as a control hybridization without DNA sample showing low background signal. Both results suggest that the fluorescent signals were generated by specific hybridization reactions. The high correlation of signal between Cy5 and Cy3 channel as well as dye-swap experiments indicated that biases between the two labeling fluorophores during competitive hybridization were negligible. Moreover, correlation between technical and biological replicates confirmed the high reproducibility of the methodology. In the screen, we used stable polyclonal cells expressing shRNA constructs targeting firefly luciferase (shLuciferase) as control. We checked the knockdown efficiency in protein or RNA level of individual shRNA constructs for 12 human HDAC genes (HDAC1~4, HDAC6~9, SIRT1~3 and SIRT5) by immunoblotting or quantitative PCR, and generated stable polyclonal query cell lines expressing two shRNA constructs with the highest knockdown efficiency for each HDAC gene. After transduction by TRC shRNA lentiviral pools, benchmark samples were harvested prior to puromycin selection, and the remaining cells were propagated under selection and harvested again after 18 population doublings as the end samples. Half-hairpin barcode library of the benchmark and end samples was recovered from genomic DNA respectively by PCR with Cy5 and Cy3-labeled primers, gel purified, and hybridized to the microarray. For each shRNA construct, the Z-score of the log2(Cy5/Cy3) was computed, and Z-score difference was calculated by subtracting the Z-score of the control sample from that of the query sample. Z-score differences larger than 1.5 and less than -1.5 were used as arbitrary thresholds to define candidate negative and positive genetic interactions, respectively.
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Project description:Dynamic acetylation of metabolic proteins has emerged as a ubiquitous post-translational modification of human metabolic proteins. However, the corresponding modifying enzymes and the functions of the modification await exploration. Using a genome-wide synthetic lethality screen, we constructed a genetic interaction network of human histone deacetylases (HDACs) and discovered many metabolic substrates of these enzymes. We further confirmed that the adenosine monophosphate-activated protein kinase (AMPK) catalytic subunit is acetylated and deacetylated by EP300 and HDAC1, respectively. Deacetylation of AMPK catalytic subunit enhances physical interaction with the upstream kinase LKB1, and leads to AMPK phosphorylation and activation. These findings highlight the importance of genetic interaction profiling to identify specific substrates of individual HDACs and elucidate how cells use protein (de)acetylation to coordinate nutrient availability and cellular energy status.
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