Homolog-based redundancy estimates.

<p><b>(A)</b> Scatter plots depicting the sensitivity of gene 1 and gene 2 for close paralog pairs between non-responsive (NR-NR, left panel), between non-responsive and responsive (NR-R, middle panel) or between responsive (R-R, right panel) mutants. <b>(B)</b> Boxplots of the average sensitivity of the same groups as in A. The difference between all three groups is statistically significant (<i>p</i>-value _{(NR-NR vs. NR-R)} = 3.14×10⁻⁹; <i>p</i>-value _{(NR-NR vs. R-R)} = 1.02×10⁻⁸; <i>p</i>-value _{(NR-R vs. R-R)} = 1.66x10^-3) based on a one-sided Mann-Whitney test. <b>(C)</b> Same as in A, except that only close paralog pairs with a significant negative SGI score are shown. <b>(D)</b> Boxplots of the average sensitivity of the same groups as in C (<i>p</i>-value _{(NR-NR vs. NR-R)} = 0.128, <i>p</i>-value _{(NR-NR vs. R-R)} = 1.4x10^-3, <i>p</i>-value _{(NR-R vs. R-R)} = 3.27x10^-3), statistical test as in B.</p

Growth condition dependency is the major cause of non-responsiveness upon genetic perturbation

<div><p>Investigating the role and interplay between individual proteins in biological processes is often performed by assessing the functional consequences of gene inactivation or removal. Depending on the sensitivity of the assay used for determining phenotype, between 66% (growth) and 53% (gene expression) of <i>Saccharomyces cerevisiae</i> gene deletion strains show no defect when analyzed under a single condition. Although it is well known that this non-responsive behavior is caused by different types of redundancy mechanisms or by growth condition/cell type dependency, it is not known what the relative contribution of these different causes is. Understanding the underlying causes of and their relative contribution to non-responsive behavior upon genetic perturbation is extremely important for designing efficient strategies aimed at elucidating gene function and unraveling complex cellular systems. Here, we provide a systematic classification of the underlying causes of and their relative contribution to non-responsive behavior upon gene deletion. The overall contribution of redundancy to non-responsive behavior is estimated at 29%, of which approximately 17% is due to homology-based redundancy and 12% is due to pathway-based redundancy. The major determinant of non-responsiveness is condition dependency (71%). For approximately 14% of protein complexes, just-in-time assembly can be put forward as a potential mechanistic explanation for how proteins can be regulated in a condition dependent manner. Taken together, the results underscore the large contribution of growth condition requirement to non-responsive behavior, which needs to be taken into account for strategies aimed at determining gene function. The classification provided here, can also be further harnessed in systematic analyses of complex cellular systems.</p></div

Homolog-based redundancy estimates.

<p><b>(A)</b> Scatter plots depicting the sensitivity of gene 1 and gene 2 for close paralog pairs between non-responsive (NR-NR, left panel), between non-responsive and responsive (NR-R, middle panel) or between responsive (R-R, right panel) mutants. <b>(B)</b> Boxplots of the average sensitivity of the same groups as in A. The difference between all three groups is statistically significant (<i>p</i>-value _{(NR-NR vs. NR-R)} = 3.14×10⁻⁹; <i>p</i>-value _{(NR-NR vs. R-R)} = 1.02×10⁻⁸; <i>p</i>-value _{(NR-R vs. R-R)} = 1.66x10^-3) based on a one-sided Mann-Whitney test. <b>(C)</b> Same as in A, except that only close paralog pairs with a significant negative SGI score are shown. <b>(D)</b> Boxplots of the average sensitivity of the same groups as in C (<i>p</i>-value _{(NR-NR vs. NR-R)} = 0.128, <i>p</i>-value _{(NR-NR vs. R-R)} = 1.4x10^-3, <i>p</i>-value _{(NR-R vs. R-R)} = 3.27x10^-3), statistical test as in B.</p

Distribution of non-responsive behavior.

<p>Percentage of non-responsive (NR; light grey) and responsive (R; dark grey) mutants within different functional categories, as well as the overall percentage. A mutant is classified as NR if three or less transcripts are changing compared to WT. A mutant is classified as R if four or more transcripts are changing. Functional categories as defined in the original study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173432#pone.0173432.ref003" target="_blank">3</a>].</p

General characteristics of non-responsive and responsive mutants.

<p><b>(A)</b> Frequency density distribution of wildtype (WT) mRNA transcript levels (A) obtained from the average of 200 WT strains, adapted from Kemmeren et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173432#pone.0173432.ref003" target="_blank">3</a>]. <i>P</i>-value indicates the difference between transcript levels in the WT strains between NR and R mutants based on a two-sided Mann-Whitney test. <b>(B)</b> Frequency density distribution of the number of proteins per WT cell [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173432#pone.0173432.ref027" target="_blank">27</a>] for NR and R mutants, adapted from Kemmeren et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173432#pone.0173432.ref003" target="_blank">3</a>]. Only detectable proteins are depicted. <i>P</i>-value indicates the difference between protein levels in NR and R mutants based on a two-sided Mann-Whitney test. <b>(C)</b> Number of NR and R mutants with a close paralog (CP) that arose from whole-genome duplication (WGD) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173432#pone.0173432.ref031" target="_blank">31</a>] or small scale duplications (SSD) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173432#pone.0173432.ref020" target="_blank">20</a>]. <b>(D)</b> Box plots showing the number of significant negative synthetic genetic interaction (SGI) scores [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173432#pone.0173432.ref022" target="_blank">22</a>](ε ≤ -0.08, <i>p</i> ≤ 0.05; left panel), number of protein-protein interactions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173432#pone.0173432.ref030" target="_blank">30</a>](PPI, middle panel) and sensitivity to different conditions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173432#pone.0173432.ref008" target="_blank">8</a>] (percentage of conditions that deletion mutants show a relative growth defect; growth defect > 0, <i>p</i> ≤ 0.05; right panel) for NR and R mutants. <i>P</i>-values are based on a two-sided Mann-Whitney test.</p

Relative contribution of redundancy and condition dependency and potential mechanisms.

<p><b>(A)</b> Flowchart showing the relative contribution of redundancy and condition dependency to non-responsiveness. Potential mechanisms are indicated for homology-based redundancy, pathway-based redundancy and “just-in-time assembly”. <b>(B)</b> Relative contribution of redundancy and condition dependency to non-responsiveness within each functional category. <b>(C)</b> Line plot showing the mRNA expression changes across 1,484 deletion mutants [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173432#pone.0173432.ref003" target="_blank">3</a>] for the Dcs1-Dcs2 heterodimer. Individual lines indicate the Dcs1 and Dcs2 subunit. The highly regulated subunit is indicated in red. <b>(D)</b> Line plot as in C for the GID complex.</p

The RNAPII CTD was critical for the association of transcription related factors.

<p>(A, B, C and D) Left. Average gene profiles of H3K36me3, Cet1, TFIIB and Elf1 at genes with decreased (top) or increased (bottom) mRNA levels upon truncation of the CTD. Right. Average occupancy scores of H3K36me3, Cet1, TFIIB and Elf1 at genes with decreased (top) (paired t-test p value 8.68e-6, 2.72e-7, 8.66e-8 and 9.17e-6 respectively) or increased (bottom) (paired t-test p value 9.34e-23, 7.82e-25, 0.136 and 4e-15 respectively) mRNA levels upon truncation of the CTD. For H3K36me3 and Efl1, the average occupancy scores were calculated for the coding region. For Cet1 and TFIIB, the average occupancy scores were calculated for the promoter, which consisted of 500 bp upstream of the start codon.</p

Design and validation of 14-3-3ζ shRNA constructs

<p><b><i>A</i></b> 14-3-3ζ specific shRNA sequences (1222 and 1854 bp) were cloned into pLentiLox3.7 vectors and <b><i>B</i></b> western blot analysis revealed that both constructs effectively reduced 14-3-3ζ protein levels in Neuro2A cells, the 1854 construct being most effective <i>in vitro. </i><b><i>C</i></b><i> In situ</i> hybridization confirmed effective knockdown of 14-3-3ζ in the CeA after infection with the 1854 shRNA expressing lentivirus. The top panels show 14-3-3ζ expression in the CeA after infection with the control or the 1854 shRNA expressing lentivirus. The bottom panels show GFP mRNA and therefore the infection site in adjacent sections. 14-3-3ζ mRNA is completely absent in the area that is infected with the 1854 shRNA expressing lentivirus.</p

Taste control experiments show that the increase in alcohol intake in mice with CeA 14-3-3ζ knockdown is not secondary to altered taste sensitivity.

<p>Control mice and mice with CeA 14-3-3ζ knockdown showed equal intake of solutions containing <i>A</i> the caloric sweet tastant sucrose and <i>B</i> the non-caloric sweet tastant saccharin. <i>C</i> Aversion for the bitter tastant quinine was also not different between controls and mice with CeA 14-3-3ζ knockdown.</p

Serial CTD truncations led to progressive steady state transcriptional defects.

<p>Expression microarrays were normalized using spiked in controls to determine global changes in mRNA levels. As no such changes were detected, the expression profiles were normalized to total mRNA levels. Differentially expressed genes were determined by p value <0.01 and fold change >1.7 compared to wild type. (A) Heatmap of genes with significantly increased (top) or decreased (bottom) mRNA levels in the <i>rpb1-CTD11</i> mutant. Groups A, B and C approximately outline subsets of genes whose expression were decreased when the CTD was truncated to 13, 12 or 11 repeats respectively. Yellow indicates genes with increased mRNA levels and blue indicates genes with decreased levels. (B) Scatterplot of profile paired correlations in gene expression and genetic interaction. Boxplot of transcriptional frequency (C) and mRNA half-life (D) showing significant differences in half-life (p value 4.54e-14) and transcriptional frequency (p value 0.0131) between genes with increased or decreased expression in the <i>rpb1-CTD11</i> mutant. Outliers are not shown. (E) Differences in enriched transcription factors between genes with increased or decreased mRNA levels.</p