Limits...
Variable Glutamine-Rich Repeats Modulate Transcription Factor Activity.

Gemayel R, Chavali S, Pougach K, Legendre M, Zhu B, Boeynaems S, van der Zande E, Gevaert K, Rousseau F, Schymkowitz J, Babu MM, Verstrepen KJ - Mol. Cell (2015)

Bottom Line: Incremental changes in the number of repeats in the yeast transcriptional regulator Ssn6 (Cyc8) result in systematic, repeat-length-dependent variation in expression of target genes that result in direct phenotypic changes.Quantitative proteomic analysis reveals that the Ssn6 repeats affect its solubility and interactions with Tup1 and other regulators.Thus, Q-rich repeats are dynamic functional domains that modulate a regulator's innate function, with the inherent risk of pathogenic repeat expansions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Systems Biology, VIB, Gaston Geenslaan 1, 3001 Heverlee, Belgium; Laboratory of Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG), Department M2S, KU Leuven, Gaston Geenslaan 1, 3001 Heverlee, Belgium.

No MeSH data available.


Related in: MedlinePlus

Correlation between Ssn6 TR2-Dependent Variation in Target mRNA, Protein Levels, Expression Noise, and Associated Phenotypes(A) Mean mRNA levels, measured by RNA-seq are expressed as fold change relative to the WT (TR2-63 variant). Mean fluorescence of the corresponding reporter was measured by flow cytometry. Data points represent mean ± SEM, n = 5. See also Figure S5.(B) Analytical flow cytometry of single-cell fluorescence distributions of SSN6 target genes in the TR2 variants. A representative histogram for flo11::YFP (promoter fusion), IMA1-YFP, and CIN5-RFP (protein fusions) in each TR2 variant is shown. Expression noise (defined by the standard deviation divided by the mean fluorescence; i.e., magnitude of variability as a percentage of expression level) (Raser and O’Shea, 2004) was calculated from the fluorescence distributions. Data points represent mean ± SD, n = 5.(C) Images show flo11::YFP fluorescence in the SSN6 TR2 variants. Cells were analyzed by live-cell fluorescence and differential interference contrast (DIC) microscopy.(D) Adhesion to plastic and flocculation intensity correlate with FLO11 expression levels. Data points represent mean ± SD, n = 2. Colony morphologies of the SSN6 TR2 variants show a graded variation in complexity on YP-sucrose. The agar invading capacity is also TR2-length dependent. Cultures were spotted on YPD plates and pictures taken after 11 days of growth at 30°C (pre-wash). The plates were then washed under water to remove non-agar-invading cells (post-wash). Growth rates in palatinose correlate with IMA1 expression. Data points represent mean ± SD, n = 3.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4543046&req=5

fig4: Correlation between Ssn6 TR2-Dependent Variation in Target mRNA, Protein Levels, Expression Noise, and Associated Phenotypes(A) Mean mRNA levels, measured by RNA-seq are expressed as fold change relative to the WT (TR2-63 variant). Mean fluorescence of the corresponding reporter was measured by flow cytometry. Data points represent mean ± SEM, n = 5. See also Figure S5.(B) Analytical flow cytometry of single-cell fluorescence distributions of SSN6 target genes in the TR2 variants. A representative histogram for flo11::YFP (promoter fusion), IMA1-YFP, and CIN5-RFP (protein fusions) in each TR2 variant is shown. Expression noise (defined by the standard deviation divided by the mean fluorescence; i.e., magnitude of variability as a percentage of expression level) (Raser and O’Shea, 2004) was calculated from the fluorescence distributions. Data points represent mean ± SD, n = 5.(C) Images show flo11::YFP fluorescence in the SSN6 TR2 variants. Cells were analyzed by live-cell fluorescence and differential interference contrast (DIC) microscopy.(D) Adhesion to plastic and flocculation intensity correlate with FLO11 expression levels. Data points represent mean ± SD, n = 2. Colony morphologies of the SSN6 TR2 variants show a graded variation in complexity on YP-sucrose. The agar invading capacity is also TR2-length dependent. Cultures were spotted on YPD plates and pictures taken after 11 days of growth at 30°C (pre-wash). The plates were then washed under water to remove non-agar-invading cells (post-wash). Growth rates in palatinose correlate with IMA1 expression. Data points represent mean ± SD, n = 3.

Mentions: We next analyzed the SSN6 TR2 variants for phenotypes associated with FLO11 and IMA1, two target genes (highlighted in Figure 3B) that underlie very specific and quantifiable phenotypes: FLO11 mediates complex colony morphology (induced by growth on sucrose) (Voordeckers et al., 2012b), invasive growth (induced by glucose starvation) (Brückner and Mösch, 2012), adhesion to plastic (Reynolds and Fink, 2001), and flocculation (Verstrepen and Klis, 2006), while IMA1 is a glucosidase required for growth on palatinose (a naturally occurring disaccharide) (Brown et al., 2010; Voordeckers et al., 2012a). All the Flo11-related phenotypes presented a common response to variation in SSN6 TR2 number: colonies showed more “wrinkled” surfaces, stronger agar invasion and plastic adhesion, and higher flocculation levels in the short SSN6 TR2 variants (Figure 4D). These phenotypes gradually decrease with increasing TR2 number. On the other hand, the Ima1-dependent growth rates on palatinose closely follow Ima1 levels in these TR2 variants (Figure 4D). Together, this analysis demonstrates that Ssn6 TR2 variation results in changes in the expression levels of its target genes and also results in corresponding changes in fitness in different nutrient environments. Moreover, these transcriptomics and phenotypic data are in keeping with the results of the genome-scale analysis showing that the targets of Q-rich TFs are enriched in metabolic processes (Figure S2B).


Variable Glutamine-Rich Repeats Modulate Transcription Factor Activity.

Gemayel R, Chavali S, Pougach K, Legendre M, Zhu B, Boeynaems S, van der Zande E, Gevaert K, Rousseau F, Schymkowitz J, Babu MM, Verstrepen KJ - Mol. Cell (2015)

Correlation between Ssn6 TR2-Dependent Variation in Target mRNA, Protein Levels, Expression Noise, and Associated Phenotypes(A) Mean mRNA levels, measured by RNA-seq are expressed as fold change relative to the WT (TR2-63 variant). Mean fluorescence of the corresponding reporter was measured by flow cytometry. Data points represent mean ± SEM, n = 5. See also Figure S5.(B) Analytical flow cytometry of single-cell fluorescence distributions of SSN6 target genes in the TR2 variants. A representative histogram for flo11::YFP (promoter fusion), IMA1-YFP, and CIN5-RFP (protein fusions) in each TR2 variant is shown. Expression noise (defined by the standard deviation divided by the mean fluorescence; i.e., magnitude of variability as a percentage of expression level) (Raser and O’Shea, 2004) was calculated from the fluorescence distributions. Data points represent mean ± SD, n = 5.(C) Images show flo11::YFP fluorescence in the SSN6 TR2 variants. Cells were analyzed by live-cell fluorescence and differential interference contrast (DIC) microscopy.(D) Adhesion to plastic and flocculation intensity correlate with FLO11 expression levels. Data points represent mean ± SD, n = 2. Colony morphologies of the SSN6 TR2 variants show a graded variation in complexity on YP-sucrose. The agar invading capacity is also TR2-length dependent. Cultures were spotted on YPD plates and pictures taken after 11 days of growth at 30°C (pre-wash). The plates were then washed under water to remove non-agar-invading cells (post-wash). Growth rates in palatinose correlate with IMA1 expression. Data points represent mean ± SD, n = 3.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543046&req=5

fig4: Correlation between Ssn6 TR2-Dependent Variation in Target mRNA, Protein Levels, Expression Noise, and Associated Phenotypes(A) Mean mRNA levels, measured by RNA-seq are expressed as fold change relative to the WT (TR2-63 variant). Mean fluorescence of the corresponding reporter was measured by flow cytometry. Data points represent mean ± SEM, n = 5. See also Figure S5.(B) Analytical flow cytometry of single-cell fluorescence distributions of SSN6 target genes in the TR2 variants. A representative histogram for flo11::YFP (promoter fusion), IMA1-YFP, and CIN5-RFP (protein fusions) in each TR2 variant is shown. Expression noise (defined by the standard deviation divided by the mean fluorescence; i.e., magnitude of variability as a percentage of expression level) (Raser and O’Shea, 2004) was calculated from the fluorescence distributions. Data points represent mean ± SD, n = 5.(C) Images show flo11::YFP fluorescence in the SSN6 TR2 variants. Cells were analyzed by live-cell fluorescence and differential interference contrast (DIC) microscopy.(D) Adhesion to plastic and flocculation intensity correlate with FLO11 expression levels. Data points represent mean ± SD, n = 2. Colony morphologies of the SSN6 TR2 variants show a graded variation in complexity on YP-sucrose. The agar invading capacity is also TR2-length dependent. Cultures were spotted on YPD plates and pictures taken after 11 days of growth at 30°C (pre-wash). The plates were then washed under water to remove non-agar-invading cells (post-wash). Growth rates in palatinose correlate with IMA1 expression. Data points represent mean ± SD, n = 3.
Mentions: We next analyzed the SSN6 TR2 variants for phenotypes associated with FLO11 and IMA1, two target genes (highlighted in Figure 3B) that underlie very specific and quantifiable phenotypes: FLO11 mediates complex colony morphology (induced by growth on sucrose) (Voordeckers et al., 2012b), invasive growth (induced by glucose starvation) (Brückner and Mösch, 2012), adhesion to plastic (Reynolds and Fink, 2001), and flocculation (Verstrepen and Klis, 2006), while IMA1 is a glucosidase required for growth on palatinose (a naturally occurring disaccharide) (Brown et al., 2010; Voordeckers et al., 2012a). All the Flo11-related phenotypes presented a common response to variation in SSN6 TR2 number: colonies showed more “wrinkled” surfaces, stronger agar invasion and plastic adhesion, and higher flocculation levels in the short SSN6 TR2 variants (Figure 4D). These phenotypes gradually decrease with increasing TR2 number. On the other hand, the Ima1-dependent growth rates on palatinose closely follow Ima1 levels in these TR2 variants (Figure 4D). Together, this analysis demonstrates that Ssn6 TR2 variation results in changes in the expression levels of its target genes and also results in corresponding changes in fitness in different nutrient environments. Moreover, these transcriptomics and phenotypic data are in keeping with the results of the genome-scale analysis showing that the targets of Q-rich TFs are enriched in metabolic processes (Figure S2B).

Bottom Line: Incremental changes in the number of repeats in the yeast transcriptional regulator Ssn6 (Cyc8) result in systematic, repeat-length-dependent variation in expression of target genes that result in direct phenotypic changes.Quantitative proteomic analysis reveals that the Ssn6 repeats affect its solubility and interactions with Tup1 and other regulators.Thus, Q-rich repeats are dynamic functional domains that modulate a regulator's innate function, with the inherent risk of pathogenic repeat expansions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Systems Biology, VIB, Gaston Geenslaan 1, 3001 Heverlee, Belgium; Laboratory of Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG), Department M2S, KU Leuven, Gaston Geenslaan 1, 3001 Heverlee, Belgium.

No MeSH data available.


Related in: MedlinePlus