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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

TR2 Variation Modulates Ssn6 Solubility and Protein Interactions(A) Identification of Ssn6 interactors. Shared and unique interacting proteins between WT Ssn6 (TR2-63) and the repeat deletion (TR2-0) or expansion (TR2-105) forms are shown. Quantification is shown of enriched or depleted proteins in pull-downs of representative TR2 variants (labeled with 13C isotope, H) relative to the WT (TR2-63) (labeled with 12C isotope, L). Data points represent mean ± SD, n = 2. p values were evaluated using the unpaired two-tailed t test. Asterisks indicate p < 0.05. See also Table S5.(B) Co-localization of the Hsp70 chaperone Ssa2 and the expanded Ssn6 (TR2-105). Live cells of TR2 variants containing Ssn6-YFP and Ssa2-RFP fusion proteins were visualized by fluorescence and DIC microscopy.(C) Ssa2 maintains Ssn6 function. Analytical flow cytometry profiles of flo11::YFP in the TR2 variants and their Δssa2 counterparts show how deletion of SSA2 affects the expression of the FLO11 target gene. See also Figure S6.(D) Sequestration of expanded Ssn6 (TR2-105) in intranuclear foci (arrowheads) (left) and increased Ssn6 aggregation in the absence of Ssa2 (right). Live cells of TR2 variants containing Ssn6-YFP fusion and their Δssa2 counterparts were visualized by fluorescence and DIC microscopy. See also Figure S6.(E) The SSN6 TR2 region modulates Ssn6 solubility. Cultures of HA-tagged Ssn6 TR2 variants were analyzed by protein aggregation assay. Soluble and insoluble fractions of Ssn6 were quantified by western blot. For each variant, the ratio of the respective fraction over the sum of the soluble and insoluble fractions was calculated. The horizontal line indicates the mean of the insoluble fraction of the WT Ssn6 (TR2-63). Data represent mean ± SD, n = 3 or n = 2. p values were evaluated using the unpaired two-tailed t test. Asterisks indicate p < 0.05, ns, not significant.(F) Disruption of the Ssn6 TR2 conformation by proline insertions. Sequencing of the Ssn6 TR2 region show the replacement of two (mutant a) or three (mutants b and c) glutamine-alanine residues by prolines (red).(G) The α-helical coiled-coil structure of TR2 is essential for Ssn6 function. Median flo11::YFP fluorescence in the QA/P mutants (a, b, c) (striped bars) is comparable to the TR2-0 variant (green bar). Data points represent mean ± SD, n = 2.
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fig5: TR2 Variation Modulates Ssn6 Solubility and Protein Interactions(A) Identification of Ssn6 interactors. Shared and unique interacting proteins between WT Ssn6 (TR2-63) and the repeat deletion (TR2-0) or expansion (TR2-105) forms are shown. Quantification is shown of enriched or depleted proteins in pull-downs of representative TR2 variants (labeled with 13C isotope, H) relative to the WT (TR2-63) (labeled with 12C isotope, L). Data points represent mean ± SD, n = 2. p values were evaluated using the unpaired two-tailed t test. Asterisks indicate p < 0.05. See also Table S5.(B) Co-localization of the Hsp70 chaperone Ssa2 and the expanded Ssn6 (TR2-105). Live cells of TR2 variants containing Ssn6-YFP and Ssa2-RFP fusion proteins were visualized by fluorescence and DIC microscopy.(C) Ssa2 maintains Ssn6 function. Analytical flow cytometry profiles of flo11::YFP in the TR2 variants and their Δssa2 counterparts show how deletion of SSA2 affects the expression of the FLO11 target gene. See also Figure S6.(D) Sequestration of expanded Ssn6 (TR2-105) in intranuclear foci (arrowheads) (left) and increased Ssn6 aggregation in the absence of Ssa2 (right). Live cells of TR2 variants containing Ssn6-YFP fusion and their Δssa2 counterparts were visualized by fluorescence and DIC microscopy. See also Figure S6.(E) The SSN6 TR2 region modulates Ssn6 solubility. Cultures of HA-tagged Ssn6 TR2 variants were analyzed by protein aggregation assay. Soluble and insoluble fractions of Ssn6 were quantified by western blot. For each variant, the ratio of the respective fraction over the sum of the soluble and insoluble fractions was calculated. The horizontal line indicates the mean of the insoluble fraction of the WT Ssn6 (TR2-63). Data represent mean ± SD, n = 3 or n = 2. p values were evaluated using the unpaired two-tailed t test. Asterisks indicate p < 0.05, ns, not significant.(F) Disruption of the Ssn6 TR2 conformation by proline insertions. Sequencing of the Ssn6 TR2 region show the replacement of two (mutant a) or three (mutants b and c) glutamine-alanine residues by prolines (red).(G) The α-helical coiled-coil structure of TR2 is essential for Ssn6 function. Median flo11::YFP fluorescence in the QA/P mutants (a, b, c) (striped bars) is comparable to the TR2-0 variant (green bar). Data points represent mean ± SD, n = 2.

Mentions: To check for changes in the Ssn6 stability and interactome, we fused the Ssn6 repeat variants (TR2-0, TR2-55, TR2-63, TR2-90, and TR2-105) to six copies of an HA epitope tag at their C terminus. Using gel-bound anti-HA antibodies, we isolated the tagged Ssn6 from soluble protein extracts. Bound proteins were separated by SDS-PAGE, followed by in-gel digestion and identification by mass spectrometry (Figure 5A, left). This qualitative approach shows that the interactomes of the repeat deletion and expansion variants are larger than that of the WT and also contain common elements (Figure 5A, left). Notably, these common interactors are involved in active chromatin remodeling (e.g., the histone variant H2AZ, Dot6, Yku80), transcription (Ppr1, Tfg1), and signaling (Pkc1, Bmh1, Ypt1). To confirm these results and to accurately quantify TR2-dependent changes in the Ssn6 interactome, we repeated the pull-down experiment and eluted the antibody-bound protein complexes. The eluted fractions were digested, and the peptide mixtures from the WT were labeled with a light isotope (12C3-propionate), while those from the TR2 variants were labeled with a heavy isotope (13C3-propionate). The labeled peptides from each TR2 variant and the WT were mixed 1:1 and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Figure 5A, right). Among the 85 proteins detected, we considered the ones identified by at least two different peptides in all five TR2 variants tested (Table S5). We find >2-fold depletion of Ssn6 and its known interaction partner Tup1 in the TR2-105 variant relative to the WT (Figure 5A, right). Concurrently, we find a significant enrichment of the Hsp70 chaperone Ssa2 in the TR2 expansion (TR2-90 and TR2-105) and deletion (TR2-0) variants. Ssa2 is a cytosolic chaperone required for protein folding, translocation, and degradation of unfolded proteins. It also partners with Hsp40 co-chaperones to promote the refolding of aggregated or unfolded proteins (Verghese et al., 2012). Interestingly, we find that Ssa2 co-localizes with expanded Ssn6 in the nucleus, but not with the WT or TR2-0 variant (Figure 5B).


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)

TR2 Variation Modulates Ssn6 Solubility and Protein Interactions(A) Identification of Ssn6 interactors. Shared and unique interacting proteins between WT Ssn6 (TR2-63) and the repeat deletion (TR2-0) or expansion (TR2-105) forms are shown. Quantification is shown of enriched or depleted proteins in pull-downs of representative TR2 variants (labeled with 13C isotope, H) relative to the WT (TR2-63) (labeled with 12C isotope, L). Data points represent mean ± SD, n = 2. p values were evaluated using the unpaired two-tailed t test. Asterisks indicate p < 0.05. See also Table S5.(B) Co-localization of the Hsp70 chaperone Ssa2 and the expanded Ssn6 (TR2-105). Live cells of TR2 variants containing Ssn6-YFP and Ssa2-RFP fusion proteins were visualized by fluorescence and DIC microscopy.(C) Ssa2 maintains Ssn6 function. Analytical flow cytometry profiles of flo11::YFP in the TR2 variants and their Δssa2 counterparts show how deletion of SSA2 affects the expression of the FLO11 target gene. See also Figure S6.(D) Sequestration of expanded Ssn6 (TR2-105) in intranuclear foci (arrowheads) (left) and increased Ssn6 aggregation in the absence of Ssa2 (right). Live cells of TR2 variants containing Ssn6-YFP fusion and their Δssa2 counterparts were visualized by fluorescence and DIC microscopy. See also Figure S6.(E) The SSN6 TR2 region modulates Ssn6 solubility. Cultures of HA-tagged Ssn6 TR2 variants were analyzed by protein aggregation assay. Soluble and insoluble fractions of Ssn6 were quantified by western blot. For each variant, the ratio of the respective fraction over the sum of the soluble and insoluble fractions was calculated. The horizontal line indicates the mean of the insoluble fraction of the WT Ssn6 (TR2-63). Data represent mean ± SD, n = 3 or n = 2. p values were evaluated using the unpaired two-tailed t test. Asterisks indicate p < 0.05, ns, not significant.(F) Disruption of the Ssn6 TR2 conformation by proline insertions. Sequencing of the Ssn6 TR2 region show the replacement of two (mutant a) or three (mutants b and c) glutamine-alanine residues by prolines (red).(G) The α-helical coiled-coil structure of TR2 is essential for Ssn6 function. Median flo11::YFP fluorescence in the QA/P mutants (a, b, c) (striped bars) is comparable to the TR2-0 variant (green bar). Data points represent mean ± SD, n = 2.
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fig5: TR2 Variation Modulates Ssn6 Solubility and Protein Interactions(A) Identification of Ssn6 interactors. Shared and unique interacting proteins between WT Ssn6 (TR2-63) and the repeat deletion (TR2-0) or expansion (TR2-105) forms are shown. Quantification is shown of enriched or depleted proteins in pull-downs of representative TR2 variants (labeled with 13C isotope, H) relative to the WT (TR2-63) (labeled with 12C isotope, L). Data points represent mean ± SD, n = 2. p values were evaluated using the unpaired two-tailed t test. Asterisks indicate p < 0.05. See also Table S5.(B) Co-localization of the Hsp70 chaperone Ssa2 and the expanded Ssn6 (TR2-105). Live cells of TR2 variants containing Ssn6-YFP and Ssa2-RFP fusion proteins were visualized by fluorescence and DIC microscopy.(C) Ssa2 maintains Ssn6 function. Analytical flow cytometry profiles of flo11::YFP in the TR2 variants and their Δssa2 counterparts show how deletion of SSA2 affects the expression of the FLO11 target gene. See also Figure S6.(D) Sequestration of expanded Ssn6 (TR2-105) in intranuclear foci (arrowheads) (left) and increased Ssn6 aggregation in the absence of Ssa2 (right). Live cells of TR2 variants containing Ssn6-YFP fusion and their Δssa2 counterparts were visualized by fluorescence and DIC microscopy. See also Figure S6.(E) The SSN6 TR2 region modulates Ssn6 solubility. Cultures of HA-tagged Ssn6 TR2 variants were analyzed by protein aggregation assay. Soluble and insoluble fractions of Ssn6 were quantified by western blot. For each variant, the ratio of the respective fraction over the sum of the soluble and insoluble fractions was calculated. The horizontal line indicates the mean of the insoluble fraction of the WT Ssn6 (TR2-63). Data represent mean ± SD, n = 3 or n = 2. p values were evaluated using the unpaired two-tailed t test. Asterisks indicate p < 0.05, ns, not significant.(F) Disruption of the Ssn6 TR2 conformation by proline insertions. Sequencing of the Ssn6 TR2 region show the replacement of two (mutant a) or three (mutants b and c) glutamine-alanine residues by prolines (red).(G) The α-helical coiled-coil structure of TR2 is essential for Ssn6 function. Median flo11::YFP fluorescence in the QA/P mutants (a, b, c) (striped bars) is comparable to the TR2-0 variant (green bar). Data points represent mean ± SD, n = 2.
Mentions: To check for changes in the Ssn6 stability and interactome, we fused the Ssn6 repeat variants (TR2-0, TR2-55, TR2-63, TR2-90, and TR2-105) to six copies of an HA epitope tag at their C terminus. Using gel-bound anti-HA antibodies, we isolated the tagged Ssn6 from soluble protein extracts. Bound proteins were separated by SDS-PAGE, followed by in-gel digestion and identification by mass spectrometry (Figure 5A, left). This qualitative approach shows that the interactomes of the repeat deletion and expansion variants are larger than that of the WT and also contain common elements (Figure 5A, left). Notably, these common interactors are involved in active chromatin remodeling (e.g., the histone variant H2AZ, Dot6, Yku80), transcription (Ppr1, Tfg1), and signaling (Pkc1, Bmh1, Ypt1). To confirm these results and to accurately quantify TR2-dependent changes in the Ssn6 interactome, we repeated the pull-down experiment and eluted the antibody-bound protein complexes. The eluted fractions were digested, and the peptide mixtures from the WT were labeled with a light isotope (12C3-propionate), while those from the TR2 variants were labeled with a heavy isotope (13C3-propionate). The labeled peptides from each TR2 variant and the WT were mixed 1:1 and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Figure 5A, right). Among the 85 proteins detected, we considered the ones identified by at least two different peptides in all five TR2 variants tested (Table S5). We find >2-fold depletion of Ssn6 and its known interaction partner Tup1 in the TR2-105 variant relative to the WT (Figure 5A, right). Concurrently, we find a significant enrichment of the Hsp70 chaperone Ssa2 in the TR2 expansion (TR2-90 and TR2-105) and deletion (TR2-0) variants. Ssa2 is a cytosolic chaperone required for protein folding, translocation, and degradation of unfolded proteins. It also partners with Hsp40 co-chaperones to promote the refolding of aggregated or unfolded proteins (Verghese et al., 2012). Interestingly, we find that Ssa2 co-localizes with expanded Ssn6 in the nucleus, but not with the WT or TR2-0 variant (Figure 5B).

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