Limits...
Stc1: a critical link between RNAi and chromatin modification required for heterochromatin integrity.

Bayne EH, White SA, Kagansky A, Bijos DA, Sanchez-Pulido L, Hoe KL, Kim DU, Park HO, Ponting CP, Rappsilber J, Allshire RC - Cell (2010)

Bottom Line: Our analyses show that Stc1 is specifically required to establish H3K9 methylation via RNAi, and interacts both with the RNAi effector Ago1, and with the chromatin-modifying CLRC complex.Moreover, tethering Stc1 to a euchromatic locus is sufficient to induce silencing and heterochromatin formation independently of RNAi.We conclude that Stc1 associates with RITS on centromeric transcripts and recruits CLRC, thereby coupling RNAi to chromatin modification.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.

Show MeSH
Stc1 Interacts with CLRC and Ago1(A) List of proteins found specifically and reproducibly associated with Stc1-FLAG by affinity purification and mass spectrometry (LC-MS/MS). Average numbers of peptides identified in each replicate are shown.(B and C) Stc1-FLAG IP followed by western analysis determining requirements for Stc1 association with Rik1-myc, GFP-Dos1, Dos2-HA, myc-Clr4, and myc-Ago1.(D) Clr4 association with Ago1 requires Stc1. Flag-Clr4 IP followed by western analysis of myc-Ago1.See also Figure S3.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2875855&req=5

fig3: Stc1 Interacts with CLRC and Ago1(A) List of proteins found specifically and reproducibly associated with Stc1-FLAG by affinity purification and mass spectrometry (LC-MS/MS). Average numbers of peptides identified in each replicate are shown.(B and C) Stc1-FLAG IP followed by western analysis determining requirements for Stc1 association with Rik1-myc, GFP-Dos1, Dos2-HA, myc-Clr4, and myc-Ago1.(D) Clr4 association with Ago1 requires Stc1. Flag-Clr4 IP followed by western analysis of myc-Ago1.See also Figure S3.

Mentions: To gain further insight into the functional relationship between Stc1 and known RNAi and chromatin components, we affinity selected Stc1-FLAG from cell lysates and identified coprecipitating proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specificity was ensured by subtracting proteins identified in immunoprecipitates (IPs) from untagged control strains (Figure S3A and Tables S3 and S4). Notably, many of the identified peptides corresponded to CLRC components: Cul4, Rik1, Dos1, and Dos2. Nedd8 peptides were also detected, consistent with Cul4 being neddylated (Figure 3A). The association of Stc1-FLAG with Rik1, Dos1, and Dos2 was confirmed by western analysis of IPs to detect Rik1-myc, GFP-Dos1, and Dos2-HA. In addition, although the CLRC component Clr4 was not detected in our LC-MS/MS analysis, association of myc-Clr4 with Stc1-FLAG was detected by IP-western (Figure 3B and Figures S3B and S3C). Interestingly, association of Stc1-FLAG with the RITS component Ago1 (myc-Ago1) was also observed, consistent with the RNAi-like (dcr1Δ) phenotype of stc1Δ cells (Figure 3C and Figure S3D). Association between Stc1-FLAG and RDRC components was not detected (Figure S3E). The finding that Stc1 associates with both CLRC and Ago1, along with the phenotype of stc1Δ cells, indicates that Stc1 may be a key bridging protein that connects the chromatin modification machinery to RNAi.


Stc1: a critical link between RNAi and chromatin modification required for heterochromatin integrity.

Bayne EH, White SA, Kagansky A, Bijos DA, Sanchez-Pulido L, Hoe KL, Kim DU, Park HO, Ponting CP, Rappsilber J, Allshire RC - Cell (2010)

Stc1 Interacts with CLRC and Ago1(A) List of proteins found specifically and reproducibly associated with Stc1-FLAG by affinity purification and mass spectrometry (LC-MS/MS). Average numbers of peptides identified in each replicate are shown.(B and C) Stc1-FLAG IP followed by western analysis determining requirements for Stc1 association with Rik1-myc, GFP-Dos1, Dos2-HA, myc-Clr4, and myc-Ago1.(D) Clr4 association with Ago1 requires Stc1. Flag-Clr4 IP followed by western analysis of myc-Ago1.See also Figure S3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875855&req=5

fig3: Stc1 Interacts with CLRC and Ago1(A) List of proteins found specifically and reproducibly associated with Stc1-FLAG by affinity purification and mass spectrometry (LC-MS/MS). Average numbers of peptides identified in each replicate are shown.(B and C) Stc1-FLAG IP followed by western analysis determining requirements for Stc1 association with Rik1-myc, GFP-Dos1, Dos2-HA, myc-Clr4, and myc-Ago1.(D) Clr4 association with Ago1 requires Stc1. Flag-Clr4 IP followed by western analysis of myc-Ago1.See also Figure S3.
Mentions: To gain further insight into the functional relationship between Stc1 and known RNAi and chromatin components, we affinity selected Stc1-FLAG from cell lysates and identified coprecipitating proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specificity was ensured by subtracting proteins identified in immunoprecipitates (IPs) from untagged control strains (Figure S3A and Tables S3 and S4). Notably, many of the identified peptides corresponded to CLRC components: Cul4, Rik1, Dos1, and Dos2. Nedd8 peptides were also detected, consistent with Cul4 being neddylated (Figure 3A). The association of Stc1-FLAG with Rik1, Dos1, and Dos2 was confirmed by western analysis of IPs to detect Rik1-myc, GFP-Dos1, and Dos2-HA. In addition, although the CLRC component Clr4 was not detected in our LC-MS/MS analysis, association of myc-Clr4 with Stc1-FLAG was detected by IP-western (Figure 3B and Figures S3B and S3C). Interestingly, association of Stc1-FLAG with the RITS component Ago1 (myc-Ago1) was also observed, consistent with the RNAi-like (dcr1Δ) phenotype of stc1Δ cells (Figure 3C and Figure S3D). Association between Stc1-FLAG and RDRC components was not detected (Figure S3E). The finding that Stc1 associates with both CLRC and Ago1, along with the phenotype of stc1Δ cells, indicates that Stc1 may be a key bridging protein that connects the chromatin modification machinery to RNAi.

Bottom Line: Our analyses show that Stc1 is specifically required to establish H3K9 methylation via RNAi, and interacts both with the RNAi effector Ago1, and with the chromatin-modifying CLRC complex.Moreover, tethering Stc1 to a euchromatic locus is sufficient to induce silencing and heterochromatin formation independently of RNAi.We conclude that Stc1 associates with RITS on centromeric transcripts and recruits CLRC, thereby coupling RNAi to chromatin modification.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.

Show MeSH