Limits...
Cohesin proteins promote ribosomal RNA production and protein translation in yeast and human cells.

Bose T, Lee KK, Lu S, Xu B, Harris B, Slaughter B, Unruh J, Garrett A, McDowell W, Box A, Li H, Peak A, Ramachandran S, Seidel C, Gerton JL - PLoS Genet. (2012)

Bottom Line: These mutations do not significantly affect chromosome segregation.We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and (35)S-methionine incorporation, all of which indicate a deficit in protein translation.Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

ABSTRACT
Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of cohesin in gene expression is not well understood. We used budding yeast strains bearing mutations analogous to the human cohesinopathy disease alleles under control of their native promoter to study gene expression. These mutations do not significantly affect chromosome segregation. Transcriptional profiling reveals that many targets of the transcriptional activator Gcn4 are induced in the eco1-W216G mutant background. The upregulation of Gcn4 was observed in many cohesin mutants, and this observation suggested protein translation was reduced. We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and (35)S-methionine incorporation, all of which indicate a deficit in protein translation. Metabolic labeling shows that the eco1-W216G and smc1-Q843Δ mutants produce less ribosomal RNA, which is expected to constrain ribosome biogenesis. Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged. Similar defects in rRNA production and protein translation are observed in a human Roberts syndrome cell line. In addition, cohesion is defective specifically at the rDNA locus in the eco1-W216G mutant, as has been previously reported for Roberts syndrome. Collectively, our data suggest that cohesin proteins normally facilitate production of ribosomal RNA and protein translation, and this is one way they can influence gene expression. Reduced translational capacity could contribute to the human cohesinopathies.

Show MeSH

Related in: MedlinePlus

Gcn4 targets and Gcn4 are elevated in cohesin mutants.A. Histogram for transcription factor binding sites from the eco1-W216G strain showing the number of genes upregulated or downregulated from Figure 1B that have a Gcn4 site (time 0), a Tbp1 site (time 0), or a Rap1 site (time 15 min). The p value is calculated by a hypergeometric test using the number of up or down regulated genes with the binding site versus the number of genes in the genome with the site. B. Strains with W303 background having the indicated mutations were transformed with the p180 reporter plasmid that contains a Gcn4-lacZ transgene. β-galactosidase levels (y axis) were measured for each strain in triplicate following growth to mid log phase in YPD+CSM. The error bars represent the standard deviation of at least three independent measurements. One asterisk indicates p less than or equal to 0.002, two asterisks indicates p<0.0001 from a Student's two tailed t test. C. β-galactosidase levels were measured using the p226 reporter. This construct has only the 4th uORF from the Gcn4 leader sequence, which confers minimal translational control. D. Strains with the BY4742 background with the indicated mutations were treated as in B. E. Gcn4 was tagged with the TAP epitope. Protein extracts from equal numbers of cells were used for Western blotting. Gcn4-TAP was detected with the α-PAP antibody. Pgk1 serves as a loading control. All samples were loaded on the same blot and subjected to the same exposure, but intervening lanes were removed. See Figure S2 for RT-qPCR confirmation of the misregulation of the Gcn4 targets SNO1 and SNZ1 in the mutants.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3375231&req=5

pgen-1002749-g002: Gcn4 targets and Gcn4 are elevated in cohesin mutants.A. Histogram for transcription factor binding sites from the eco1-W216G strain showing the number of genes upregulated or downregulated from Figure 1B that have a Gcn4 site (time 0), a Tbp1 site (time 0), or a Rap1 site (time 15 min). The p value is calculated by a hypergeometric test using the number of up or down regulated genes with the binding site versus the number of genes in the genome with the site. B. Strains with W303 background having the indicated mutations were transformed with the p180 reporter plasmid that contains a Gcn4-lacZ transgene. β-galactosidase levels (y axis) were measured for each strain in triplicate following growth to mid log phase in YPD+CSM. The error bars represent the standard deviation of at least three independent measurements. One asterisk indicates p less than or equal to 0.002, two asterisks indicates p<0.0001 from a Student's two tailed t test. C. β-galactosidase levels were measured using the p226 reporter. This construct has only the 4th uORF from the Gcn4 leader sequence, which confers minimal translational control. D. Strains with the BY4742 background with the indicated mutations were treated as in B. E. Gcn4 was tagged with the TAP epitope. Protein extracts from equal numbers of cells were used for Western blotting. Gcn4-TAP was detected with the α-PAP antibody. Pgk1 serves as a loading control. All samples were loaded on the same blot and subjected to the same exposure, but intervening lanes were removed. See Figure S2 for RT-qPCR confirmation of the misregulation of the Gcn4 targets SNO1 and SNZ1 in the mutants.

Mentions: The gene expression data was further analyzed to determine whether the genes that were misregulated in the eco1-W216G mutant had any enrichment for particular transcription factor binding sites in their promoter regions. In the promoters of genes that were upregulated at the time 0 timepoint, we found a significant enrichment for Gcn4 and Tbp1/Spt15 binding sites (Figure 2A). Gcn4 is a transcriptional activator that activates the expression of many classes of genes, including stress and amino acid biosynthesis genes. Tbp1/Spt15, or TATA binding protein, is an evolutionarily conserved general transcription factor that interacts with other factors to form transcription preinitiation complexes at promoters. SNO1 and SNZ1 have been reported to be upregulated in pol III mutants [22] in a Gcn4-dependent manner [25]. These genes were found to be upregulated in the microarray data. The misregulation of SNO1 and SNZ1 was confirmed by RT-qPCR (SNO1, 3-fold, SNZ1, 9-fold, eco1-W216G at time 0, Figure S2). In the promoters of genes that were downregulated at time 0 there were fewer than average Gcn4 and Tbp1/Spt15 binding sites.


Cohesin proteins promote ribosomal RNA production and protein translation in yeast and human cells.

Bose T, Lee KK, Lu S, Xu B, Harris B, Slaughter B, Unruh J, Garrett A, McDowell W, Box A, Li H, Peak A, Ramachandran S, Seidel C, Gerton JL - PLoS Genet. (2012)

Gcn4 targets and Gcn4 are elevated in cohesin mutants.A. Histogram for transcription factor binding sites from the eco1-W216G strain showing the number of genes upregulated or downregulated from Figure 1B that have a Gcn4 site (time 0), a Tbp1 site (time 0), or a Rap1 site (time 15 min). The p value is calculated by a hypergeometric test using the number of up or down regulated genes with the binding site versus the number of genes in the genome with the site. B. Strains with W303 background having the indicated mutations were transformed with the p180 reporter plasmid that contains a Gcn4-lacZ transgene. β-galactosidase levels (y axis) were measured for each strain in triplicate following growth to mid log phase in YPD+CSM. The error bars represent the standard deviation of at least three independent measurements. One asterisk indicates p less than or equal to 0.002, two asterisks indicates p<0.0001 from a Student's two tailed t test. C. β-galactosidase levels were measured using the p226 reporter. This construct has only the 4th uORF from the Gcn4 leader sequence, which confers minimal translational control. D. Strains with the BY4742 background with the indicated mutations were treated as in B. E. Gcn4 was tagged with the TAP epitope. Protein extracts from equal numbers of cells were used for Western blotting. Gcn4-TAP was detected with the α-PAP antibody. Pgk1 serves as a loading control. All samples were loaded on the same blot and subjected to the same exposure, but intervening lanes were removed. See Figure S2 for RT-qPCR confirmation of the misregulation of the Gcn4 targets SNO1 and SNZ1 in the mutants.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375231&req=5

pgen-1002749-g002: Gcn4 targets and Gcn4 are elevated in cohesin mutants.A. Histogram for transcription factor binding sites from the eco1-W216G strain showing the number of genes upregulated or downregulated from Figure 1B that have a Gcn4 site (time 0), a Tbp1 site (time 0), or a Rap1 site (time 15 min). The p value is calculated by a hypergeometric test using the number of up or down regulated genes with the binding site versus the number of genes in the genome with the site. B. Strains with W303 background having the indicated mutations were transformed with the p180 reporter plasmid that contains a Gcn4-lacZ transgene. β-galactosidase levels (y axis) were measured for each strain in triplicate following growth to mid log phase in YPD+CSM. The error bars represent the standard deviation of at least three independent measurements. One asterisk indicates p less than or equal to 0.002, two asterisks indicates p<0.0001 from a Student's two tailed t test. C. β-galactosidase levels were measured using the p226 reporter. This construct has only the 4th uORF from the Gcn4 leader sequence, which confers minimal translational control. D. Strains with the BY4742 background with the indicated mutations were treated as in B. E. Gcn4 was tagged with the TAP epitope. Protein extracts from equal numbers of cells were used for Western blotting. Gcn4-TAP was detected with the α-PAP antibody. Pgk1 serves as a loading control. All samples were loaded on the same blot and subjected to the same exposure, but intervening lanes were removed. See Figure S2 for RT-qPCR confirmation of the misregulation of the Gcn4 targets SNO1 and SNZ1 in the mutants.
Mentions: The gene expression data was further analyzed to determine whether the genes that were misregulated in the eco1-W216G mutant had any enrichment for particular transcription factor binding sites in their promoter regions. In the promoters of genes that were upregulated at the time 0 timepoint, we found a significant enrichment for Gcn4 and Tbp1/Spt15 binding sites (Figure 2A). Gcn4 is a transcriptional activator that activates the expression of many classes of genes, including stress and amino acid biosynthesis genes. Tbp1/Spt15, or TATA binding protein, is an evolutionarily conserved general transcription factor that interacts with other factors to form transcription preinitiation complexes at promoters. SNO1 and SNZ1 have been reported to be upregulated in pol III mutants [22] in a Gcn4-dependent manner [25]. These genes were found to be upregulated in the microarray data. The misregulation of SNO1 and SNZ1 was confirmed by RT-qPCR (SNO1, 3-fold, SNZ1, 9-fold, eco1-W216G at time 0, Figure S2). In the promoters of genes that were downregulated at time 0 there were fewer than average Gcn4 and Tbp1/Spt15 binding sites.

Bottom Line: These mutations do not significantly affect chromosome segregation.We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and (35)S-methionine incorporation, all of which indicate a deficit in protein translation.Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

ABSTRACT
Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of cohesin in gene expression is not well understood. We used budding yeast strains bearing mutations analogous to the human cohesinopathy disease alleles under control of their native promoter to study gene expression. These mutations do not significantly affect chromosome segregation. Transcriptional profiling reveals that many targets of the transcriptional activator Gcn4 are induced in the eco1-W216G mutant background. The upregulation of Gcn4 was observed in many cohesin mutants, and this observation suggested protein translation was reduced. We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and (35)S-methionine incorporation, all of which indicate a deficit in protein translation. Metabolic labeling shows that the eco1-W216G and smc1-Q843Δ mutants produce less ribosomal RNA, which is expected to constrain ribosome biogenesis. Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged. Similar defects in rRNA production and protein translation are observed in a human Roberts syndrome cell line. In addition, cohesion is defective specifically at the rDNA locus in the eco1-W216G mutant, as has been previously reported for Roberts syndrome. Collectively, our data suggest that cohesin proteins normally facilitate production of ribosomal RNA and protein translation, and this is one way they can influence gene expression. Reduced translational capacity could contribute to the human cohesinopathies.

Show MeSH
Related in: MedlinePlus