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High-throughput genetic and gene expression analysis of the RNAPII-CTD reveals unexpected connections to SRB10/CDK8.

Aristizabal MJ, Negri GL, Benschop JJ, Holstege FC, Krogan NJ, Kobor MS - PLoS Genet. (2013)

Bottom Line: Truncating the CTD altered RNAPII occupancy, leading to not only decreases, but also increases in mRNA levels.This suggested a positive role of Cdk8 in relationship to RNAPII, which contrasted with the observed negative role at the activated INO1 gene.Here, loss of CDK8 suppressed the reduced mRNA expression and RNAPII occupancy levels of CTD truncation mutants.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
The C-terminal domain (CTD) of RNA polymerase II (RNAPII) is composed of heptapeptide repeats, which play a key regulatory role in gene expression. Using genetic interaction, chromatin immunoprecipitation followed by microarrays (ChIP-on-chip) and mRNA expression analysis, we found that truncating the CTD resulted in distinct changes to cellular function. Truncating the CTD altered RNAPII occupancy, leading to not only decreases, but also increases in mRNA levels. The latter were largely mediated by promoter elements and in part were linked to the transcription factor Rpn4. The mediator subunit Cdk8 was enriched at promoters of these genes, and its removal not only restored normal mRNA and RNAPII occupancy levels, but also reduced the abnormally high cellular amounts of Rpn4. This suggested a positive role of Cdk8 in relationship to RNAPII, which contrasted with the observed negative role at the activated INO1 gene. Here, loss of CDK8 suppressed the reduced mRNA expression and RNAPII occupancy levels of CTD truncation mutants.

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Genome-wide occupancy profiles of RNAPII identified a direct effect for the CTD in transcription regulation.(A) Chromosome plots of relative Rpb3 occupancy revealed similar profiles between wild type and rpb1-CTD11 mutants. Rpb3 occupancy differences were observed in the rpb1-CTD11 mutant at genes identified to have significantly increased (YNL037C - top) or decreased (YDR033W - bottom) mRNA levels. Light gray boxes depict ORFs and dark gray boxes depict ARSs. (B) Average gene profile of Rpb3 in genes with increased (left) or decreased (right) mRNA levels upon truncation of the CTD. (C) Average Rpb3 occupancy scores at coding regions with increased (left) (p value 3.36e-7) or decreased (right) (p value 2.98e-22) mRNA levels revealed an intimate link between Rpb3 binding and expression levels.
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pgen-1003758-g003: Genome-wide occupancy profiles of RNAPII identified a direct effect for the CTD in transcription regulation.(A) Chromosome plots of relative Rpb3 occupancy revealed similar profiles between wild type and rpb1-CTD11 mutants. Rpb3 occupancy differences were observed in the rpb1-CTD11 mutant at genes identified to have significantly increased (YNL037C - top) or decreased (YDR033W - bottom) mRNA levels. Light gray boxes depict ORFs and dark gray boxes depict ARSs. (B) Average gene profile of Rpb3 in genes with increased (left) or decreased (right) mRNA levels upon truncation of the CTD. (C) Average Rpb3 occupancy scores at coding regions with increased (left) (p value 3.36e-7) or decreased (right) (p value 2.98e-22) mRNA levels revealed an intimate link between Rpb3 binding and expression levels.

Mentions: The measured gene expression changes in CTD truncation mutants could result from either effects on the synthesis or stability of the mRNA. To differentiate between these two possibilities, we measured RNAPII occupancy genome-wide and determined if the changes in gene expression correlated with alterations in RNAPII occupancy (Complete dataset can be found in array-express, code E-MTAB-1341). Specifically, we measured RNAPII in rpb1-CTD11 and wild type cells by chromatin immunoprecipitation followed by hybridization on a whole genome tiled microarray (ChIP-on-chip) using an antibody specific to the RNAPII subunit Rpb3. Despite the use of different platforms, antibodies and normalization methods, the obtained genome-wide Rpb3 occupancy profiles obtained in wild type cells were highly correlated with those previously published by several groups (Figure S2) [35]–[39]. Furthermore, the occupancy maps revealed highly correlated profiles between rpb1-CTD11 and wild type cells (Spearman's rho 0.85), agreeing with the limited transcriptional differences detected by the expression analysis. Nonetheless, our Rpb3 occupancy plots showed clear RNAPII occupancy differences along genes that were identified as either having increased or decreased mRNA levels in the rpb1-CTD11 mutant (Figure 3A and B). Accordingly, plotting the average Rpb3 occupancy scores of the differentially regulated genes in rpb1-CTD11 versus wild type cells revealed that the genes with increased mRNA levels had a significant increase in Rpb3 binding levels along their coding regions while the genes with decreased mRNA levels had a significant decrease (one-tailed t-test p value 2.98e-22 and 3.36e-7, respectively), thus suggesting a direct effect of truncating the CTD on RNAPII levels and mRNA synthesis at specific loci (Figure 3C).


High-throughput genetic and gene expression analysis of the RNAPII-CTD reveals unexpected connections to SRB10/CDK8.

Aristizabal MJ, Negri GL, Benschop JJ, Holstege FC, Krogan NJ, Kobor MS - PLoS Genet. (2013)

Genome-wide occupancy profiles of RNAPII identified a direct effect for the CTD in transcription regulation.(A) Chromosome plots of relative Rpb3 occupancy revealed similar profiles between wild type and rpb1-CTD11 mutants. Rpb3 occupancy differences were observed in the rpb1-CTD11 mutant at genes identified to have significantly increased (YNL037C - top) or decreased (YDR033W - bottom) mRNA levels. Light gray boxes depict ORFs and dark gray boxes depict ARSs. (B) Average gene profile of Rpb3 in genes with increased (left) or decreased (right) mRNA levels upon truncation of the CTD. (C) Average Rpb3 occupancy scores at coding regions with increased (left) (p value 3.36e-7) or decreased (right) (p value 2.98e-22) mRNA levels revealed an intimate link between Rpb3 binding and expression levels.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3757075&req=5

pgen-1003758-g003: Genome-wide occupancy profiles of RNAPII identified a direct effect for the CTD in transcription regulation.(A) Chromosome plots of relative Rpb3 occupancy revealed similar profiles between wild type and rpb1-CTD11 mutants. Rpb3 occupancy differences were observed in the rpb1-CTD11 mutant at genes identified to have significantly increased (YNL037C - top) or decreased (YDR033W - bottom) mRNA levels. Light gray boxes depict ORFs and dark gray boxes depict ARSs. (B) Average gene profile of Rpb3 in genes with increased (left) or decreased (right) mRNA levels upon truncation of the CTD. (C) Average Rpb3 occupancy scores at coding regions with increased (left) (p value 3.36e-7) or decreased (right) (p value 2.98e-22) mRNA levels revealed an intimate link between Rpb3 binding and expression levels.
Mentions: The measured gene expression changes in CTD truncation mutants could result from either effects on the synthesis or stability of the mRNA. To differentiate between these two possibilities, we measured RNAPII occupancy genome-wide and determined if the changes in gene expression correlated with alterations in RNAPII occupancy (Complete dataset can be found in array-express, code E-MTAB-1341). Specifically, we measured RNAPII in rpb1-CTD11 and wild type cells by chromatin immunoprecipitation followed by hybridization on a whole genome tiled microarray (ChIP-on-chip) using an antibody specific to the RNAPII subunit Rpb3. Despite the use of different platforms, antibodies and normalization methods, the obtained genome-wide Rpb3 occupancy profiles obtained in wild type cells were highly correlated with those previously published by several groups (Figure S2) [35]–[39]. Furthermore, the occupancy maps revealed highly correlated profiles between rpb1-CTD11 and wild type cells (Spearman's rho 0.85), agreeing with the limited transcriptional differences detected by the expression analysis. Nonetheless, our Rpb3 occupancy plots showed clear RNAPII occupancy differences along genes that were identified as either having increased or decreased mRNA levels in the rpb1-CTD11 mutant (Figure 3A and B). Accordingly, plotting the average Rpb3 occupancy scores of the differentially regulated genes in rpb1-CTD11 versus wild type cells revealed that the genes with increased mRNA levels had a significant increase in Rpb3 binding levels along their coding regions while the genes with decreased mRNA levels had a significant decrease (one-tailed t-test p value 2.98e-22 and 3.36e-7, respectively), thus suggesting a direct effect of truncating the CTD on RNAPII levels and mRNA synthesis at specific loci (Figure 3C).

Bottom Line: Truncating the CTD altered RNAPII occupancy, leading to not only decreases, but also increases in mRNA levels.This suggested a positive role of Cdk8 in relationship to RNAPII, which contrasted with the observed negative role at the activated INO1 gene.Here, loss of CDK8 suppressed the reduced mRNA expression and RNAPII occupancy levels of CTD truncation mutants.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
The C-terminal domain (CTD) of RNA polymerase II (RNAPII) is composed of heptapeptide repeats, which play a key regulatory role in gene expression. Using genetic interaction, chromatin immunoprecipitation followed by microarrays (ChIP-on-chip) and mRNA expression analysis, we found that truncating the CTD resulted in distinct changes to cellular function. Truncating the CTD altered RNAPII occupancy, leading to not only decreases, but also increases in mRNA levels. The latter were largely mediated by promoter elements and in part were linked to the transcription factor Rpn4. The mediator subunit Cdk8 was enriched at promoters of these genes, and its removal not only restored normal mRNA and RNAPII occupancy levels, but also reduced the abnormally high cellular amounts of Rpn4. This suggested a positive role of Cdk8 in relationship to RNAPII, which contrasted with the observed negative role at the activated INO1 gene. Here, loss of CDK8 suppressed the reduced mRNA expression and RNAPII occupancy levels of CTD truncation mutants.

Show MeSH
Related in: MedlinePlus