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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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Regulation of Rpn4 levels partly mediated the suppression of rpb1-CTD11 defects by loss of CDK8.(A) Cdk8 occupied the promoters of genes whose expression increased in the rpb1-CTD11 mutant regardless of CTD length. (B) Boxplot comparing average Cdk8 occupancy scores at the promoters of genes whose expression increased in the rpb1-CTD11 mutant (increased) to all other genes in the genome (not increased). Significantly higher Cdk8 occupancy occurred at the promoters of genes with increased expression levels in both the wild type and the rpb1-CTD11 mutant. (C) The sensitivity of rpb1-CTD11, cdk8Δ, rpn4Δ single, double and triple mutants in the W303 background was tested by plating ten-fold serial dilutions on YPD media at 16, 30 and 37°C and YPD media containing the indicated concentrations of hydroxyurea or formamide. Deletion of RPN4 abolished the suppression. (D) Immunoblot of Rpn4 protein levels identified an increase of Rpn4 in rpb1-CTD11 mutants that was reduced upon deletion of CDK8. Pgk1 was used as a loading control. (E) Cdk8 regulated the stability of Rpn4 in vivo. Rpn4 protein stability was measured at the indicated time points under wild type and cdk8Δ conditions. Pgk1 was used as a loading control.
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pgen-1003758-g008: Regulation of Rpn4 levels partly mediated the suppression of rpb1-CTD11 defects by loss of CDK8.(A) Cdk8 occupied the promoters of genes whose expression increased in the rpb1-CTD11 mutant regardless of CTD length. (B) Boxplot comparing average Cdk8 occupancy scores at the promoters of genes whose expression increased in the rpb1-CTD11 mutant (increased) to all other genes in the genome (not increased). Significantly higher Cdk8 occupancy occurred at the promoters of genes with increased expression levels in both the wild type and the rpb1-CTD11 mutant. (C) The sensitivity of rpb1-CTD11, cdk8Δ, rpn4Δ single, double and triple mutants in the W303 background was tested by plating ten-fold serial dilutions on YPD media at 16, 30 and 37°C and YPD media containing the indicated concentrations of hydroxyurea or formamide. Deletion of RPN4 abolished the suppression. (D) Immunoblot of Rpn4 protein levels identified an increase of Rpn4 in rpb1-CTD11 mutants that was reduced upon deletion of CDK8. Pgk1 was used as a loading control. (E) Cdk8 regulated the stability of Rpn4 in vivo. Rpn4 protein stability was measured at the indicated time points under wild type and cdk8Δ conditions. Pgk1 was used as a loading control.

Mentions: To understand the mechanism underlying the restoration of the transcription and RNAPII recruitment changes in the rpb1-CTD11 mutant upon loss of CDK8, we first tried to understand the role of Cdk8 in regulating these genes. To determine if Cdk8 played a direct regulatory role at these genes, we generated a genome-wide map of Cdk8 occupancy under wild type conditions (Complete dataset can be found in array-express, code E-MTAB-1379). The average gene occupancy of Cdk8 showed clear enrichment at promoters, although we did identify Cdk8 binding to a small number of ORFs (Figure S5) [22], [23], [46]. Focusing on CTD-length dependent genes, we observed Cdk8 occupancy at the promoters of genes with increased mRNA levels in the rpb1-CTD11 mutant (Figure 8A), while very little Cdk8 was observed at the set of genes with decreased levels (data not shown). Importantly, Cdk8 occupancy was not significantly altered in strains with a truncated CTD (Figure 8A). In both situations, the preferential association of Cdk8 with the genes having increased expression was significant even when compared to all genes in the genome (one-tailed, unpaired t-test p-value 0.0001079 for wild-type and 0.005898 for rpb1-CTD11, respectively), thus supporting a direct regulatory role for Cdk8 at these loci (Figure 8B). However, despite its significant association and robust effect on normalizing the expression levels of this set of genes, our gene expression analysis clearly showed that Cdk8 was not the sole regulator of these genes as these were generally normal in cdk8Δ mutants (Figure 6A) [47].


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)

Regulation of Rpn4 levels partly mediated the suppression of rpb1-CTD11 defects by loss of CDK8.(A) Cdk8 occupied the promoters of genes whose expression increased in the rpb1-CTD11 mutant regardless of CTD length. (B) Boxplot comparing average Cdk8 occupancy scores at the promoters of genes whose expression increased in the rpb1-CTD11 mutant (increased) to all other genes in the genome (not increased). Significantly higher Cdk8 occupancy occurred at the promoters of genes with increased expression levels in both the wild type and the rpb1-CTD11 mutant. (C) The sensitivity of rpb1-CTD11, cdk8Δ, rpn4Δ single, double and triple mutants in the W303 background was tested by plating ten-fold serial dilutions on YPD media at 16, 30 and 37°C and YPD media containing the indicated concentrations of hydroxyurea or formamide. Deletion of RPN4 abolished the suppression. (D) Immunoblot of Rpn4 protein levels identified an increase of Rpn4 in rpb1-CTD11 mutants that was reduced upon deletion of CDK8. Pgk1 was used as a loading control. (E) Cdk8 regulated the stability of Rpn4 in vivo. Rpn4 protein stability was measured at the indicated time points under wild type and cdk8Δ conditions. Pgk1 was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003758-g008: Regulation of Rpn4 levels partly mediated the suppression of rpb1-CTD11 defects by loss of CDK8.(A) Cdk8 occupied the promoters of genes whose expression increased in the rpb1-CTD11 mutant regardless of CTD length. (B) Boxplot comparing average Cdk8 occupancy scores at the promoters of genes whose expression increased in the rpb1-CTD11 mutant (increased) to all other genes in the genome (not increased). Significantly higher Cdk8 occupancy occurred at the promoters of genes with increased expression levels in both the wild type and the rpb1-CTD11 mutant. (C) The sensitivity of rpb1-CTD11, cdk8Δ, rpn4Δ single, double and triple mutants in the W303 background was tested by plating ten-fold serial dilutions on YPD media at 16, 30 and 37°C and YPD media containing the indicated concentrations of hydroxyurea or formamide. Deletion of RPN4 abolished the suppression. (D) Immunoblot of Rpn4 protein levels identified an increase of Rpn4 in rpb1-CTD11 mutants that was reduced upon deletion of CDK8. Pgk1 was used as a loading control. (E) Cdk8 regulated the stability of Rpn4 in vivo. Rpn4 protein stability was measured at the indicated time points under wild type and cdk8Δ conditions. Pgk1 was used as a loading control.
Mentions: To understand the mechanism underlying the restoration of the transcription and RNAPII recruitment changes in the rpb1-CTD11 mutant upon loss of CDK8, we first tried to understand the role of Cdk8 in regulating these genes. To determine if Cdk8 played a direct regulatory role at these genes, we generated a genome-wide map of Cdk8 occupancy under wild type conditions (Complete dataset can be found in array-express, code E-MTAB-1379). The average gene occupancy of Cdk8 showed clear enrichment at promoters, although we did identify Cdk8 binding to a small number of ORFs (Figure S5) [22], [23], [46]. Focusing on CTD-length dependent genes, we observed Cdk8 occupancy at the promoters of genes with increased mRNA levels in the rpb1-CTD11 mutant (Figure 8A), while very little Cdk8 was observed at the set of genes with decreased levels (data not shown). Importantly, Cdk8 occupancy was not significantly altered in strains with a truncated CTD (Figure 8A). In both situations, the preferential association of Cdk8 with the genes having increased expression was significant even when compared to all genes in the genome (one-tailed, unpaired t-test p-value 0.0001079 for wild-type and 0.005898 for rpb1-CTD11, respectively), thus supporting a direct regulatory role for Cdk8 at these loci (Figure 8B). However, despite its significant association and robust effect on normalizing the expression levels of this set of genes, our gene expression analysis clearly showed that Cdk8 was not the sole regulator of these genes as these were generally normal in cdk8Δ mutants (Figure 6A) [47].

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