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Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism.

Lu H, Xue Y, Xue Y, Yu GK, Arias C, Lin J, Fong S, Faure M, Weisburd B, Ji X, Mercier A, Sutton J, Luo K, Gao Z, Zhou Q - Elife (2015)

Bottom Line: Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing.While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment.Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy.

No MeSH data available.


Related in: MedlinePlus

i-CDK9 causes widespread promoter-proximal pausing by Pol II and the biggest decrease in expression for genes involved in regulation of transcription and RNA metabolic process.(A) Schematic diagram illustrating calculation of the Pol II traveling ratio (TR). (B) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq under the various conditions as indicated. The pie charts below describe the percentages of genes with 1.5-fold increase, 1.5-fold decrease, or no change in TR after exposure to i-CDK9 for 2 or 8 hr as compared to DMSO. (C) Occupancy of Pol II as revealed by ChIP-seq across 4 representative genes with increased TR after CDK9 inhibition. The read coverage is shown for the entire gene plus a margin on either side equal to 7% of the gene length. (D) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq. The genes are grouped by expression changes induced by i-CDK9. Up: the 138 genes that showed at least twofold increase in expression after exposure to i-CDK9 or 8 hr. Other: genes whose expression was either unaffected or affected less than twofold by i-CDK9. (E) Enrichment of GO biological processes by DAVID. Only top 4 gene sets are shown for top 500 genes with the biggest increase in TR at 8 hr treatment with i-CDK9 (top) and top 500 genes with the largest decrease in gene expression at 8 hr i-CDK9 treatment (bottom).DOI:http://dx.doi.org/10.7554/eLife.06535.006
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fig2: i-CDK9 causes widespread promoter-proximal pausing by Pol II and the biggest decrease in expression for genes involved in regulation of transcription and RNA metabolic process.(A) Schematic diagram illustrating calculation of the Pol II traveling ratio (TR). (B) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq under the various conditions as indicated. The pie charts below describe the percentages of genes with 1.5-fold increase, 1.5-fold decrease, or no change in TR after exposure to i-CDK9 for 2 or 8 hr as compared to DMSO. (C) Occupancy of Pol II as revealed by ChIP-seq across 4 representative genes with increased TR after CDK9 inhibition. The read coverage is shown for the entire gene plus a margin on either side equal to 7% of the gene length. (D) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq. The genes are grouped by expression changes induced by i-CDK9. Up: the 138 genes that showed at least twofold increase in expression after exposure to i-CDK9 or 8 hr. Other: genes whose expression was either unaffected or affected less than twofold by i-CDK9. (E) Enrichment of GO biological processes by DAVID. Only top 4 gene sets are shown for top 500 genes with the biggest increase in TR at 8 hr treatment with i-CDK9 (top) and top 500 genes with the largest decrease in gene expression at 8 hr i-CDK9 treatment (bottom).DOI:http://dx.doi.org/10.7554/eLife.06535.006

Mentions: To investigate the global impact of CDK9 inhibition on transcriptional elongation by Pol II, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was performed to examine the genome-wide occupancy of Pol II before and after HeLa cells were treated with i-CDK9. Similar to the situation reported in other cell types (Zeitlinger et al., 2007; Rahl et al., 2010; Liu et al., 2013), among the total of 11,197 genes with detectable Pol peaks, 7805 (70%) showed a traveling ratio (TR; also called the pausing index; (Zeitlinger et al., 2007; Rahl et al., 2010) greater than 2.0 in the control DMSO-treated HeLa cells. Since TR is defined as the relative ratio of Pol II density in the promoter-proximal region vs the gene body (Figure 2A), the above numbers suggest that the majority of the genes were experiencing promoter-proximal pausing by Pol II in the control cells. Importantly, upon treatment with i-CDK9 for 2 and 8 hr, 6339 (56.6%) and 7658 (68.4%) genes displayed an increase of at least 1.5-fold in Pol II TR, respectively (Figure 2B), indicating significantly elevated promoter-proximal pausing by Pol II upon CDK9 inhibition. Figure 2C shows four representative genes (SMUG1, TMEM115, SEC13 and CSNK1D) with significantly elevated Pol II TR upon 8 hr of i-CDK9 treatment.10.7554/eLife.06535.006Figure 2.i-CDK9 causes widespread promoter-proximal pausing by Pol II and the biggest decrease in expression for genes involved in regulation of transcription and RNA metabolic process.


Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism.

Lu H, Xue Y, Xue Y, Yu GK, Arias C, Lin J, Fong S, Faure M, Weisburd B, Ji X, Mercier A, Sutton J, Luo K, Gao Z, Zhou Q - Elife (2015)

i-CDK9 causes widespread promoter-proximal pausing by Pol II and the biggest decrease in expression for genes involved in regulation of transcription and RNA metabolic process.(A) Schematic diagram illustrating calculation of the Pol II traveling ratio (TR). (B) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq under the various conditions as indicated. The pie charts below describe the percentages of genes with 1.5-fold increase, 1.5-fold decrease, or no change in TR after exposure to i-CDK9 for 2 or 8 hr as compared to DMSO. (C) Occupancy of Pol II as revealed by ChIP-seq across 4 representative genes with increased TR after CDK9 inhibition. The read coverage is shown for the entire gene plus a margin on either side equal to 7% of the gene length. (D) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq. The genes are grouped by expression changes induced by i-CDK9. Up: the 138 genes that showed at least twofold increase in expression after exposure to i-CDK9 or 8 hr. Other: genes whose expression was either unaffected or affected less than twofold by i-CDK9. (E) Enrichment of GO biological processes by DAVID. Only top 4 gene sets are shown for top 500 genes with the biggest increase in TR at 8 hr treatment with i-CDK9 (top) and top 500 genes with the largest decrease in gene expression at 8 hr i-CDK9 treatment (bottom).DOI:http://dx.doi.org/10.7554/eLife.06535.006
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4490784&req=5

fig2: i-CDK9 causes widespread promoter-proximal pausing by Pol II and the biggest decrease in expression for genes involved in regulation of transcription and RNA metabolic process.(A) Schematic diagram illustrating calculation of the Pol II traveling ratio (TR). (B) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq under the various conditions as indicated. The pie charts below describe the percentages of genes with 1.5-fold increase, 1.5-fold decrease, or no change in TR after exposure to i-CDK9 for 2 or 8 hr as compared to DMSO. (C) Occupancy of Pol II as revealed by ChIP-seq across 4 representative genes with increased TR after CDK9 inhibition. The read coverage is shown for the entire gene plus a margin on either side equal to 7% of the gene length. (D) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq. The genes are grouped by expression changes induced by i-CDK9. Up: the 138 genes that showed at least twofold increase in expression after exposure to i-CDK9 or 8 hr. Other: genes whose expression was either unaffected or affected less than twofold by i-CDK9. (E) Enrichment of GO biological processes by DAVID. Only top 4 gene sets are shown for top 500 genes with the biggest increase in TR at 8 hr treatment with i-CDK9 (top) and top 500 genes with the largest decrease in gene expression at 8 hr i-CDK9 treatment (bottom).DOI:http://dx.doi.org/10.7554/eLife.06535.006
Mentions: To investigate the global impact of CDK9 inhibition on transcriptional elongation by Pol II, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was performed to examine the genome-wide occupancy of Pol II before and after HeLa cells were treated with i-CDK9. Similar to the situation reported in other cell types (Zeitlinger et al., 2007; Rahl et al., 2010; Liu et al., 2013), among the total of 11,197 genes with detectable Pol peaks, 7805 (70%) showed a traveling ratio (TR; also called the pausing index; (Zeitlinger et al., 2007; Rahl et al., 2010) greater than 2.0 in the control DMSO-treated HeLa cells. Since TR is defined as the relative ratio of Pol II density in the promoter-proximal region vs the gene body (Figure 2A), the above numbers suggest that the majority of the genes were experiencing promoter-proximal pausing by Pol II in the control cells. Importantly, upon treatment with i-CDK9 for 2 and 8 hr, 6339 (56.6%) and 7658 (68.4%) genes displayed an increase of at least 1.5-fold in Pol II TR, respectively (Figure 2B), indicating significantly elevated promoter-proximal pausing by Pol II upon CDK9 inhibition. Figure 2C shows four representative genes (SMUG1, TMEM115, SEC13 and CSNK1D) with significantly elevated Pol II TR upon 8 hr of i-CDK9 treatment.10.7554/eLife.06535.006Figure 2.i-CDK9 causes widespread promoter-proximal pausing by Pol II and the biggest decrease in expression for genes involved in regulation of transcription and RNA metabolic process.

Bottom Line: Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing.While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment.Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy.

No MeSH data available.


Related in: MedlinePlus