Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism: Pol II occupancy profiling by ChIP-Seq in HeLa cell line in the presence or absence of CDK9 inhibition
ABSTRACT: CDK9 is the kinase subunit of P-TEFb that enables RNA polymerase (Pol) II to transit from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to the 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 the simultaneous inhibition of CDK9 and MYC can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy. ChIP-seq of Pol II in HeLa cells before or after i-CDk9 treatment
Project description:CDK9 is the kinase subunit of P-TEFb that enables RNA polymerase (Pol) II to transit from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to the 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 the simultaneous inhibition of CDK9 and MYC can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy. We used microarrays to examine the global impact on gene expression by imhibiting CDK9 at different time durations. HeLa cell lines treated with CDK9 inhibitor at different time points
Project description:Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation. Comparison of the chromatin occupancy of  PAF1, CDC73, LEO1, CTR9, total Pol II, and CTD serine 2-phosphorylated Pol II by ChIP-seq in THP1 cells;  PAF1, Pol II, Pol II (ser-5p), CDK12, and CDK9 by ChIP-seq in control and PAF1 knockdown cells;  LEO1 and Pol II by ChIP-seq in control and flavopiridol treated THP1 cells.
Project description:Most metazoan promoters have RNA polymerase II (Pol II) paused slightly downstream of the transcription start site by NELF and DSIF. This pausing keeps these promoters available for rapid induction by P-TEFb, whose activity causes NELF to dissociate and Pol II and DSIF to elongate on the gene. ChIP-Seq data was generated for Pol II, NELF, and DSIF in HeLa cells and used to look at pausing downstream of unannotated promoters. 3x ChIP-Seq (Pol II, NELF, DSIF) in HeLa cells
Project description:Chromatin is highly condensed and transcriptionally repressed during mitosis. Although it is established that some general transcription factors are inactivated by phosphorylation at mitosis, many details of mitotic transcriptional repression and its underlying mechanisms are largely unknown. Here, we provide evidence that as cells enter mitosis, genes with transcriptionally engaged RNA Polymerase II (Pol II) can continue transcription until the end of the gene to clear Pol II from mitotic chromatin. Using ChIP-Seq, we find that the transcriptional reinitiation process is globally impaired in early mitosis (prophase/prometaphase), with loss of TFIIB occupancy and nucleosome-free regions at promoters. Pretreatment of nocodazole-arrested mitotic cells with the P-TEFb inhibitor flavopiridol prevents the release of promoter-proximal engaged Pol II. Global nascent RNA sequencing and RNA fluorescence in situ hybridization (FISH) of individual genes demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Chemical and mutational inhibition of P-TEFb in mitosis leads to delays in the progression of cell division. Together, our study reveals a novel mechanism for mitotic transcriptional repression whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division. ChIP-Seq of Pol II of different forms, TFIIB, H3K4me3 in human HeLa cells at different cell cycle stages. ChIP-Seq of Pol II in HeLa mitotic cells with or without CDK9 inhibitor flavopiridol pretreatment. Nascent RNA-seq in asynchronous and arrested mitotic cells.
Project description:Recent studies have found that promoter-proximal pausing occurs at most Pol2-regulated genes. DSIF and NELF function as negative elongation factors and promote Pol2 pausing. P-TEFb, whose enzymatic activity lies in the kinase Cdk9, positively regulates the transition into productive elongation by phosphorylating subunits in DSIF, NELF and Pol2. To gain insights into the interplay between these factors in regulating transcriptional pause release, we tested if knockdown of either pausing factor could bypass P-TEFb function. We find that P-TEFb function is still required for transcriptional elongation after DSIF or NELF knockdown. mES cells were infected with a shRNA hairpin to knockdown Spt5, Spt4, NelfE or control. The resulting cells were then treated with flavopiridol (1uM for 60 minutes). DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. A sample of whole cell extract was sequenced and used as the background to determine enrichment. ChIP was performed using an antibody against total RNA Pol2 (Rpb1 N-terminus, Santa Cruz sc-899).
Project description:The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to ensure proper mRNA maturation and prevent interference between neighboring genes. Pol II slowing downstream of the cleavage and polyadenylation signal (CPS) leads to recruitment of cleavage and polyadenylation factors and termination, but how this chain of events is initiated remains unclear. In a chemical-genetic screen we identified protein phosphatase 1 (PP1) isoforms as substrates of human positive transcription elongation factor b (P-TEFb), the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 complex. Here we show that Cdk9 and PP1 govern phosphorylation of the conserved transcription factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2. Sites phosphorylated by Cdk9 in the Spt5 carboxy-terminal domain (CTD) are dephosphorylated by Dis2 in vitro, and Cdk9 inhibition in vivo leads to rapid Spt5 dephosphorylation that is retarded by concurrent Dis2 inactivation. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the CPS, prior to or concomitant with Pol II pausing. A Dis2-inactivating mutation stabilizes Spt5 phosphorylation (pSpt5) on chromatin, promotes transcription beyond the normal termination zone detected by precision run-on transcription and sequencing (PRO-seq), and is suppressed by ablation of Cdk9 target sites in Spt5. These results support a model whereby the transition of Pol II from elongation to termination is regulated by opposing activities of Cdk9 and Dis2 towards their common substrate Spt5—a bistable switch analogous to a Cdk1-PP1 module that controls exit from mitosis. Overall design: PRO-seq: Data represent 20 different treatments of various S. pombe strains performed in biological replicates. There are processed files for both replicates and combined replicate data for each experiment. ChIP-seq: Data represent 6 different ChIP-seq experiments in S. pombe, performed in biological replicates. There are raw and processed files for all biological replicates. ChIP-seq: Data represent 16 different ChIP-seq experiments in S. pombe, performed in biological replicates. There are raw and processed files for all biological replicates. PRO-seq: Experiment was done in five different S. pombe strains as biological duplicates. There are processed files for both replicates and combined replicate data for each sample.
Project description:CDK7 phosphorylates the RNA polymerase II (pol II) CTD and activates the P-TEFb- associated kinase, CDK9, but its regulatory roles remain obscure. Using human CDK7 analog-sensitive (CDK7as) cells, we observed reduced capping enzyme recruitment, increased pol II promoter-proximal pausing, and defective termination at gene 3'-ends upon CDK7 inhibition. We also found that CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5'-ends and H3K36me3 was displaced toward gene 3'-ends in CDK7as cells. Together, these results implicate a CDK7-dependent "CTD code" that regulates epigenetic marks in addition to RNA processing and pol II pausing. Overall design: WT and analogue sensitive Cdk7as mutant cells were treated with the ATP analogue NM-PP1 that specifically inhibits the Cdk7as mutant kinase. Using ChIP-seq and RNA-seq we tested the effects of Cdk7 inactivation on pol II distribution along genes, CTD Ser2 and Ser5 phosphorylation, capping enzyme recruitment, histone H3K4 and H3K36 methylation and mRNA expression
Project description:Recruitment of the RNA Polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA binding transcription factors is well recognized as a key regulatory step in gene expression. We describe here evidence that promoter-proximal pausing is a general feature of transcription by Pol II in embryonic stem (ES) cells, and thus an additional step where regulation of gene expression may occur. We report here that c-Myc, which occupies a third of actively transcribed genes in ES cells and is a key regulator of cellular proliferation, binds P-TEFb and contributes to release of promoter-proximal paused Pol II at these genes. ChIP-seq data for Pol II and additional factors controlling pause release in mouse ES cells.
Project description:Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a ‘multi-omics’ approach to show that the duration of polymerase pausing generally limits the frequency of transcription initiation in human cells (‘pause-initiation limit’). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. We show that CDK9 activity decreases the pause duration but also increases the initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription. Overall design: We performed mNET-seq reactions including empigen BB detergent treatment during immunoprecipitation in two biological replicates of human Raji B lymphocyte (CDK9as) cells treated for 15 min with DMSO (control) or 5 µM 1-NA-PP1 (CDK9as inhibitor). We performed TT-seq reactions including RNA spike-ins in two biological replicates of human Raji B lymphocyte (wild type, or CDK9as) cells treated for 15 min with DMSO (control) or 5 µM 1-NA-PP1 (CDK9as inhibitor), including 5 minutes of 4sU labeling. Raji B lymphocyte (CDK9as) cells were engineered using the CRISPR/Cas9 system and contain a homozygous mutation (F103A) at the CDK9 gene loci.
Project description:The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes. two treatements and one control