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Expression profiling of HeLa cell line treated with CDK9 inhibitor

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. 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

ORGANISM(S): Homo sapiens

SUBMITTER: Zhenhai Gao

PROVIDER: E-GEOD-60952 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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

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. ChIP-seq of Pol II in HeLa cells before or after i-CDk9 treatment

2015-06-17 | E-GEOD-60953 | biostudies-arrayexpress

2015-06-17 | GSE60953 | GEO

Expression profiling of HeLa cell line treated with CDK9 inhibitor

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.

2015-06-17 | GSE60952 | GEO

SR Proteins Collaborate with 7SK and Promoter-Associated Nascent RNA to Release Paused Polymerase

Project description:RNAP II is frequently paused near gene promoters in mammals and its transition to productive elongation requires active recruitment of P-TEFb, a cyclin-dependent kinase for RNAP II and other key transcription elongation factors. A fraction of P-TEFb is sequestered in an inhibitory complex containing the 7SK noncoding RNA, but it has been unclear how P-TEFb is switched from the 7SK complex to RNAP II during transcription activation. We report that SRSF2 (also known as SC35, an SR splicing factor) is part of the 7SK complex assembled at gene promoters and plays a direct role in transcription pause release. We demonstrate RNA-dependent, coordinated release of SRSF2 and P-TEFb from the 7SK complex and transcription activation via SRSF2 binding to promoter-associated nascent RNA. These findings reveal an unanticipated SR protein function, a role for promoter-proximal nascent RNA in gene activation, and an analogous mechanism to HIV Tat/TAR for activating cellular genes. SR ChIP-seq, Pol II ChIP-seq, and Gro-seq

2013-05-09 | E-GEOD-45517 | biostudies-arrayexpress

Integrative Proteomics Identifies MePCE as a Substrate of CDK9 for Releasing P-TEFb from 7SK snRNP Under Genotoxic Stress

Project description:The dynamic phosphorylation process regulated by the positive transcription elongation factor b (P-TEFb), a CDK9-cyclin T heterodimer, is critical for transcription elongation of numerous mammalian genes. Most P-TEFb in cells are normally sequestered in the 7SK snRNP, and their release from this complex is well-controlled to maintain proper cell homeostasis and growth. The comprehensive profiling of CDK9 substrates is essential for understanding diverse functions of P-TEFb in controlling gene expression. Here, integrative proteomics methodologies are developed to globally identify the potential substrates of CDK9 in vivo. Our results show that the 7SK methylphosphate capping enzyme MePCE is directly phosphorylated by CDK9 within the 7SK snRNP, which is known to exhibit little kinase activity toward other known CDK9 substrates. Upon ultraviolet radiation, MePCE becomes phosphorylated, leading to the dissociation of 7SK snRNP and release of P-TEFb to activate transcription. Meanwhile, the phosphorylation of MePCE does not affect its methyltransferase activity toward 7SK RNA. Our data suggest that even though CDK9 in 7SK snRNP has little kinase activity toward external substrates, it can phosphorylate the snRNP integral component MePCE, which is required to release P-TEFb. This novel inside-to-outside mode of P-TEFb activation may serve as a critical checkpoint in regulating gene transcription under stressful conditions.

2024-07-03 | PXD053627 |

Total RNA Pol2 ChIP-seq in mES cells treated with flavopiridol (Cdk9 inhibitor) after DSIF and NELF knockdown

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).

2011-01-19 | E-GEOD-26658 | biostudies-arrayexpress

Transcriptional Pause Release Is a Rate-Limiting Step for Somatic Cell Reprogramming (RNA-seq)

Project description:Reactivation of the pluripotency network during somatic cell reprogramming by exogenous transcription factors involves chromatin remodeling and the recruitment of RNA polymerase II (Pol II) to target loci. Here, we report that Pol II is engaged at pluripotency promoters in reprogramming but remains paused and inefficiently released. We also show that bromodomain-containing protein 4 (BRD4) stimulates productive transcriptional elongation of pluripotency genes by dissociating the pause release factor P-TEFb from an inactive complex containing HEXIM1. Consequently, BRD4 overexpression enhances reprogramming efficiency and HEXIM1 suppresses it, whereas Brd4 and Hexim1 knockdown do the opposite. We further demonstrate that the reprogramming factor KLF4 helps recruit P-TEFb to pluripotency promoters. Our work thus provides a mechanism for explaining the reactivation of pluripotency genes in reprogramming and unveils an unanticipated role for KLF4 in transcriptional pause release. Examination of differential gene expression after overexpression of Cdk9-DN at 4 time points of somatic cell reprogramming

2014-10-07 | E-GEOD-59813 | biostudies-arrayexpress

JMJD5 couples with CDK9 to release the paused RNA polymerase II

Project description:More than 30% of genes in higher eukaryotes are regulated by RNA Polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS) thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxy-terminal domain of Pol II together with phosphorylation of Ser5 within the second repeat, HR-Ser2p(1,2)-Ser5p(2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

2020-06-27 | GSE153322 | GEO

Total RNA Pol2 ChIP-seq in mES cells treated with flavopiridol (Cdk9 inhibitor) after DSIF and NELF knockdown

2011-01-19 | GSE26658 | GEO

Genome-wide map of RNA polymerase II, NELF, and DSIF in HeLa 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

2014-02-01 | E-GEOD-53008 | biostudies-arrayexpress

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