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DRB-PRO-Seq for measuring Pol II transcriptional elongation rate alterations upon inhibition of analog sensitive CDK12 and/or CDK13

ABSTRACT: DRB-PRO-Seq for measuring Pol II transcriptional elongation rate alterations upon inhibition of analog sensitive CDK12 and/or CDK13

PROVIDER: PRJNA593231 | ENA |

REPOSITORIES: ENA

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DRB-PRO-Seq for measuring Pol II transcriptional elongation rate alterations upon inhibition of analog sensitive CDK12 and/or CDK13

Project description:We performed PRO-Seq to determine Pol II elongation rates by measruing the distance of Pol II travels during 25 minutes after Pol II being relased from DRB induced pausing. We allowed Pol II to release in the presence of 1-NM-PP1 or DMSO after DRB being wahsed out in order to assess the effects of CDK12 and/or CDK13 inhibtion on transcriptional elongation rates.

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DRB-PRO-Seq for measuring Pol II transcriptional elongation rate alteration up on CDK12 and CDK13 inhibition by THZ531

Project description:We performed PRO-Seq to measure the Pol II elongation rate by measruing the distance of Pol II travels at viarous time points after relased from DRB induced pausing. We compared DMSO/DRB or THZ531/DRB co-treated sample to assess the effect of CDK12 and CDK13 inhibtion by THZ531 on transcriptional elongation rate.

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RNA-Seq for validating the RNA expression of wild type and analog sensitive CDK12 and CDK13 in analog sensitive MV4;11 cell lines that generated by CRISPR HDR

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CDK12 and CDK13

Project description:CDK12 and CDK13

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Project description:The RNA polymerase II (POLII) driven transcription cycle is tightly regulated at distinct checkpoints through cyclin dependent kinases (CDKs) and their cognate Cyclins. The molecular events underpinning transcriptional elongation and processivity and CDK-Cyclins involved remain poorly understood. Using CRISPR-CAS9 homology-directed-repair we generated analog-sensitive-kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with DNA-damage and cellular growth signaling pathways respectively, with minimal effects on cell viability. In contrast, dual-kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including wide-spread use of alternative 3’ polyadenylation sites. At the molecular level dual-kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define significant redundancy between CDK12 and CDK13, and identify both as fundamental regulators of global POLII processivity and transcription elongation.

2020-04-30 | PXD015453 | Pride

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Project description:We used 3' RNA-Seq to determine the effects of analog senstive CDK12, CDK13 and dual inhibition on gene expression and polyadenatltion site usages, and to test the redundancy between CDK12 and CDK13 in maintaining global transcrition on the tramcrotpomte level.

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DRB-PRO-Seq for measuring Pol II transcriptional elongation rate alteration up on CDK12 and CDK13 inhibition by THZ531

Project description:DRB-PRO-Seq for measuring Pol II transcriptional elongation rate alteration up on CDK12 and CDK13 inhibition by THZ531

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Project description:Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12–cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.

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