Dataset Information

A PP2A-Integrator complex fine-tunes transcription by opposing CDK9

ABSTRACT: In this study, we discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonises phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates and amplification of acute cell growth and pro-inflammatory transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that finely-tuned gene expression relies on the balance of kinase and phosphatase activity throughout the transcription cycle.

INSTRUMENT(S): Q Exactive HF, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Sarah Welsh

LAB HEAD: Alessandro Gardini

PROVIDER: PXD025060 | Pride | 2022-08-12

REPOSITORIES: Pride

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Publications

The PP2A-Integrator-CDK9 axis fine-tunes transcription and can be targeted therapeutically in cancer.

Vervoort Stephin J SJ Welsh Sarah A SA Devlin Jennifer R JR Barbieri Elisa E Knight Deborah A DA Offley Sarah S Bjelosevic Stefan S Costacurta Matteo M Todorovski Izabela I Kearney Conor J CJ Sandow Jarrod J JJ Fan Zheng Z Blyth Benjamin B McLeod Victoria V Vissers Joseph H A JHA Pavic Karolina K Martin Ben P BP Gregory Gareth G Demosthenous Elena E Zethoven Magnus M Kong Isabella Y IY Hawkins Edwin D ED Hogg Simon J SJ Kelly Madison J MJ Newbold Andrea A Simpson Kaylene J KJ Kauko Otto O Harvey Kieran F KF Ohlmeyer Michael M Westermarck Jukka J Gray Nathanael N Gardini Alessandro A Johnstone Ricky W RW

Cell 20210517 12

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphoryl ...[more]

PMID: 34004147

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Project description:CarD is an essential mycobacterial protein that we had previously shown to bind the RNA polymerase (RNAP) and affect the transcriptional profile of M. smegmatis and Mycobacterium tuberculosis. For this reason, we suspected that CarD was directly regulating transcriptional complexes but we did not know at what stage of CarD was functioning and at which genes CarD interacted with the RNAP. To determine in which stage of the transcription cycle (initiation, elongation, or termination) CarD acts, we used Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. Specific antibodies targeting core RNAPb, RNAPσ, or a hemagglutinin (HA) epitope fused to CarD (CarD-HA) were used to co-immunoprecipitate associated DNA. CarD-HA was immunoprecipitated from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain and unfused HA was immunoprecipitated from the Mc2155 attB::pmsg431 strain with monoclonal antibodies specific for HA (Sigma). RNAP β and σ were immunoprecipitated from M. smegmatis ΔcarD attB::tetcarD-HA with monoclonal antibodies specific for these subunits (Neoclone, Madison, WI; 8RB13 for β, 2G10 for σ). Co-precipitated DNA was sequenced using a SOLiD sequencer (Life Technologies), which provided sufficient reads for 100-fold coverage of the genome. The number of sequence reads per base pair was normalized to the total number of reads and expressed as a log2 value. The reads per base pair from the HA-alone sample served as the background and was subtracted from the other datasets. We found that CarD was never present on the genome in the absence of RNAP. However, whereas RNAP core enzyme was found throughout transcribed regions of the genome, CarD was primarily associated with promoter regions and highly correlated with RNAPσ. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. The genome sequences associated with M. smegmatis CarD, RNAPb, and RNAPs were determined by ChIP-seq analysis. Samples were done in duplicate, except for RNAPs. And sequencing was performed using a SOLiD sequencer (Life Technologies).

Dataset Information

A PP2A-Integrator complex fine-tunes transcription by opposing CDK9

Publications

The PP2A-Integrator-CDK9 axis fine-tunes transcription and can be targeted therapeutically in cancer.

Similar Datasets

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