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Lurbinectedin specifically triggers the degradation of phosphorylated RNA Polymerase II and the formation of DNA breaks in cancer cells

ABSTRACT: We have defined the mechanism of action of lurbinectedin, a marine-derived drug exhibiting a potent anti-tumorigenic activity across several cancer cell lines and tumor xenografts. This drug currently undergoing clinical evaluation in ovarian, breast and small-cell lung cancer patients inhibits the transcription process through (1) its binding to CG rich sequences, mainly located around the promoter of protein coding genes; (2) the irreversible stalling of elongating RNA polymerase II (Pol II) on the DNA template and its specific degradation by the ubiquitin/proteasome machinery and (3) the generation of DNA breaks. The finding that inhibition of Pol II phosphorylation prevents its degradation and the formation of DNA breaks after drug treatment underscores the connection between transcription elongation and DNA repair. Our results not only help to better understand the high specificity of this drug in cancer therapy but also improve our understanding of an important transcription regulation mechanism.

ORGANISM(S): Homo sapiens

PROVIDER: GSE77071 | GEO | 2017/01/13

SECONDARY ACCESSION(S): PRJNA309387

REPOSITORIES: GEO

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Tyrosine 1 phosphorylated RNA polymerase II transcribes PROMPTs to promote proximal promoter pausing and induce global transcriptional repression in response to DNA damage

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2024-02-13 | GSE244353 | GEO

Phosphorylated MED1 Links Transcription Recycling and Cancer Growth

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Chd1 regulates repair of promoter-proximal DNA breaks to sustain hypertranscription in embryonic stem cells

Project description:Stem and progenitor cells undergo a global elevation of nascent transcription, or hypertranscription, during key developmental transitions involving rapid cell proliferation. The chromatin remodeler Chd1 binds to RNA Pol I and II genes and is required for hypertranscription in embryonic stem (ES) cells in vitro and the early post-implantation epiblast in vivo. Biochemically, Chd1 has been shown to facilitate transcription at least in part by removing nucleosomal barriers to elongation, but its mechanism of action in stem cells remains poorly understood. Here we report a novel role for Chd1 in the repair of promoter-proximal endogenous double-stranded DNA breaks (DSBs) in ES cells. An unbiased proteomics approach revealed that Chd1 interacts with several DNA repair factors including ATM, Parp1, Kap1 and Topoisomerase 2. We show that wild-type ES cells display high levels of phosphorylated H2AX and Kap1 at chromatin, notably at rDNA in the nucleolus, in a Chd1-dependent manner. Loss of Chd1 leads to an extensive accumulation of DSBs at Chd1 target Pol II genes and rDNA. Genes prone to DNA breaks in Chd1-null ES cells tend to be longer genes with GC-rich promoters, a more labile nucleosomal structure and roles in chromatin organization, transcription and signaling. These results reveal a vulnerability of hypertranscribing stem cells to endogenous DNA breaks, with important implications for developmental and cancer biology.

2021-06-14 | PXD024604 | Pride

Homo sapiens

Project description:Lurbinectedin specifically triggers the degradation of phosphorylated RNA Polymerase II and the formation of DNA breaks in cancer cells

| PRJNA309387 | ENA

DNA damage-induced transcription stress triggers the genome-wide degradation of promoter-paused Pol II

Project description:The precise regulation of RNA Polymerase II (Pol II) transcription after genotoxic stress is crucial for proper execution of the DNA damage-induced stress response. While stalling of Pol II on transcription-blocking lesions (TBLs) blocks transcript elongation and initiates DNA repair in cis, TBLs additionally elicit a response in trans that regulates transcription genome-wide. Here we uncover that after an initial elongation block in cis, TBLs trigger the genome-wide VCP-mediated proteasomal degradation of promoter-paused Pol II in trans. This degradation is mechanistically distinct from processing of TBL-stalled Pol II, is signaled via GSK3, and contributes to the TBL-induced transcription block, even in transcription-coupled repair-deficient cells. Thus, our data reveal the targeted degradation of promoter-bound Pol II as an important new component of the transcription stress response that enables the genome-wide adaptation of transcription to genotoxic stress.

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Project description:DNA breaks at RNA Pol II pausing sites

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Screening HIF-1 Cofactors in Breast Cancer Cells by RIME

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2022-02-17 | PXD024373 | Pride

Targeted protein degradation reveals Pol II heterogeneity and functional diversity

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2022-08-09 | PXD035866 |

Mechanistic basis for selective Mediator-dependence of cell type-specifying transcription

Project description:The Mediator complex routes signals from DNA-binding transcription factors to RNA polymerase (Pol) II. Despite its pivotal position, mechanistic understanding of Mediator in human cells remains incomplete. Here, we quantified Mediator-controlled Pol II kinetics by coupling rapid subunit degradation with orthogonal experimental readouts. Consistent with a model of condensate-driven transcription initiation, large clusters of hypo-phosphorylated Pol II rapidly disassembled upon Mediator degradation. This was accompanied by a selective and pronounced disruption of cell type-specifying transcriptional circuits, whose constituent genes featured exceptionally high rates of Pol II turnover. Remarkably, transcriptional output of most other genes was largely unaffected by acute Mediator ablation. Maintenance of transcriptional activity at these genes was linked to an unexpected, CDK9-dependent compensatory feedback loop that elevated Pol II pause release rates genome-wide. Collectively, our work positions human Mediator as a globally acting coactivator that selectively safeguards the functionality of cell type-specifying transcriptional networks.

2021-09-08 | PXD017611 | Pride

Structural basis of human transcription-DNA repair coupling

Project description:Transcription-coupled DNA repair (TCR) efficiently removes bulky DNA lesions from transcribed parts of the genome and constitutes a major defence against DNA damage by UV irradiation. TCR begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CsB, the E3 ubiquitin ligase complex CRL4CsA, and the UV-stimulated scaffold protein A (UVSSA). Here we provide five high-resolution structures of Pol II transcription complexes with bound TCR factors and complementary biochemical data. Together with published work, our results converge on a model for the molecular mechanism of transcription-DNA repair coupling. In this model, Pol II stalling triggers the formation of an alternative transcription elongation complex that we call ECact. In ECact, CsB has replaced the elongation factor DSIF, binds the PAF1 complex, and repositions upstream DNA such that it contacts the elongation factor SPT6. The CsB translocase now pulls on the upstream DNA template, thereby pushing Pol II forward. If the lesion cannot be bypassed, Pol II gets stably stalled. CRL4CsA spans over the Pol II clamp to reach the Pol II jaw and direct ubiquitination of RPB1 residue lysine-1268. This triggers the recruitment of TFIIH to UVSSA, which is located at downstream DNA, and enables nucleotide excision repair. Finally, large-scale conformational changes in CRL4CsA enable ubiquitination of CsB and the release of TCR factors, thereby allowing Pol II to resume elongation and continue transcription over repaired DNA.

2022-02-17 | PXD025328 | Pride

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