Project description:Various modes of DNA repair counteract genotoxic DNA double-strand breaks (DSBs) to maintain genome stability. Recent findings suggest that the human DNA damage response (DDR) utilises damage-induced small RNA for efficient repair of DSBs. However, production and processing of RNA is poorly understood. Here we show that localised induction of DSBs triggers phosphorylation of RNA polymerase II (RNAPII) on carboxy-terminal domain (CTD) residue tyrosine-1 in an Mre11-Rad50-Nbs1 (MRN) complex-dependent manner. CTD Tyr1-phosphorylated RNAPII synthetises, strand-specific, damage-responsive transcripts (DARTs). DART synthesis occurs via formation of transient RNA-DNA hybrid (R-loop) intermediates. Impaired R-loop formation attenuates DART synthesis, impairs recruitment of repair factors and delays the DDR. Collectively, we provide mechanistic insight in RNA-dependent DSB repair.
Project description: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.
Project description:One major class of anti-cancer drugs targets topoisomerase II to induce DNA double-strand breaks and cell death of fast growing cells. Here, we compare three members of this class - the antracyclines doxorubicin and aclarubicin, and a chemically unrelated compound, etoposide. Aclarubicin does not induce DNA breaks. We define a new activity for the antracyclines: unsupported histone eviction from ´open´ or loosely packed chromosomal areas reflecting exon and promoter regions. Comparison of histone H3K4me3 of cells post topoisomerase II inhibitors treatment to un-treated ones by ChIP-seq. Comparison of phosphorylated histone H2AX of cells post topoisomerase II inhibitors doxorubicin and etoposide treatment to un-treated ones by ChIP-seq.
Project description:Topoisomerase II (topo II) is essential for disentangling newly-replicated chromosomes. DNA unlinking involves the physical passage of one DNA duplex through another and depends on the transient formation of double-strand DNA breaks, a step exploited by frontline chemotherapies to kill cancer cells. Although anti-topo II drugs are efficacious, they also elicit cytotoxic side effects in normal cells; insights into how topo II is regulated in different cellular contexts is essential to improve their targeted use. Using chemical fractionation and mass spectrometry, we have discovered that topo II is subject to metabolic control through the TCA cycle. We show that TCA metabolites stimulate topo II activity in vitro and that levels of TCA flux modulate cellular sensitivity to anti-topo II drugs in vivo. Our works reveals an unanticipated connection between the control of DNA topology and cellular metabolism, a finding with important ramifications for the clinical use of anti-topo II therapies.
Project description:In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of initiating Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the Pol IIB form and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability.
Project description:In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of initiating Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the Pol IIB form and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability. This study was performed in a human Raji cell line. It contains ChIP-seq data for H3K36me3 (two replicates), H3K4me1 (two replicates), H3K4me3 (two replicates), Pol II (three replicates), Ser2P (two replicates), Ser5P (two replicates), Ser7P (two replicates), Tyr1P 3D12 (two replicates) and Tyr1P 8G5 (one replicate). MNase-experiment for nucleosomes was performed in paired-end sequencing on one replicate, 4 replicates for the input genomic DNA was used and one replicate was generated for the short strand specific RNA experiment.
Project description:Phf5a regulates transcription elongation in mouse embryonic stem cells (ESCs), through regulation of the Paf1 complex. In this study we assayed for genome-wide localization of Ser-5-phosphorylated RNA polymerase II and Ser-2-phosphorylated RNA polymerase II in mouse ESCs under conditions of shControl and shPhf5a knockdown. These results revealed that downregualtion of Phf5a results in the increase of the initiating form of RNA polymerase II (Ser5-phosphorylated) and in the aberrant loss of the elongating form of RNA polymerase II (Ser2-phosphorylated) of pluripotency genes in ESCs.
Project description:Kras is the most commonly mutated oncogene in human cancer and mutant Kras is responsible for over 90% of pancreatic ductal adnocarcinoma (PDAC), the most leath cancer. Here, we identified that RNA polymerase II associated factor 1 complex (PAF1C) is specifically required for the survival of PDAC but not normal adult panreatic cells. We show that PAF1C maintains cancer cell genomic stability by restraining the over accumulation of enhancer RNAs (eRNAs) driven by mutant Kras. Loss of PAF1C leads to cancer-specific lengthening and accumulation of eRNAs on chromatin and therby abbarrent R-loop formation, TC-NER pathway activation and double stranded DNA breaks, which in turn trigger cell death. We also demonstrate that the sepcific demand for PAF1C by cancer cells is due to the global activation of enhancers and thus eRNA transcription during tumorigenesis. The work provides a novel insight in how transcription addiction is caused during tumorigenesis.
Project description:Kras is the most commonly mutated oncogene in human cancer and mutant Kras is responsible for over 90% of pancreatic ductal adnocarcinoma (PDAC), the most leath cancer. Here, we identified that RNA polymerase II associated factor 1 complex (PAF1C) is specifically required for the survival of PDAC but not normal adult panreatic cells. We show that PAF1C maintains cancer cell genomic stability by restraining the over accumulation of enhancer RNAs (eRNAs) driven by mutant Kras. Loss of PAF1C leads to cancer-specific lengthening and accumulation of eRNAs on chromatin and therby abbarrent R-loop formation, TC-NER pathway activation and double stranded DNA breaks, which in turn trigger cell death. We also demonstrate that the sepcific demand for PAF1C by cancer cells is due to the global activation of enhancers and thus eRNA transcription during tumorigenesis. The work provides a novel insight in how transcription addiction is caused during tumorigenesis.
Project description:Kras is the most commonly mutated oncogene in human cancer and mutant Kras is responsible for over 90% of pancreatic ductal adnocarcinoma (PDAC), the most leath cancer. Here, we identified that RNA polymerase II associated factor 1 complex (PAF1C) is specifically required for the survival of PDAC but not normal adult panreatic cells. We show that PAF1C maintains cancer cell genomic stability by restraining the over accumulation of enhancer RNAs (eRNAs) driven by mutant Kras. Loss of PAF1C leads to cancer-specific lengthening and accumulation of eRNAs on chromatin and therby abbarrent R-loop formation, TC-NER pathway activation and double stranded DNA breaks, which in turn trigger cell death. We also demonstrate that the sepcific demand for PAF1C by cancer cells is due to the global activation of enhancers and thus eRNA transcription during tumorigenesis. The work provides a novel insight in how transcription addiction is caused during tumorigenesis.