ABSTRACT
Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects initiation, development, and survival of this tumor type. Using patient-derived xenografts of KRAS- and MYC-driven pancreatic carcinoma, we show that coinhibition of topoisomerase 1 (TOP1) and bromodomain-containing protein 4 (BRD4) synergistically induces tumor regression by targeting promoter pause release. Comparing the nascent transcriptome with the recruitment of elongation and termination factors, we found that coinhibition of TOP1 and BRD4 disrupts recruitment of transcription termination factors. Thus, RNA polymerases transcribe downstream of genes for hundreds of kilobases leading to readthrough transcription. This occurs during replication, perturbing replisome progression and inducing DNA damage. The synergistic effect of TOP1 + BRD4 inhibition is specific to cancer cells leaving normal cells unaffected, highlighting the tumor's vulnerability to transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of transcription and replication.
Subject(s)
Pancreatic Neoplasms , Transcription Factors , Humans , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , DNA Topoisomerases, Type I/metabolism , Pancreatic NeoplasmsABSTRACT
Posttranscriptional modifications of mRNA have emerged as regulators of gene expression. Although pseudouridylation is the most abundant, its biological role remains poorly understood. Here, we demonstrate that the pseudouridine synthase dyskerin associates with RNA polymerase II, binds to thousands of mRNAs, and is responsible for their pseudouridylation, an action that occurs in chromatin and does not appear to require a guide RNA with full complementarity. In cells lacking dyskerin, mRNA pseudouridylation is reduced, while at the same time, de novo protein synthesis is enhanced, indicating that this modification interferes with translation. Accordingly, mRNAs with fewer pseudouridines due to knockdown of dyskerin are translated more efficiently. Moreover, mRNA pseudouridylation is severely reduced in patients with dyskeratosis congenita caused by inherited mutations in the gene encoding dyskerin (i.e., DKC1). Our findings demonstrate that pseudouridylation by dyskerin modulates mRNA translatability, with important implications for both normal development and disease.
Subject(s)
Nuclear Proteins , RNA-Binding Proteins , Humans , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Cell Cycle Proteins/metabolismABSTRACT
DNA topoisomerase 1 (TOP11) inhibitors are mainstays of anticancer therapy. These drugs trap TOP1 on DNA, stabilizing the TOP1-cleavage complex (TOP1-cc). The accumulation of TOP1-ccs perturbs DNA replication fork progression, leading to DNA breaks and cell death. By analyzing the genomic occupancy and activity of TOP1, we show that cells adapt to treatment with multiple doses of TOP1 inhibitor by promoting the degradation of TOP1-ccs, allowing cells to better tolerate subsequent doses of TOP1 inhibitor. The E3-RING Cullin 3 ligase in complex with the BTBD1 and BTBD2 adaptor proteins promotes TOP1-cc ubiquitination and subsequent proteasomal degradation. NEDDylation of Cullin 3 activates this pathway, and inhibition of protein NEDDylation or depletion of Cullin 3 sensitizes cancer cells to TOP1 inhibitors. Collectively, our data uncover a previously unidentified NEDD8-Cullin 3 pathway involved in the adaptive response to TOP1 inhibitors, which can be targeted to improve the efficacy of TOP1 drugs in cancer therapy.
Subject(s)
Topoisomerase I Inhibitors , Ubiquitin-Protein Ligases , Topoisomerase I Inhibitors/pharmacology , Ubiquitination , Ubiquitin-Protein Ligases/metabolismABSTRACT
TOP1 CAD-seq enables mapping of TOP1 sites of covalent engagement with DNA. The procedure depends upon enrichment of DNA-covalent adducts using chaotropic salts and immunoprecipitation with an antibody specific for TOP1. Here, we describe a step-by-step protocol compatible with Illumina sequencing and bioinformatic pipeline for preliminary data analysis. Compared to other approaches for the genomic study of topoisomerases, TOP1 CAD-seq provides information about active TOP1 engaged on the DNA, taking advantage of low background due to absence of crosslinking. For complete details on the use and execution of this protocol, please refer to Das et al. (2022).
Subject(s)
DNA Adducts , DNA , DNA Topoisomerases, Type I/genetics , HumansABSTRACT
High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.
Subject(s)
DNA Topoisomerases, Type II/metabolism , Neoplasms/enzymology , Poly-ADP-Ribose Binding Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Transcription, Genetic , Animals , DNA Replication , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/metabolism , DNA Topoisomerases, Type II/genetics , DNA, Neoplasm/biosynthesis , DNA, Neoplasm/genetics , DNA, Superhelical/biosynthesis , DNA, Superhelical/genetics , Enzyme Activation , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , K562 Cells , Multienzyme Complexes , Neoplasms/genetics , Neoplasms/pathology , Poly-ADP-Ribose Binding Proteins/genetics , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins c-myc/genetics , RatsABSTRACT
Deregulated expression of MYC family oncogenes occurs frequently in human cancer and is often associated with aggressive disease and poor prognosis. While MYC is a highly warranted target, it has been considered "undruggable," and no specific anti-MYC drugs are available in the clinic. We recently identified molecules named MYCMIs that inhibit the interaction between MYC and its essential partner MAX. Here we show that one of these molecules, MYCMI-7, efficiently and selectively inhibits MYC:MAX and MYCN:MAX interactions in cells, binds directly to recombinant MYC, and reduces MYC-driven transcription. In addition, MYCMI-7 induces degradation of MYC and MYCN proteins. MYCMI-7 potently induces growth arrest/apoptosis in tumor cells in a MYC/MYCN-dependent manner and downregulates the MYC pathway on a global level as determined by RNA sequencing. Sensitivity to MYCMI-7 correlates with MYC expression in a panel of 60 tumor cell lines and MYCMI-7 shows high efficacy toward a collection of patient-derived primary glioblastoma and acute myeloid leukemia (AML) ex vivo cultures. Importantly, a variety of normal cells become G1 arrested without signs of apoptosis upon MYCMI-7 treatment. Finally, in mouse tumor models of MYC-driven AML, breast cancer, and MYCN-amplified neuroblastoma, treatment with MYCMI-7 downregulates MYC/MYCN, inhibits tumor growth, and prolongs survival through apoptosis with few side effects. In conclusion, MYCMI-7 is a potent and selective MYC inhibitor that is highly relevant for the development into clinically useful drugs for the treatment of MYC-driven cancer. Significance: Our findings demonstrate that the small-molecule MYCMI-7 binds MYC and inhibits interaction between MYC and MAX, thereby hampering MYC-driven tumor cell growth in culture and in vivo while sparing normal cells.
Subject(s)
Neuroblastoma , Animals , Mice , Humans , N-Myc Proto-Oncogene Protein/genetics , Cell Line, Tumor , Neuroblastoma/drug therapy , Cell Proliferation , Cell CycleABSTRACT
As cells enter mitosis, chromatin compacts to facilitate chromosome segregation yet remains transcribed. Transcription supercoils DNA to levels that can impede further progression of RNA polymerase II (RNAPII) unless it is removed by DNA topoisomerase 1 (TOP1). Using ChIP-seq on mitotic cells, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity by RNAPII during elongation allowed RNAPII clearance from genes in prometaphase and enabled chromosomal segregation. Disruption of the TOP1-RNAPII interaction impaired RNAPII spiking at promoters and triggered defects in the post-mitotic transcription program. This program includes factors necessary for cell growth, and cells with impaired TOP1-RNAPII interaction are more sensitive to inhibitors of mTOR signaling. We conclude that TOP1 is necessary for assisting transcription during mitosis with consequences for growth and gene expression long after mitosis is completed. In this sense, TOP1 ensures that cellular memory is preserved in subsequent generations.
Subject(s)
Cell Proliferation , Chromatin Assembly and Disassembly , Colorectal Neoplasms/enzymology , DNA Topoisomerases, Type I/metabolism , G1 Phase , Mitosis , RNA Polymerase II/metabolism , Transcription, Genetic , Cell Proliferation/drug effects , Chromatin Immunoprecipitation Sequencing , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , DNA Topoisomerases, Type I/genetics , G1 Phase/drug effects , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , MTOR Inhibitors/pharmacology , Mitosis/drug effects , RNA Polymerase II/geneticsABSTRACT
Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 co-immunoprecipitated and deubiquitinated TOP2, and stabilized SUMOylated TOP2 trapped in cleavage complexes (TOP2ccs), which halted the DNA damage response to TOP2-induced double strand DNA breaks and promoted cell survival. Induction of the productive virus cycle in epithelial and lymphoid cell lines carrying recombinant EBV encoding the active enzyme was accompanied by TOP2 deubiquitination, accumulation of TOP2ccs and resistance to Etoposide toxicity. The protective effect of BPLF1 was dependent on the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that releases DNA-trapped TOP2 and promotes error-free DNA repair. These findings highlight a previously unrecognized function of BPLF1 in supporting a non-proteolytic pathway for TOP2ccs debulking that favors cell survival and virus production.
Subject(s)
DNA Topoisomerases, Type II/metabolism , Epstein-Barr Virus Infections/metabolism , Viral Regulatory and Accessory Proteins/metabolism , HEK293 Cells , HeLa Cells , HumansABSTRACT
The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post-replication. The translesion synthesis polymerase Polη is required for damage-induced cohesion through a hitherto unknown mechanism. Since Polη is functionally associated with transcription, and transcription triggers de novo cohesion in Schizosaccharomyces pombe, we hypothesized that transcription facilitates damage-induced cohesion in Saccharomyces cerevisiae. Here, we show dysregulated transcriptional profiles in the Polη null mutant (rad30Δ), where genes involved in chromatin assembly and positive transcription regulation were downregulated. In addition, chromatin association of RNA polymerase II was reduced at promoters and coding regions in rad30Δ compared to WT cells, while occupancy of the H2A.Z variant (Htz1) at promoters was increased in rad30Δ cells. Perturbing histone exchange at promoters inactivated damage-induced cohesion, similarly to deletion of the RAD30 gene. Conversely, altering regulation of transcription elongation suppressed the deficient damage-induced cohesion in rad30Δ cells. Furthermore, transcription inhibition negatively affected formation of damage-induced cohesion. These results indicate that the transcriptional deregulation of the Polη null mutant is connected with its reduced capacity to establish damage-induced cohesion. This also suggests a linkage between regulation of transcription and formation of damage-induced cohesion after replication.
Subject(s)
Cell Cycle Proteins/biosynthesis , Chromosomal Proteins, Non-Histone/biosynthesis , DNA-Directed DNA Polymerase/genetics , RNA Polymerase II/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/enzymology , Transcription, Genetic , Chromatin/metabolism , DNA-Directed DNA Polymerase/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Fungal , Genes, Fungal , Mutation , Promoter Regions, Genetic , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/genetics , TATA Box , CohesinsABSTRACT
Here, we present a strategy to map and quantify the interactions between Myc and chromatin using a calibrated Myc ChIP-seq approach. We recommend the use of an internal spike-in control for post-sequencing normalization to enable detection of broad changes in Myc binding as can occur under conditions with varied Myc abundance. We also highlight a range of bioinformatic analyses that can dissect the downstream effects of Myc binding. These methods include peak calling, mapping Myc onto an integrated metagenome, juxtaposing ChIP-seq data with matching RNA-seq data, and identifying gene ontologies enriched for genes with high Myc binding. Our aim is to provide a guided strategy, from cell harvest through to bioinformatic analysis, to elucidate the global effects of Myc on transcription.
Subject(s)
Chromatin Immunoprecipitation Sequencing/methods , Chromatin Immunoprecipitation/methods , Proto-Oncogene Proteins c-myc/genetics , Binding Sites , Chromatin/genetics , Computational Biology/methods , DNA/genetics , DNA-Binding Proteins , Genes, myc , High-Throughput Nucleotide Sequencing , Humans , Proto-Oncogene Proteins c-myc/metabolism , Sequence Analysis, DNA/methodsABSTRACT
Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation.
Subject(s)
Cell Cycle Proteins/metabolism , Cyclin A2/metabolism , Cytoplasm/metabolism , G2 Phase/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , S Phase/genetics , Signal Transduction/genetics , CDC2 Protein Kinase/deficiency , CDC2 Protein Kinase/genetics , Cell Nucleus/metabolism , Chromatin/metabolism , Cyclin A2/genetics , Cyclin-Dependent Kinase 2/deficiency , Cyclin-Dependent Kinase 2/genetics , DNA Damage/genetics , Enzyme Activation/genetics , HeLa Cells , Humans , Mitosis/genetics , Phosphorylation/genetics , Protein Binding , Transfection , Polo-Like Kinase 1ABSTRACT
The protooncogene MYC regulates a variety of cellular processes, including proliferation and metabolism. Maintaining MYC at homeostatic levels is critical to normal cell function; overexpression drives many cancers. MYC stability is regulated through phosphorylation: phosphorylation at Thr58 signals degradation while Ser62 phosphorylation leads to its stabilization and functional activation. The bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator with intrinsic kinase and histone acetyltransferase (HAT) activities that activates transcription of key protooncogenes, including MYC We report that BRD4 phosphorylates MYC at Thr58, leading to MYC ubiquitination and degradation, thereby regulating MYC target genes. Importantly, BRD4 degradation, but not inhibition, results in increased levels of MYC protein. Conversely, MYC inhibits BRD4's HAT activity, suggesting that MYC regulates its own transcription by limiting BRD4-mediated chromatin remodeling of its locus. The MYC stabilizing kinase, ERK1, regulates MYC levels directly and indirectly by inhibiting BRD4 kinase activity. These findings demonstrate that BRD4 negatively regulates MYC levels, which is counteracted by ERK1 activation.
Subject(s)
Cell Cycle Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Transcription Factors/metabolism , Acetylation , Cell Nucleus/metabolism , Chromatin/metabolism , Dipeptides/pharmacology , Gene Expression Regulation/drug effects , HeLa Cells , Heterocyclic Compounds, 3-Ring/pharmacology , Histones/metabolism , Humans , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Protein Binding , Protein Stability/drug effects , Proto-Oncogene Proteins c-myc/genetics , UbiquitinationABSTRACT
How spatial chromosome organization influences genome integrity is still poorly understood. Here, we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CCCTC-binding factor (CTCF) and cohesin-bound sites at the bases of chromatin loops, and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hotspots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability and are key contributors to the occurrence of genome rearrangements that drive cancer.
Subject(s)
DNA Topoisomerases, Type II/genetics , Genomic Instability/genetics , Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Poly-ADP-Ribose Binding Proteins/genetics , Translocation, Genetic/genetics , CCCTC-Binding Factor/genetics , Carcinogenesis/genetics , Cell Line, Tumor , Chromatin/chemistry , Chromatin/genetics , Chromosomes/chemistry , Chromosomes/genetics , DNA/genetics , DNA Breaks, Double-Stranded , Humans , Leukemia/genetics , Leukemia/pathologyABSTRACT
Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.
Subject(s)
Adenosine Triphosphate/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Genome , Animals , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Line , Chondroitin Sulfate Proteoglycans/genetics , Chondroitin Sulfate Proteoglycans/metabolism , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosomes/metabolism , DNA-Binding Proteins , Humans , Mice , Mutagenesis , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , CohesinsABSTRACT
Although our knowledge of chromatin organization has advanced significantly in recent years, much about the relationships between different features of genome architecture is still unknown. Folding of mammalian genomes into spatial domains is thought to depend on architectural proteins, other DNA-binding proteins, and different forms of RNA. In addition, emerging evidence points towards the possibility that the three-dimensional organisation of the genome is controlled by DNA topology. In this scenario, cohesin, CCCTC-binding factor (CTCF), transcription, DNA supercoiling, and topoisomerases are integrated to dictate different layers of genome organization, and the contribution of all four to gene control is an important direction of future studies. In this perspective, we review recent studies that give new insight on how DNA supercoiling shape chromatin structure.
Subject(s)
Cell Cycle Proteins/chemistry , Chromatin/chemistry , Chromosomal Proteins, Non-Histone/chemistry , DNA Topoisomerases/chemistry , DNA, Superhelical/chemistry , Nucleic Acid Conformation , Animals , CCCTC-Binding Factor/chemistry , CCCTC-Binding Factor/metabolism , Cell Cycle Proteins/metabolism , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Topoisomerases/metabolism , DNA, Superhelical/metabolism , Genome , Humans , RNA/chemistry , RNA/metabolism , Transcription, Genetic , CohesinsABSTRACT
Proteins manipulating intracellular DNA necessarily impart torsional stress, which redistributes across the DNA. Overtwisting and undertwisting of the double helix result in the manifestation of positive and negative DNA supercoiling. A growing body of evidence indicates that DNA topology is an important player in the key regulatory steps of genome function, highlighting the need for biochemical techniques to detect dynamic changes in the DNA structure. Psoralen binding to DNA in vivo is proportional to the level of supercoiling, providing an excellent probe for the topological state of nuclear DNA. Here we describe a psoralen-based methodology to detect transcription-induced DNA supercoiling genome-wide. The DNA samples generated with this approach can be hybridized to microarray platforms or high-throughput sequenced to provide a topological snapshot of the whole genome.
Subject(s)
DNA, Superhelical/genetics , Ficusin/pharmacology , Photosensitizing Agents/pharmacology , Transcription, Genetic , Cell Line , DNA, Superhelical/chemistry , Genome, Human , Humans , Models, Molecular , Nucleic Acid ConformationABSTRACT
Here, we present two approaches to map DNA double-strand breaks (DSBs) and single-strand breaks (SSBs) in the genome of human cells. We named these methods respectively DSB-Seq and SSB-Seq. We tested the DSB and SSB-Seq in HCT1116, human colon cancer cells, and validated the results using the topoisomerase 2 (Top2)-poisoning agent etoposide (ETO). These methods are powerful tools for the direct detection of the physiological and pathological "breakome" of the DNA in human cells.
Subject(s)
Chromosome Mapping , DNA Breaks, Double-Stranded , High-Throughput Nucleotide Sequencing , Chromosome Mapping/methods , Computational Biology/methods , DNA/chemistry , DNA/genetics , DNA/isolation & purification , DNA Breaks, Double-Stranded/drug effects , DNA Damage , DNA Topoisomerases, Type II/metabolism , DNA, Single-Stranded , Etoposide/pharmacology , High-Throughput Nucleotide Sequencing/methods , Sequence Analysis, DNAABSTRACT
In response to stresses, cells often halt normal cellular processes, yet stress-specific pathways must bypass such inhibition to generate effective responses. We investigated how cells redistribute global transcriptional activity in response to DNA damage. We show that an oscillatory increase of p53 levels in response to double-strand breaks drives a counter-oscillatory decrease of MYC levels. Using RNA sequencing (RNA-seq) of newly synthesized transcripts, we found that p53-mediated reduction of MYC suppressed general transcription, with the most highly expressed transcripts reduced to a greater extent. In contrast, upregulation of p53 targets was relatively unaffected by MYC suppression. Reducing MYC during the DNA damage response was important for cell-fate regulation, as counteracting MYC repression reduced cell-cycle arrest and elevated apoptosis. Our study shows that global inhibition with specific activation of transcriptional pathways is important for the proper response to DNA damage; this mechanism may be a general principle used in many stress responses.
Subject(s)
Breast Neoplasms/genetics , DNA Breaks, Double-Stranded , Proto-Oncogene Proteins c-myc/genetics , Transcription, Genetic , Transcriptome , Tumor Suppressor Protein p53/genetics , Apoptosis , Binding Sites , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , CRISPR-Cas Systems , Cell Cycle Checkpoints , Female , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , MCF-7 Cells , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins c-myc/metabolism , RNA Interference , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Signal Transduction , Time Factors , Transfection , Tumor Suppressor Protein p53/metabolismABSTRACT
50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.