ABSTRACT
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
We report a mechanism through which the transcription machinery directly controls topoisomerase 1 (TOP1) activity to adjust DNA topology throughout the transcription cycle. By comparing TOP1 occupancy using chromatin immunoprecipitation sequencing (ChIP-seq) versus TOP1 activity using topoisomerase 1 sequencing (TOP1-seq), a method reported here to map catalytically engaged TOP1, TOP1 bound at promoters was discovered to become fully active only after pause-release. This transition coupled the phosphorylation of the carboxyl-terminal-domain (CTD) of RNA polymerase II (RNAPII) with stimulation of TOP1 above its basal rate, enhancing its processivity. TOP1 stimulation is strongly dependent on the kinase activity of BRD4, a protein that phosphorylates Ser2-CTD and regulates RNAPII pause-release. Thus the coordinated action of BRD4 and TOP1 overcame the torsional stress opposing transcription as RNAPII commenced elongation but preserved negative supercoiling that assists promoter melting at start sites. This nexus between transcription and DNA topology promises to elicit new strategies to intercept pathological gene expression.
Subject(s)
DNA Topoisomerases, Type I/metabolism , DNA/metabolism , RNA Polymerase II/metabolism , Transcription, Genetic , DNA/chemistry , DNA Topoisomerases, Type I/genetics , Gene Knockdown Techniques , Humans , Promoter Regions, Genetic , RNA Polymerase II/chemistry , RNA Polymerase II/isolation & purification , Transcription Elongation, Genetic , Transcription Factors/isolation & purification , Transcription Initiation SiteABSTRACT
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
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
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
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.
Subject(s)
B-Lymphocytes/metabolism , Cell Cycle , Cell Nucleus/metabolism , Chromatin Assembly and Disassembly , Chromatin/metabolism , Histones/metabolism , Lymphocyte Activation , Proto-Oncogene Proteins c-myc/metabolism , Acetyl Coenzyme A/metabolism , Acetylation , Adenosine Triphosphate/metabolism , Animals , B-Lymphocytes/immunology , Cell Line , Chromatin/chemistry , Chromatin/genetics , DNA Methylation , Epigenesis, Genetic , Genotype , Histones/chemistry , Immunity, Humoral , Methylation , Mice, Inbred C57BL , Mice, Knockout , Nucleic Acid Conformation , Phenotype , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-myc/chemistry , Proto-Oncogene Proteins c-myc/genetics , Single Molecule Imaging , Structure-Activity Relationship , Time Factors , Transcription, GeneticABSTRACT
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
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 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
The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEß). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEß) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.
Subject(s)
Cyclin-Dependent Kinases/genetics , DNA Repair , Transcription Factors, TFII/genetics , Trichothiodystrophy Syndromes/genetics , Amino Acid Sequence , Cyclin-Dependent Kinases/metabolism , DNA Damage , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Gene Silencing , Humans , Infant , Male , Molecular Sequence Data , Mutation, Missense , Pedigree , Phosphorylation , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Transcription Factor TFIIH/genetics , Transcription Factor TFIIH/metabolism , Transcription Factors, TFII/metabolism , Xeroderma Pigmentosum Group D Protein/genetics , Xeroderma Pigmentosum Group D Protein/metabolism , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
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
The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins in vivo. Formaldehyde cross-linking of DNA and proteins is a critical step required to trap their interactions inside the cells before immunoprecipitation and analysis. Yet insufficient attention has been given to variables that might give rise to artifacts in this procedure, such as the duration of cross-linking. We analyzed the dependence of the ChIP signal on the duration of formaldehyde cross-linking time for two proteins: DNA topoisomerase 1 (Top1) that is functionally associated with the double helix in vivo, especially with active chromatin, and green fluorescent protein (GFP) that has no known bona fide interactions with DNA. With short time of formaldehyde fixation, only Top1 immunoprecipation efficiently recovered DNA from active promoters, whereas prolonged fixation augmented non-specific recovery of GFP dramatizing the need to optimize ChIP protocols to minimize the time of cross-linking, especially for abundant nuclear proteins. Thus, ChIP is a powerful approach to study the localization of protein on the genome when care is taken to manage potential artifacts.
Subject(s)
Chromatin Immunoprecipitation/methods , Chromatin/chemistry , Cross-Linking Reagents/chemistry , DNA Topoisomerases, Type I/chemistry , DNA/chemistry , Formaldehyde/chemistry , Green Fluorescent Proteins/chemistry , Cell Line, Tumor , DNA-Binding Proteins/chemistry , HCT116 Cells , Humans , Time FactorsABSTRACT
Genomic DNA is under constant assault by endogenous and exogenous DNA damaging agents. DNA breakage can represent a major threat to genome integrity but can also be necessary for genome function. Here we present approaches to map DNA double-strand breaks (DSBs) and single-strand breaks (SSBs) at the genome-wide scale by two methods called DSB- and SSB-Seq, respectively. We tested these methods in human colon cancer cells and validated the results using the Topoisomerase II (Top2)-poisoning agent etoposide (ETO). Our results show that the combination of ETO treatment with break-mapping techniques is a powerful method to elaborate the pattern of Top2 enzymatic activity across the genome.
Subject(s)
DNA Breaks, Double-Stranded , DNA Breaks, Single-Stranded , DNA Topoisomerases, Type II/metabolism , DNA/metabolism , Chromosome Mapping , HCT116 Cells , Humans , Sequence Analysis, DNAABSTRACT
Through dynamic changes in structure resulting from DNA-protein interactions and constraints given by the structural features of the double helix, chromatin accommodates and regulates different DNA-dependent processes. All DNA transactions (such as transcription, DNA replication and chromosomal segregation) are necessarily linked to strong changes in the topological state of the double helix known as torsional stress or supercoiling. As virtually all DNA transactions are in turn affected by the torsional state of DNA, these changes have the potential to serve as regulatory signals detected by protein partners. This two-way relationship indicates that DNA dynamics may contribute to the regulation of many events occurring during cell life. In this review we will focus on the role of DNA supercoiling in the cellular processes, with particular emphasis on transcription. Besides giving an overview on the multiplicity of factors involved in the generation and dissipation of DNA torsional stress, we will discuss recent studies which give new insight into the way cells use DNA dynamics to perform functions otherwise not achievable. This article is part of a Special Issue entitled: Chromatin in time and space.
Subject(s)
DNA, Superhelical/physiology , Transcription, Genetic , Animals , Chromatin/genetics , Chromatin/metabolism , Chromatin Assembly and Disassembly , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Gene Expression Regulation , Humans , Promoter Regions, GeneticABSTRACT
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
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
Top1 inhibition by camptothecin (CPT) perturbs RNA polymerase II (Pol II) density at promoters and along transcribed genes suggesting an involvement of Top1 in Pol II pausing. Here, we demonstrate that Top1 inhibition favors Pol II escape from a promoter-proximal pausing site of the human HIF-1alpha gene in living cells. Interestingly, alternative splicing at exon 11 was markedly altered in nascent HIF-1alpha mRNAs, and chromatin structure was also affected with enhanced histone acetylation and reduced nucleosome density in a manner dependent on cdk activity. Moreover, CPT increases transcription of a novel long RNA (5'aHIF1alpha), antisense to human HIF-1alpha mRNA, and a known antisense RNA at the 3'-end of the gene, while decreasing mRNA levels under normoxic and hypoxic conditions. The effects require Top1, but are independent from Top1-induced replicative DNA damage. Chromatin RNA immunoprecipitation results showed that CPT can activate antisense transcription mediated by cyclin-dependent kinase (cdk) activity. Thus, Top1 inhibition can trigger a transcriptional stress, involving antisense transcription and increased chromatin accessibility, which is dependent on cdk activity and deregulated Pol II pausing. A changed balance of antisense transcripts and mRNAs may then lead to altered regulation of HIF-1alpha activity in human cancer cells.