Showing papers on "Transcription (biology) published in 2019"
TL;DR: Advances in RNA-sequencing technologies and methods over the past decade are discussed and adaptations that are enabling a fuller understanding of RNA biology are outlined, from when and where an RNA is expressed to the structures it adopts.
Abstract: Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.
947 citations
TL;DR: This review highlights recent work suggesting that R-loops can be problematic to cells as blocks to efficient transcription and replication that trigger the DNA damage response and compares the available next-generation sequencing-based approaches to map R-loop genome wide.
411 citations
TL;DR: This Review discusses the various approaches that are being explored to target transcription factors in cancer, with many of the inhibitors developed from such approaches now advancing to early clinical trials.
Abstract: Mutated or dysregulated transcription factors represent a unique class of drug targets that mediate aberrant gene expression, including blockade of differentiation and cell death gene expression programmes, hallmark properties of cancers. Transcription factor activity is altered in numerous cancer types via various direct mechanisms including chromosomal translocations, gene amplification or deletion, point mutations and alteration of expression, as well as indirectly through non-coding DNA mutations that affect transcription factor binding. Multiple approaches to target transcription factor activity have been demonstrated, preclinically and, in some cases, clinically, including inhibition of transcription factor-cofactor protein-protein interactions, inhibition of transcription factor-DNA binding and modulation of levels of transcription factor activity by altering levels of ubiquitylation and subsequent proteasome degradation or by inhibition of regulators of transcription factor expression. In addition, several new approaches to targeting transcription factors have recently emerged including modulation of auto-inhibition, proteolysis targeting chimaeras (PROTACs), use of cysteine reactive inhibitors, targeting intrinsically disordered regions of transcription factors and combinations of transcription factor inhibitors with kinase inhibitors to block the development of resistance. These innovations in drug development hold great promise to yield agents with unique properties that are likely to impact future cancer treatment.
409 citations
TL;DR: In this paper, the authors investigated whether the phosphorylation of the C-terminal domain of the RNA polymerase II (PolII) C-interaction subunit regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing.
Abstract: The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex1–4. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus5,6. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain7–12. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers7,8, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites9–12. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference. RNA polymerase II with a hypophosphorylated C-terminal domain preferentially incorporates into mediator condensates, and with a hyperphosphorylated C-terminal domain into splicing-factor condensates, revealing phosphorylation as a regulatory mechanism in condensate preference.
404 citations
TL;DR: The ability to perform spatially resolved, genome-wide RNA profiling with high detection efficiency and accuracy by MERFISH could help address a wide array of questions ranging from the regulation of gene expression in cells to the development of cell fate and organization in tissues.
Abstract: The expression profiles and spatial distributions of RNAs regulate many cellular functions. Image-based transcriptomic approaches provide powerful means to measure both expression and spatial information of RNAs in individual cells within their native environment. Among these approaches, multiplexed error-robust fluorescence in situ hybridization (MERFISH) has achieved spatially resolved RNA quantification at transcriptome scale by massively multiplexing single-molecule FISH measurements. Here, we increased the gene throughput of MERFISH and demonstrated simultaneous measurements of RNA transcripts from ∼10,000 genes in individual cells with ∼80% detection efficiency and ∼4% misidentification rate. We combined MERFISH with cellular structure imaging to determine subcellular compartmentalization of RNAs. We validated this approach by showing enrichment of secretome transcripts at the endoplasmic reticulum, and further revealed enrichment of long noncoding RNAs, RNAs with retained introns, and a subgroup of protein-coding mRNAs in the cell nucleus. Leveraging spatially resolved RNA profiling, we developed an approach to determine RNA velocity in situ using the balance of nuclear versus cytoplasmic RNA counts. We applied this approach to infer pseudotime ordering of cells and identified cells at different cell-cycle states, revealing ∼1,600 genes with putative cell cycle-dependent expression and a gradual transcription profile change as cells progress through cell-cycle stages. Our analysis further revealed cell cycle-dependent and cell cycle-independent spatial heterogeneity of transcriptionally distinct cells. We envision that the ability to perform spatially resolved, genome-wide RNA profiling with high detection efficiency and accuracy by MERFISH could help address a wide array of questions ranging from the regulation of gene expression in cells to the development of cell fate and organization in tissues.
402 citations
TL;DR: It is reported that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally, and it is shown that m 6A modifications are enriched in the vicinity of H3K 36me3 peaks, and are reduced globally when cellular H 3K36 me3 is depleted.
Abstract: DNA and histone modifications have notable effects on gene expression1. Being the most prevalent internal modification in mRNA, the N6-methyladenosine (m6A) mRNA modification is as an important post-transcriptional mechanism of gene regulation2–4 and has crucial roles in various normal and pathological processes5–12. However, it is unclear how m6A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally. We show that m6A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m6A methyltransferase complex (MTC), which in turn facilitates the binding of the m6A MTC to adjacent RNA polymerase II, thereby delivering the m6A MTC to actively transcribed nascent RNAs to deposit m6A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m6A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m6A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation. METTL14 recognizes the trimethyl mark on lysine 36 of histone H3 that directs m6A modifications co-transcriptionally.
383 citations
TL;DR: Allele-specific single-cell RNA sequencing provides insights into transcription kinetics, with data indicating that core promoter sequences affect burst size, whereas enhancers mainly affect burst frequency.
Abstract: Mammalian gene expression is inherently stochastic1,2, and results in discrete bursts of RNA molecules that are synthesized from each allele3–7. Although transcription is known to be regulated by promoters and enhancers, it is unclear how cis-regulatory sequences encode transcriptional burst kinetics. Characterization of transcriptional bursting, including the burst size and frequency, has mainly relied on live-cell4,6,8 or single-molecule RNA fluorescence in situ hybridization3,5,8,9 recordings of selected loci. Here we determine transcriptome-wide burst frequencies and sizes for endogenous mouse and human genes using allele-sensitive single-cell RNA sequencing. We show that core promoter elements affect burst size and uncover synergistic effects between TATA and initiator elements, which were masked at mean expression levels. Notably, we provide transcriptome-wide evidence that enhancers control burst frequencies, and demonstrate that cell-type-specific gene expression is primarily shaped by changes in burst frequencies. Together, our data show that burst frequency is primarily encoded in enhancers and burst size in core promoters, and that allelic single-cell RNA sequencing is a powerful model for investigating transcriptional kinetics. Allele-specific single-cell RNA sequencing provides insights into transcription kinetics, with data indicating that core promoter sequences affect burst size, whereas enhancers mainly affect burst frequency.
379 citations
TL;DR: Structural and microscopy studies of gene transcription underpin a model in which phosphorylation controls the shuttling of RNA polymerase II between promoter and gene-body condensates to regulate transcription initiation and elongation.
Abstract: The regulated transcription of genes determines cell identity and function. Recent structural studies have elucidated mechanisms that govern the regulation of transcription by RNA polymerases during the initiation and elongation phases. Microscopy studies have revealed that transcription involves the condensation of factors in the cell nucleus. A model is emerging for the transcription of protein-coding genes in which distinct transient condensates form at gene promoters and in gene bodies to concentrate the factors required for transcription initiation and elongation, respectively. The transcribing enzyme RNA polymerase II may shuttle between these condensates in a phosphorylation-dependent manner. Molecular principles are being defined that rationalize transcriptional organization and regulation, and that will guide future investigations. Structural and microscopy studies of gene transcription underpin a model in which phosphorylation controls the shuttling of RNA polymerase II between promoter and gene-body condensates to regulate transcription initiation and elongation.
356 citations
TL;DR: In this paper, optical reconstruction of chromatin architecture (ORCA) is used to trace the DNA path in single cells with nanoscale accuracy and genomic resolution reaching two kilobases.
Abstract: The establishment of cell types during development requires precise interactions between genes and distal regulatory sequences. We have a limited understanding of how these interactions look in three dimensions, vary across cell types in complex tissue, and relate to transcription. Here we describe optical reconstruction of chromatin architecture (ORCA), a method that can trace the DNA path in single cells with nanoscale accuracy and genomic resolution reaching two kilobases. We used ORCA to study a Hox gene cluster in cryosectioned Drosophila embryos and labelled around 30 RNA species in parallel. We identified cell-type-specific physical borders between active and Polycomb-repressed DNA, and unexpected Polycomb-independent borders. Deletion of Polycomb-independent borders led to ectopic enhancer-promoter contacts, aberrant gene expression, and developmental defects. Together, these results illustrate an approach for high-resolution, single-cell DNA domain analysis in vivo, identify domain structures that change with cell identity, and show that border elements contribute to the formation of physical domains in Drosophila.
305 citations
TL;DR: It is proposed, that the transcription compartment is part of the regulatory architecture of embryonic nuclei and offers a transcriptionally competent environment to facilitate early escape from repression before global genome activation.
Abstract: Most metazoan embryos commence development with rapid, transcriptionally silent cell divisions, with genome activation delayed until the mid-blastula transition (MBT). However, a set of genes escapes global repression and gets activated before MBT. Here we describe the formation and the spatio-temporal dynamics of a pair of distinct transcription compartments, which encompasses the earliest gene expression in zebrafish. 4D imaging of pri-miR430 and zinc-finger-gene activities by a novel, native transcription imaging approach reveals transcriptional sharing of nuclear compartments, which are regulated by homologous chromosome organisation. These compartments carry the majority of nascent-RNAs and active Polymerase II, are chromatin-depleted and represent the main sites of detectable transcription before MBT. Transcription occurs during the S-phase of increasingly permissive cleavage cycles. It is proposed, that the transcription compartment is part of the regulatory architecture of embryonic nuclei and offers a transcriptionally competent environment to facilitate early escape from repression before global genome activation. Transcription is globally repressed in early stage of embryo development, but a set of genes including pri-miR-430 and zinc finger genes is known to escape the repression. Here the authors image the very first transcriptional activities in the living zebra fish embryo, demonstrating a cell cycle-coordinated polymerase II transcription compartment.
259 citations
TL;DR: It is shown that DNA sequences encoding TF binding site number, density, and affinity above sharply defined thresholds drive condensation of TFs and coactivators, which helps to understand how the genome can scaffold transcriptional condensates at specific loci and how the universal phenomenon of phase separation might regulate this process.
TL;DR: It is proposed that the assembly of DSB-induced transcriptional promoters drives RNA synthesis, which stimulates phase separation of DDR factors in the shape of foci, and that dilncRNAs drive molecular crowding of DDR proteins into foci that exhibit liquid–liquid phase- separation condensate properties.
Abstract: Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II are necessary for DNA-damage-response (DDR) focus formation. We demonstrate that induction of DSBs results in the assembly of functional promoters that include a complete RNA polymerase II preinitiation complex, MED1 and CDK9. Absence or inactivation of these factors causes a reduction in DDR foci both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins, such as 53BP1, into foci that exhibit liquid-liquid phase-separation condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis, which stimulates phase separation of DDR factors in the shape of foci.
TL;DR: Recent studies reveal that, although not required for higher-level genome organization, transcription has a role in the formation and stabilization of genomic subdomains and enhancer–promoter interactions.
Abstract: The spatial organization of the genome into compartments and topologically associated domains can have an important role in the regulation of gene expression. But could gene expression conversely regulate genome organization? Here, we review recent studies that assessed the requirement of transcription and/or the transcription machinery for the establishment or maintenance of genome topology. The results reveal different requirements at different genomic scales. Transcription is generally not required for higher-level genome compartmentalization, has only moderate effects on domain organization and is not sufficient to create new domain boundaries. However, on a finer scale, transcripts or transcription does seem to have a role in the formation of subcompartments and subdomains and in stabilizing enhancer-promoter interactions. Recent evidence suggests a dynamic, reciprocal interplay between fine-scale genome organization and transcription, in which each is able to modulate or reinforce the activity of the other.
TL;DR: A large-scale RBP ChIP-seq analysis reveals widespread RBP presence in active chromatin regions in the human genome, and proposes that various RBPs may enhance network interaction through harnessing regulatory RNAs to control transcription.
TL;DR: Functional experiments and bioinformatics analysis revealed that SATB2-AS1 inhibits CRC cell metastasis and regulates TH1-type chemokines expression and immune cell density in CRC.
Abstract: Emerging studies suggest that long non-coding RNAs (lncRNAs) play crucial roles in colorectal cancer (CRC). Here, we report a lncRNA, SATB2-AS1, which is specifically expressed in colorectal tissue and is significantly reduced in CRC. We systematically elucidated its functions and possible molecular mechanisms in CRC. LncRNA expression in CRC was analyzed by RNA-sequencing and RNA microarrays. The expression level of SATB2-AS1 in tissues was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH). The functional role of SATB2-AS1 in CRC was investigated by a series of in vivo and in vitro assays. RNA pull-down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), chromatin isolation by RNA purification (ChIRP), Bisulfite Sequencing PCR (BSP) and bioinformatics analysis were utilized to explore the potential mechanisms of SATB2-AS1. SATB2-AS1 is specifically expressed in colorectal tissues and downregulated in CRC. Survival analysis indicates that decreased SATB2-AS1 expression is associated with poor survival. Functional experiments and bioinformatics analysis revealed that SATB2-AS1 inhibits CRC cell metastasis and regulates TH1-type chemokines expression and immune cell density in CRC. Mechanistically, SATB2-AS1 directly binds to WDR5 and GADD45A, cis-activating SATB2 (Special AT-rich binding protein 2) transcription via mediating histone H3 lysine 4 tri-methylation (H3K4me3) deposition and DNA demethylation of the promoter region of SATB2. This study reveals the functions of SATB2-AS1 in CRC tumorigenesis and progression, suggesting new biomarkers and therapeutic targets in CRC.
TL;DR: Peppers, a series of monomeric, bright and stable FRs with a broad range of emission maxima spanning from cyan to red, are developed and it is believed these FRs will be useful tools for live imaging of cellular RNAs.
Abstract: Fluorescent RNAs (FRs), aptamers that bind and activate fluorescent dyes, have been used to image abundant cellular RNA species. However, limitations such as low brightness and limited availability of dye/aptamer combinations with different spectral characteristics have limited use of these tools in live mammalian cells and in vivo. Here, we develop Peppers, a series of monomeric, bright and stable FRs with a broad range of emission maxima spanning from cyan to red. Peppers allow simple and robust imaging of diverse RNA species in live cells with minimal perturbation of the target RNA's transcription, localization and translation. Quantification of the levels of proteins and their messenger RNAs in single cells suggests that translation is governed by normal enzyme kinetics but with marked heterogeneity. We further show that Peppers can be used for imaging genomic loci with CRISPR display, for real-time tracking of protein-RNA tethering, and for super-resolution imaging. We believe these FRs will be useful tools for live imaging of cellular RNAs.
TL;DR: It is shown how direct RNA-seq data can be used to define transcription initiation and RNA cleavage sites associated with all polyadenylated viral RNAs and demonstrated that low level read-through transcription produces a novel class of chimeric HSV-1 transcripts.
Abstract: Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns. Direct RNA sequencing (direct RNA-seq) using nanopore arrays offers an exciting alternative whereby individual polyadenylated RNAs are sequenced directly, without the recoding and amplification biases inherent to other sequencing methodologies. Here we use direct RNA-seq to profile the herpes simplex virus type 1 (HSV-1) transcriptome during productive infection of primary cells. We show how direct RNA-seq data can be used to define transcription initiation and RNA cleavage sites associated with all polyadenylated viral RNAs and demonstrate that low level read-through transcription produces a novel class of chimeric HSV-1 transcripts, including a functional mRNA encoding a fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L. Thus, direct RNA-seq offers a powerful method to characterize the changing transcriptional landscape of viruses with complex genomes. Here, Depledge et al. use nanopore arrays for direct RNA sequencing to profile the HSV-1 transcriptome in productively infected cells. Sequencing of individual RNAs reveals a highly complex viral transcriptome including mRNAs encoding new viral fusion proteins derived by read-through transcription.
TL;DR: TBL detection by the stalling of RNA polymerase II is highly efficient but may interfere with repair, and overall with transcription and replication, Consequently, TC-NER deregulation causes hereditary disorders with complex genotype–phenotype correlations.
Abstract: The spatiotemporal control of RNA polymerase II (Pol II)-mediated gene transcription is tightly and intricately regulated. In addition, preservation of the integrity of the DNA template is required so as to ensure unperturbed transcription, particularly since DNA is continually challenged by different types of damaging agents that can form transcription-blocking DNA lesions (TBLs), which impede transcription elongation and cause transcription stress. To overcome the highly cytotoxic effects of TBLs, an intricate cellular response has evolved, in which the transcription-coupled nucleotide excision repair (TC-NER) pathway has a central role in removing TBLs specifically from the transcribed strand. Damage detection by stalling of the transcribing Pol II is highly efficient, but a stalled Pol II complex may create an even bigger problem by interfering with repair of the lesions, and overall with transcription and replication. In this Review, we discuss the effects of different types of DNA damage on Pol II, important concepts of transcription stress, the manner in which TBLs are removed by TC-NER and how different tissues respond to TBLs. We also discuss the role of TBLs in ageing and the complex genotype–phenotype correlations of TC-NER hereditary disorders. Transcription-blocking DNA lesions (TBLs) cause transcription stress and are repaired by transcription-coupled nucleotide excision repair (TC-NER). TBL detection by the stalling of RNA polymerase II is highly efficient but may interfere with repair, and overall with transcription and replication. Consequently, TC-NER deregulation causes hereditary disorders with complex genotype–phenotype correlations.
TL;DR: A robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells, is reported, which enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.
Abstract: We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9-mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.
TL;DR: The cytotopic structuromes substantially expand RNA structural information and enable detailed investigation of the central role of RNA structure in linking transcription, translation and RNA decay, and validate a novel role for the RNA-binding protein LIN28A as an N6-methyladenosine modification ‘anti-reader’.
Abstract: RNA structure is intimately connected to each step of gene expression. Recent advances have enabled transcriptome-wide maps of RNA secondary structure, called 'RNA structuromes'. However, previous whole-cell analyses lacked the resolution to unravel the landscape and also the regulatory mechanisms of RNA structural changes across subcellular compartments. Here we reveal the RNA structuromes in three compartments, chromatin, nucleoplasm and cytoplasm, in human and mouse cells. The cytotopic structuromes substantially expand RNA structural information and enable detailed investigation of the central role of RNA structure in linking transcription, translation and RNA decay. We develop a resource with which to visualize the interplay of RNA-protein interactions, RNA modifications and RNA structure and predict both direct and indirect reader proteins of RNA modifications. We also validate a novel role for the RNA-binding protein LIN28A as an N6-methyladenosine modification 'anti-reader'. Our results highlight the dynamic nature of RNA structures and its functional importance in gene regulation.
TL;DR: Investigation of the functions of a new circRNA circ-DONSON in GC progression found it promotes GC progression through recruiting the NURF complex to initiate SOX4 expression and suppressed the proliferation, migration and invasion of GC cells while promoting apoptosis.
Abstract: Circular RNAs (circRNAs) are a novel type of noncoding RNAs and play important roles in tumorigenesis, including gastric cancer (GC). However, the functions of most circRNAs remain poorly understood. In our study, we aimed to investigate the functions of a new circRNA circ-DONSON in GC progression. The expression of circ-DONSON in gastric cancer tissues and adjacent normal tissues was analyzed by bioinformatics method, qRT-PCR, Northern blotting and in situ hybridization (ISH). The effects of circ-DONSON on GC cell proliferation, apoptosis, migration and invasion were measured by using CCK8, colony formation, EdU, immunofluorescence (IF), FACS and Transwell assays. qRT-PCR and Western blotting were utilized to validate how circ-DONSON regulates SOX4 expression. ChIP, DNA fluorescence in situ hybridization (DNA-FISH) and DNA accessibility assays were used to investigate how circ-DONSON regulates SOX4 transcription. The interaction between circ-DONSON and NURF complex was evaluated by mass spectrum, RNA immunoprecipitation (RIP), pulldown and EMSA assays. Xenograft mouse model was used to analyze the effect of circ-DONSON on GC growth in vivo. Elevated expression of circ-DONSON was observed in GC tissues and positively associated with advanced TNM stage and unfavorable prognosis. Silencing of circ-DONSON significantly suppressed the proliferation, migration and invasion of GC cells while promoting apoptosis. circ-DONSON was localized in the nucleus, recruited the NURF complex to SOX4 promoter and initiated its transcription. Silencing of the NURF complex subunit SNF2L, BPTF or RBBP4 similarly attenuated GC cell growth and increased apoptosis. circ-DONSON knockdown inhibited GC growth in vivo. circ-DONSON promotes GC progression through recruiting the NURF complex to initiate SOX4 expression.
TL;DR: It is demonstrated that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5' ends of mRNAs.
TL;DR: The genome-wide transcriptional profile resulting from RNA polymerase (RNAP) binding by ppGpp, a transcription regulator found throughout the bacterial domain of life, is presented, greatly expands the understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects ofppGpp.
Abstract: The second messenger nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability. ppGpp binds to two sites on RNA polymerase (RNAP) in Escherichia coli, but it has also been reported to bind to many other proteins. To determine the role of the RNAP binding sites in the genome-wide effects of ppGpp on transcription, we used RNA-seq to analyze transcripts produced in response to elevated ppGpp levels in strains with/without the ppGpp binding sites on RNAP. We examined RNAs rapidly after ppGpp production without an accompanying nutrient starvation. This procedure enriched for direct effects of ppGpp on RNAP rather than for indirect effects on transcription resulting from starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of ppGpp induction depended on ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp.
TL;DR: The data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation.
Abstract: Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation.
TL;DR: It is proposed that GADD45A is an epigenetic R-loop reader that recruits the demethylation machinery to promoter CGIs and mediates local DNA dem methylation by recruiting TET1.
Abstract: R-loops are DNA-RNA hybrids enriched at CpG islands (CGIs) that can regulate chromatin states1-8. How R-loops are recognized and interpreted by specific epigenetic readers is unknown. Here we show that GADD45A (growth arrest and DNA damage protein 45A) binds directly to R-loops and mediates local DNA demethylation by recruiting TET1 (ten-eleven translocation 1). Studying the tumor suppressor TCF21 (ref. 9), we find that antisense long noncoding (lncRNA) TARID (TCF21 antisense RNA inducing promoter demethylation) forms an R-loop at the TCF21 promoter. Binding of GADD45A to the R-loop triggers local DNA demethylation and TCF21 expression. TARID transcription, R-loop formation, DNA demethylation, and TCF21 expression proceed sequentially during the cell cycle. Oxidized DNA demethylation intermediates are enriched at genomic R-loops and their levels increase upon RNase H1 depletion. Genomic profiling in embryonic stem cells identifies thousands of R-loop-dependent TET1 binding sites at CGIs. We propose that GADD45A is an epigenetic R-loop reader that recruits the demethylation machinery to promoter CGIs.
TL;DR: Since LINC01123 is upregulated in NSCLC, correlates with prognosis, and controls proliferation and aerobic glycolysis by a positive feedback loop with c-Myc, it is expected to be a potential biomarker and therapeutic target forNSCLC.
Abstract: Long non-coding RNAs (lncRNAs) have been associated with non-small cell lung cancer (NSCLC), but the underlying molecular mechanisms of their specific roles in mediating aerobic glycolysis have been poorly explored. Next-generation RNA sequencing assay was performed to identify the differentially expressed RNAs between NSCLC tissues with high 18F-fluorodeoxyglucose (FDG) uptake and their adjacent normal lung tissues. LINC01123 expression in NSCLC tissues was measured by real-time PCR and in situ hybridization (ISH) assay. The biological role of LINC01123 in cell growth and aerobic glycolysis capability was determined by performing functional experiments in vitro and in vivo. Further, the transcription of LINC01123 was explored by bioinformatics analysis, dual-luciferase reporter assay, and chromatin immunoprecipitation (ChIP) assay. RNA immunoprecipitation (RIP) and luciferase analyses were used to confirm the predicted competitive endogenous RNA (ceRNA) mechanisms between LINC01123 and c-Myc. Three hundred sixty-four differentially expressed genes were identified in RNA-seq assay, and LINC01123 was one of the most overexpressed lncRNAs. Further validation in expanded NSCLC cohorts confirmed that LINC01123 was upregulated in 92 paired NSCLC tissues and associated with poor survival. Functional assays showed that LINC01123 promoted NSCLC cell proliferation and aerobic glycolysis. Mechanistic investigations revealed that LINC01123 was a direct transcriptional target of c-Myc. Meanwhile, LINC01123 increased c-Myc mRNA expression by sponging miR-199a-5p. In addition, rescue experiments showed that LINC01123 functioned as an oncogene depending on miR-199a-5p and c-Myc. Since LINC01123 is upregulated in NSCLC, correlates with prognosis, and controls proliferation and aerobic glycolysis by a positive feedback loop with c-Myc, it is expected to be a potential biomarker and therapeutic target for NSCLC.
TL;DR: This Review provides an overview of different approaches for measuring nascent RNA synthesis and discusses the complementary strengths of these different methods and how they have contributed to a mechanistic understanding of transcription regulation.
Abstract: The programmes that direct an organism's development and maintenance are encoded in its genome. Decoding of this information begins with regulated transcription of genomic DNA into RNA. Although transcription and its control can be tracked indirectly by measuring stable RNAs, it is only by directly measuring nascent RNAs that the immediate regulatory changes in response to developmental, environmental, disease and metabolic signals are revealed. Multiple complementary methods have been developed to quantitatively track nascent transcription genome-wide at nucleotide resolution, all of which have contributed novel insights into the mechanisms of gene regulation and transcription-coupled RNA processing. Here we critically evaluate the array of strategies used for investigating nascent transcription and discuss the recent conceptual advances they have provided.
TL;DR: An alternative pathway for PARPi-mediated growth control in BRCA1/2-intact breast cancer cells involving rDNA transcription and ribosome biogenesis is identified, evident in xenografts in mice and human breast cancer patient samples.
TL;DR: SR-4835 is reported, a highly selective dual inhibitor of CDK12 and CDK13, which disables triple-negative breast cancer (TNBC) cells and provokes a "BRCAness" phenotype that results in deficiencies in DNA damage repair, promoting synergy with DNA-damaging chemotherapy and PARP inhibitors.
TL;DR: A mathematical model of regulation is derived that relates transcription, chromosome structure, and the cell's ability to sense changes in estrogen and predicts that hypervariability is largely dynamic and does not reflect a stable biological state.