Showing papers on "Transcription (biology) published in 2017"
TL;DR: This work systematically analyzed binding specificities of full-length transcription factors and extended DNA binding domains to unmethylated and CpG-methylated DNA by using methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment).
Abstract: INTRODUCTION Nearly all cells in the human body share the same primary genome sequence consisting of four nucleotide bases. One of the bases, cytosine, is commonly modified by methylation of its 5 position in CpG dinucleotides (mCpG). Most CpG dinucleotides in the human genome are methylated, but the level of CpG methylation varies with genetic location (promoter versus gene body), whether genes are active versus silenced, and cell type. Research has shown that the maintenance of a particular cellular state after cell division is dependent on faithful transmission of methylated CpGs, as well as inheritance of the mother cells’ repertoire of transcription factors by the daughter cells. These two mechanisms of epigenetic inheritance are linked to each other; the binding of transcription factors can be affected by cytosine methylation, and cytosine methylation can, in turn, be added or removed by proteins that associate with transcription factors. RATIONALE The genetic and epigenetic language, which imparts when and where genes are expressed, is understood at a conceptual level. However, a more detailed understanding is needed of the genomic regulatory mechanism by which methylated cytosines affect transcription factor binding. Because cytosine methylation changes DNA structure, it has the potential to affect binding of all transcription factors. However, a systematic analysis of binding of a large collection of transcription factors to all possible DNA sequences has not previously been conducted. RESULTS To globally characterize the effect of cytosine methylation on transcription factor binding, we systematically analyzed binding specificities of full-length transcription factors and extended DNA binding domains to unmethylated and CpG-methylated DNA by using methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment). We evaluated binding of 542 transcription factors and identified a large number of previously uncharacterized transcription factor recognition motifs. Binding of most major classes of transcription factors, including bHLH, bZIP, and ETS, was inhibited by mCpG. In contrast, transcription factors such as homeodomain, POU, and NFAT proteins preferred to bind methylated DNA. This class of binding was enriched in factors with central roles in embryonic and organismal development. The observed binding preferences were validated using several orthogonal methods, including bisulfite-SELEX and protein-binding microarrays. In addition, the preference of the pluripotency factor OCT4 to bind to a mCpG-containing motif was confirmed by chromatin immunoprecipitation analysis in mouse embryonic stem cells with low or high levels of CpG methylation (due to deficiency in all enzymes that methylate cytosines or contribute to their removal, respectively). Crystal structure analysis of the homeodomain proteins HOXB13, CDX1, CDX2, and LHX4 revealed three key residues that contribute to the preference of this developmentally important family of transcription factors for mCpG. The preference for binding to mCpG was due to direct hydrophobic interactions with the 5-methyl group of methylcytosine. In contrast, inhibition of binding of other transcription factors to methylated sequences was found to be caused by steric hindrance. CONCLUSION Our work constitutes a global analysis of the effect of cytosine methylation on DNA binding specificities of human transcription factors. CpG methylation can influence binding of most transcription factors to DNA—in some cases negatively and in others positively. Our finding that many developmentally important transcription factors prefer to bind to mCpG sites can inform future analyses of the role of DNA methylation on cell differentiation, chromatin reprogramming, and transcriptional regulation.
846 citations
TL;DR: It is shown that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation, with implications for intragenics hypomethylation in cancer.
Abstract: In mammals, DNA methylation occurs mainly at CpG dinucleotides. Methylation of the promoter suppresses gene expression, but the functional role of gene-body DNA methylation in highly expressed genes has yet to be clarified. Here we show that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation. Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment to the gene body by H3K36me3. Furthermore, the spurious transcripts can either be degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins. Elongating RNA polymerase II therefore triggers an epigenetic crosstalk mechanism that involves SetD2, H3K36me3, Dnmt3b and DNA methylation to ensure the fidelity of gene transcription initiation, with implications for intragenic hypomethylation in cancer.
520 citations
TL;DR: Recent progress in elucidating the molecular mechanisms by which lncRNAs modulate gene expression is reviewed, including the act of lnc RNA transcription rather than the lncRNA product that appears to be regulatory.
Abstract: It has recently become apparent that RNA, itself the product of transcription, is a major regulator of the transcriptional process. In particular, long noncoding RNAs (lncRNAs), which are so numerous in eukaryotes, function in many cases as transcriptional regulators. These RNAs function through binding to histone-modifying complexes, to DNA binding proteins (including transcription factors), and even to RNA polymerase II. In other cases, it is the act of lncRNA transcription rather than the lncRNA product that appears to be regulatory. We review recent progress in elucidating the molecular mechanisms by which lncRNAs modulate gene expression and future opportunities in this research field.
473 citations
TL;DR: Mouse DUX and human DUX4 are proposed as major drivers of the cleavage or 2C state, which is strongly resembling that of mouse 2C embryos.
Abstract: To better understand transcriptional regulation during human oogenesis and preimplantation development, we defined stage-specific transcription, which highlighted the cleavage stage as being highly distinctive. Here, we present multiple lines of evidence that a eutherian-specific multicopy retrogene, DUX4, encodes a transcription factor that activates hundreds of endogenous genes (for example, ZSCAN4, KDM4E and PRAMEF-family genes) and retroviral elements (MERVL/HERVL family) that define the cleavage-specific transcriptional programs in humans and mice. Remarkably, mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells, measured here by the reactivation of '2C' genes and repeat elements, the loss of POU5F1 (also known as OCT4) protein and chromocenters, and the conversion of the chromatin landscape (as assessed by transposase-accessible chromatin using sequencing (ATAC-seq)) to a state strongly resembling that of mouse 2C embryos. Thus, we propose mouse DUX and human DUX4 as major drivers of the cleavage or 2C state.
448 citations
TL;DR: It is proposed that DHX9 acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression.
Abstract: In the absence of DHX9, circular RNAs accumulate and transcription and translation are dysregulated—effects that are exacerbated by concomitant depletion of the RNA-editing enzyme ADAR. In the human genome, there are more than a million copies of the Alu transposable element. Movement of Alu elements is a common source of mutations, but as insertions usually occur in non-coding regions, they are often without discernible effect. Alu elements located near one another in an inverted orientation will form secondary structures that may affect various nuclear processes. Asifa Akhtar and colleagues find that the RNA helicase, DHX9, binds transcribed ‘IRAlus’ (inverted repeat Alu elements). In the absence of DHX9, circular RNAs accumulate, and transcription and translation are dysregulated. These effects are further exacerbated by co-depletion of DHX9 and ADAR p150, an interferon-inducible RNA modification enzyme. The authors conclude that these proteins protect against transposon insertion, which can have deleterious effects on gene expression. Transposable elements are viewed as ‘selfish genetic elements’, yet they contribute to gene regulation and genome evolution in diverse ways1. More than half of the human genome consists of transposable elements2. Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome2. Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability3. Alu elements are the main targets of the RNA-editing enzyme ADAR4 and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC5, but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant6 nuclear RNA helicase7, binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression.
367 citations
TL;DR: A novel signal transduction pathway in neurons whereby ApoE activates a non-canonical MAP kinase cascade that enhances APP transcription and amyloid-β synthesis is described.
352 citations
TL;DR: These studies establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy and show that BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition.
306 citations
TL;DR: The results suggest that enhancer H3K4me1 provides only a minor contribution to the long-range coactivator function of Mll3/4, and downregulated genes exhibit reduced polymerase levels in gene bodies, but not at promoters, suggestive of pause-release defects.
297 citations
TL;DR: Progress in super-resolution imaging, genome-wide application of RNA sequencing, chromatin immunoprecipitation and chromosome conformation capture and in theoretical modelling of 3D genome organization enables the exploration of the relationship between cell mechanics, 3D chromatin configurations and transcription, thereby shedding new light on how mechanical forces regulate gene expression.
Abstract: It is well established that cells sense chemical signals from their local microenvironment and transduce them to the nucleus to regulate gene expression programmes. Although a number of experiments have shown that mechanical cues can also modulate gene expression, the underlying mechanisms are far from clear. Nevertheless, we are now beginning to understand how mechanical cues are transduced to the nucleus and how they influence nuclear mechanics, genome organization and transcription. In particular, recent progress in super-resolution imaging, in genome-wide application of RNA sequencing, chromatin immunoprecipitation and chromosome conformation capture and in theoretical modelling of 3D genome organization enables the exploration of the relationship between cell mechanics, 3D chromatin configurations and transcription, thereby shedding new light on how mechanical forces regulate gene expression.
279 citations
TL;DR: It is demonstrated that CBP binds directly to RNAs in vivo and in–vitro, and an RNA binding region in the HAT domain of CBP-a regulatory motif unique to CBP/p300-allows RNA to stimulate CBP's HAT activity.
275 citations
TL;DR: It is proposed that DDR signalling sites, in addition to sharing a common pool of proteins, individually host a unique set of site-specific RNAs necessary for DDR activation.
Abstract: The DNA damage response (DDR) preserves genomic integrity. Small non-coding RNAs termed DDRNAs are generated at DNA double-strand breaks (DSBs) and are critical for DDR activation. Here we show that active DDRNAs specifically localize to their damaged homologous genomic sites in a transcription-dependent manner. Following DNA damage, RNA polymerase II (RNAPII) binds to the MRE11-RAD50-NBS1 complex, is recruited to DSBs and synthesizes damage-induced long non-coding RNAs (dilncRNAs) from and towards DNA ends. DilncRNAs act both as DDRNA precursors and by recruiting DDRNAs through RNA-RNA pairing. Together, dilncRNAs and DDRNAs fuel DDR focus formation and associate with 53BP1. Accordingly, inhibition of RNAPII prevents DDRNA recruitment, DDR activation and DNA repair. Antisense oligonucleotides matching dilncRNAs and DDRNAs impair site-specific DDR focus formation and DNA repair. We propose that DDR signalling sites, in addition to sharing a common pool of proteins, individually host a unique set of site-specific RNAs necessary for DDR activation.
TL;DR: Techniques that allow the temporal coordination of co-transcriptional splicing to occur on similar timescales are discussed, including spliceosome assembly, transcription elongation and other co- transcriptional events that allow for spatially constrained splicing.
Abstract: Several macromolecular machines collaborate to produce eukaryotic messenger RNA. RNA polymerase II (Pol II) translocates along genes that are up to millions of base pairs in length and generates a flexible RNA copy of the DNA template. This nascent RNA harbours introns that are removed by the spliceosome, which is a megadalton ribonucleoprotein complex that positions the distant ends of the intron into its catalytic centre. Emerging evidence that the catalytic spliceosome is physically close to Pol II in vivo implies that transcription and splicing occur on similar timescales and that the transcription and splicing machineries may be spatially constrained. In this Review, we discuss aspects of spliceosome assembly, transcription elongation and other co-transcriptional events that allow the temporal coordination of co-transcriptional splicing.
TL;DR: How TF synergy is mediated at the level of DNA binding and after binding, the role of cofactors and the post-translational modifications they catalyze, and different models of enhancer-core-promoter communication are reviewed.
TL;DR: By using a ribozyme to co-transcriptionally cleave nascent RNA, it is demonstrated that a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop, providing a topological solution for RNA invasion into duplex DNA and suggesting an order for R- loop initiation and elongation in an opposite direction to that previously proposed.
TL;DR: The RNA-binding protein hnRNPK is identified as the principal XR-PID binding factor required to recruit PCGF3/5-PRC1, providing important insights into mechanisms of chromatin modification by non-coding RNA.
TL;DR: Current knowledge on lncRNAs is reviewed and the most recent discoveries of the underlying mechanisms related to their formation are highlighted, highlighting their potential as key regulators in gene expression networks and generating new RNA species with unexpected formats.
TL;DR: It is demonstrated that dynamic looping events are regulatory rather than structural in nature and widespread coordination of dynamic enhancer activity at preformed and acquired DNA loops is uncovered.
TL;DR: Recent advances in the design and delivery of therapies to target HTT RNA and DNA are expected to improve their efficacy, safety, tolerability, and duration of effect in future studies.
Abstract: Summary No disease-slowing treatment exists for Huntington's disease, but its monogenic inheritance makes it an appealing candidate for the development of therapies targeting processes close to its genetic cause. Huntington's disease is caused by CAG repeat expansions in the HTT gene, which encodes the huntingtin protein; development of therapies to target HTT transcription and the translation of its mRNA is therefore an area of intense investigation. Huntingtin-lowering strategies include antisense oligonucleotides and RNA interference targeting mRNA, and zinc finger transcriptional repressors and CRISPR-Cas9 methods aiming to reduce transcription by targeting DNA. An intrathecally delivered antisense oligonucleotide that aims to lower huntingtin is now well into its first human clinical trial, with other antisense oligonucleotides expected to enter trials in the next 1–2 years and virally delivered RNA interference and zinc finger transcriptional repressors in advanced testing in animal models. Recent advances in the design and delivery of therapies to target HTT RNA and DNA are expected to improve their efficacy, safety, tolerability, and duration of effect in future studies.
TL;DR: Large-scale changes in nuclear architecture were associated with the deposition of activating epigenetic marks across the loop domain, plausibly facilitating phase separation and indicating how, during developmental progression and tumor suppression, non-coding transcription orchestrates chromatin folding and compartmentalization to direct with high precision enhancer-promoter communication.
TL;DR: By mapping global transcription start site (TSS) and chromatin dynamics, this work observed the cryptic transcription of thousands of treatment-induced non-annotated TSSs (TINATs) following DNMTi and HDACi treatment.
Abstract: Several mechanisms of action have been proposed for DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi), primarily based on candidate-gene approaches. However, less is known about their genome-wide transcriptional and epigenomic consequences. By mapping global transcription start site (TSS) and chromatin dynamics, we observed the cryptic transcription of thousands of treatment-induced non-annotated TSSs (TINATs) following DNMTi and HDACi treatment. The resulting transcripts frequently splice into protein-coding exons and encode truncated or chimeric ORFs translated into products with predicted abnormal or immunogenic functions. TINAT transcription after DNMTi treatment coincided with DNA hypomethylation and gain of classical promoter histone marks, while HDACi specifically induced a subset of TINATs in association with H2AK9ac, H3K14ac, and H3K23ac. Despite this mechanistic difference, both inhibitors convergently induced transcription from identical sites, as we found TINATs to be encoded in solitary long terminal repeats of the ERV9/LTR12 family, which are epigenetically repressed in virtually all normal cells.
TL;DR: The tomato transcription factor (TF) MYC2 and its downstream MTFs form a hierarchical transcriptional cascade during jasmonate-mediated plant immunity that initiates and amplifies transcriptional output.
Abstract: The hormone jasmonate (JA), which functions in plant immunity, regulates resistance to pathogen infection and insect attack through triggering genome-wide transcriptional reprogramming in plants. We show that the basic helix-loop-helix transcription factor (TF) MYC2 in tomato (Solanum lycopersicum) acts downstream of the JA receptor to orchestrate JA-mediated activation of both the wounding and pathogen responses. Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with RNA sequencing (RNA-seq) assays, we identified 655 MYC2-targeted JA-responsive genes. These genes are highly enriched in Gene Ontology categories related to TFs and the early response to JA, indicating that MYC2 functions at a high hierarchical level to regulate JA-mediated gene transcription. We also identified a group of MYC2-targeted TFs (MTFs) that may directly regulate the JA-induced transcription of late defense genes. Our findings suggest that MYC2 and its downstream MTFs form a hierarchical transcriptional cascade during JA-mediated plant immunity that initiates and amplifies transcriptional output. As proof of concept, we showed that during plant resistance to the necrotrophic pathogen Botrytis cinerea, MYC2 and the MTF JA2-Like form a transcription module that preferentially regulates wounding-responsive genes, whereas MYC2 and the MTF ETHYLENE RESPONSE FACTOR.C3 form a transcription module that preferentially regulates pathogen-responsive genes.
TL;DR: In this paper, the authors define systematic differences in transcription and RNA processing between protein-coding and lincRNA genes in human HeLa cells, based on a range of nascent transcriptomic approaches applied to different nuclear fractions, including mammalian native elongating transcript sequencing.
TL;DR: Using the recently developed ChIP-nexus method, it is found that Pol II pausing inhibits new initiation and proposed that paused Pol II helps prevent new initiation between transcription bursts, which may reduce noise.
Abstract: RNA polymerase II (Pol II) pauses downstream of the transcription initiation site before beginning productive elongation This pause is a key component of metazoan gene expression regulation Some promoters have a strong disposition for Pol II pausing and often mediate faster, more synchronous changes in expression This requires multiple rounds of transcription and thus cannot rely solely on pause release However, it is unclear how pausing affects the initiation of new transcripts during consecutive rounds of transcription Using our recently developed ChIP-nexus method, we find that Pol II pausing inhibits new initiation We propose that paused Pol II helps prevent new initiation between transcription bursts, which may reduce noise
TL;DR: It is shown that, in Drosophila, transcription of PIWI-interacting RNA (piRNA) clusters—small RNA source loci in animal gonads—is enforced through RNA polymerase II pre-initiation complex formation within repressive heterochromatin.
Abstract: Nuclear small RNA pathways safeguard genome integrity by establishing transcription-repressing heterochromatin at transposable elements. This inevitably also targets the transposon-rich source loci of the small RNAs themselves. How small RNA source loci are efficiently transcribed while transposon promoters are potently silenced is not understood. Here we show that, in Drosophila, transcription of PIWI-interacting RNA (piRNA) clusters-small RNA source loci in animal gonads-is enforced through RNA polymerase II pre-initiation complex formation within repressive heterochromatin. This is accomplished through Moonshiner, a paralogue of a basal transcription factor IIA (TFIIA) subunit, which is recruited to piRNA clusters via the heterochromatin protein-1 variant Rhino. Moonshiner triggers transcription initiation within piRNA clusters by recruiting the TATA-box binding protein (TBP)-related factor TRF2, an animal TFIID core variant. Thus, transcription of heterochromatic small RNA source loci relies on direct recruitment of the core transcriptional machinery to DNA via histone marks rather than sequence motifs, a concept that we argue is a recurring theme in evolution.
TL;DR: It is demonstrated that mouse DUX and human DUX4 are major drivers of gene activation during the cleavage or 2C state, and may have substantial implications for early embryo development, human infertility and recurrent pregnancy loss, cancer biology, and FSHD research.
Abstract: To better understand transcriptional regulation during human oogenesis and preimplantation development, we defined stage-specific transcription, which highlighted the cleavage stage as being highly distinctive. Here, we present multiple lines of evidence that a eutherian-specific multicopy retrogene, DUX4, encodes a transcription factor that activates hundreds of endogenous genes (for example, ZSCAN4, KDM4E and PRAMEF-family genes) and retroviral elements (MERVL/HERVL family) that define the cleavage-specific transcriptional programs in humans and mice. Remarkably, mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells, measured here by the reactivation of '2C' genes and repeat elements, the loss of POU5F1 (also known as OCT4) protein and chromocenters, and the conversion of the chromatin landscape (as assessed by transposase-accessible chromatin using sequencing (ATAC-seq)) to a state strongly resembling that of mouse 2C embryos. Thus, we propose mouse DUX and human DUX4 as major drivers of the cleavage or 2C state.
TL;DR: In this paper, super-enhancers drive the biogenesis of master miRNAs crucial for cell identity by enhancing both transcription and Drosha/DGCR8-mediated primary miRNA processing.
TL;DR: It is proposed that the cell’s transcription pattern is largely retained at a low level through mitosis, whereas the amplitude of transcription observed in interphase is reestablished during mitotic exit.
Abstract: Although the genome is generally thought to be transcriptionally silent during mitosis, technical limitations have prevented sensitive mapping of transcription during mitosis and mitotic exit. Thus, the means by which the interphase expression pattern is transduced to daughter cells have been unclear. We used 5-ethynyluridine to pulse-label transcripts during mitosis and mitotic exit and found that many genes exhibit transcription during mitosis, as confirmed with fluorescein isothiocyanate–uridine 5′-triphosphate labeling, RNA fluorescence in situ hybridization, and quantitative reverse transcription polymerase chain reaction. The first round of transcription immediately after mitosis primarily activates genes involved in the growth and rebuilding of daughter cells, rather than cell type–specific functions. We propose that the cell’s transcription pattern is largely retained at a low level through mitosis, whereas the amplitude of transcription observed in interphase is reestablished during mitotic exit.
TL;DR: Corn, a genetically encoded fluorescent RNA reporter suitable for quantifying RNA transcription in cells, is developed, and direct imaging of Pol III transcripts containing a photostable RNA-fluorophore complex is provided.
Abstract: Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. This is particularly challenging when measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this issue, we developed Corn, a genetically encoded fluorescent RNA reporter suitable for quantifying RNA transcription in cells. Corn binds and induces fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, which resembles the fluorophore found in red fluorescent protein (RFP). Notably, Corn shows high photostability, enabling quantitative fluorescence imaging of mTOR-dependent Pol III transcription. We found that, unlike actinomycin D, mTOR inhibitors resulted in heterogeneous transcription suppression in individual cells. Quantitative imaging of Corn-tagged Pol III transcript levels revealed distinct Pol III transcription 'trajectories' elicited by mTOR inhibition. Together, these studies provide an approach for quantitative measurement of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA-fluorophore complex.
TL;DR: In addition to sensing DNA damage and replication stress, RPA is a sensor of R loops and a regulator of RNaseH1, extending the versatile role of RPA in suppression of genomic instability.
TL;DR: A box H/ACA small nucleolar RNA-ended long noncoding RNA (lncRNA) that enhances pre-rRNA transcription (SLERT) and its regulatory role in DDX21 ring-shaped arrangements acting on Pol I complexes is revealed.