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Regulation of gene expression is linked to the organization of the genome. With age, chromatin alterations occur on all levels of genome organization, accompanied by changes in the gene expression profile. However, little is known about the changes in the level of transcriptional regulation. Here, we used a multi‐omics approach and integrated ATAC‐, RNA‐ and NET‐seq to identify age‐related changes in the chromatin landscape of murine liver and to investigate how these are linked to transcriptional regulation. We provide the first systematic inventory of the connection between aging, chromatin accessibility, and transcriptional regulation in a whole tissue. Aging in murine liver is characterized by an increase in chromatin accessibility at promoter regions, but not in an increase in transcriptional output. Instead, aging is accompanied by a decrease in promoter‐proximal pausing of RNA polymerase II (Pol II), while initiation of transcription is not decreased as assessed by RNA polymerase mapping using CUT&RUN. Based on the data reported, we propose that these age‐related changes in transcriptional regulation are due to a reduced stability of the pausing complex. Synopsis Profiling the chromatin landscape and nascent transcriptome in murine liver reveals an age‐related increase in chromatin accessibility at promoters and enhancers and a decrease in promoter‐proximal pausing of RNA polymerase II due to decreased pausing complex stability. Chromatin accessibility at promoters and enhancers increases with age in murine liver tissue. This increase in chromatin accessibility is not accompanied by major transcriptional alterations, neither at nascent nor steady‐state level. Promoter‐proximal Pol II pausing strongly decreases with age likely due to a decrease in the stability of the pausing complex. Graphical Abstract Profiling the chromatin landscape and nascent transcriptome in murine liver reveals an age‐related increase in chromatin accessibility at promoters and enhancers and a decrease in promoter‐proximal pausing of RNA polymerase II due to decreased pausing complex stability.

Eukaryotic genomes are rich in transcription units encoding \"long noncoding RNAs\" (lncRNAs). The purpose of all this transcription is unclear since most lncRNAs are quickly targeted for destruction during synthesis or shortly thereafter. As debates continue over the functional significance of many specific lncRNAs, support grows for the notion that the act of transcription rather than the RNA product itself is functionally important in many cases. Indeed, this alternative mechanism might better explain how low-abundance lncRNAs transcribed from noncoding DNA function in organisms. Here, we highlight some of the recently emerging features that distinguish coding from noncoding transcription and discuss how these differences might have important implications for the functional consequences of noncoding transcription.

Background Phosphorylation and O-GlcNAcylation are the key modifications regulating RNA Polymerase II (RNA Pol II)-driven transcription. Transcriptional kinases, cyclin-dependent kinase 7 (CDK7), CDK9 and CDK12 phosphorylate RNA Pol II, whereas O-GlcNAcylation is added by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Currently, no study has systematically evaluated how inhibiting each of these enzyme activities impacts the assembly of the appropriate protein complexes on the polymerase and the maturation of mRNA. Methods Here, we systematically evaluate remodeling of RNA Pol II interactome and effects on the nascent mRNA maturation by using mass spectrometry and SLAM-seq, respectively. For validation, we rely predominantly on analysis of intronic polyadenylation (IPA) sites, mitochondrial flux assays (Seahorse), western blotting and patient data. Results We show that OGT / OGA inhibition reciprocally affect protein recruitment to RNA Pol II, and appropriate O-GlcNAcylation levels are required for optimal function of the RNA Pol II complex. These paradoxical effects are explained through IPA, because despite being prematurely poly-adenylated, these mRNAs are scored as mature in SLAM-seq. Unlike previously proposed, we show that, similar to inhibition of CDK12, also targeting CDK9 stimulates transcription of short genes at the cost of long genes. However, our systematic proteomic- and IPA-analysis revealed that these effects are mediated by distinct molecular mechanisms: CDK9 inhibition leads to a failure of recruiting Integrator complex to RNA Pol II, and we then show that depletion of Integrator subunits phenocopy the gene length-dependent effects. In contrast, CDK12 inhibition triggers IPA. Finally, we show that dynamic O-GlcNAcylation predominantly interplays with CDK9: OGT inhibition augments CDK9 inhibitor effects on mRNA maturation due to defects in transcription elongation, while OGA inhibition rescues mRNA maturation failure caused by targeting CDK9, but induces IPA. Conclusion We show that dynamic O-GlcNAcylation is a negative regulator of mRNA biosynthesis and propose that the addition and removal of the modification serve as quality control-steps to ascertain successful generation of mature mRNAs. Our work identifies unprecedented redundancy in the regulation of RNA Pol II, which increases resilience towards transcriptional stress, and also underscores the difficulty of targeting transcription to control cancer. Graphical Abstract

Background The dynamic process of transcription termination produces transient RNA intermediates that are difficult to distinguish from each other via short-read sequencing methods. Results Here, we use single-molecule nascent RNA sequencing to characterize the various forms of transient RNAs during termination at genome-wide scale in wildtype Arabidopsis and in atxrn3 , fpa , and met1 mutants. Our data reveal a wide range of termination windows among genes, ranging from ~ 50 nt to over 1000 nt. We also observe efficient termination before downstream tRNA genes, suggesting that chromatin structure around the promoter region of tRNA genes may block pol II elongation. 5′ Cleaved readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Consistent with previous reports, we also observe long chimeric transcripts with cryptic splicing in fpa mutant; but loss of CG DNA methylation has no obvious impact on termination in the met1 mutant. Conclusions Our method is applicable to establish a comprehensive termination landscape in a broad range of species.

A substantial fraction of the metazoan transcriptome undergoes circadian oscillations in many cells and tissues. Based on the transcription feedback loops important for circadian timekeeping, it is commonly assumed that this mRNA cycling reflects widespread transcriptional regulation. To address this issue, we directly measured the circadian dynamics of mouse liver transcription using Nascent-Seq (genome-wide sequencing of nascent RNA). Although many genes are rhythmically transcribed, many rhythmic mRNAs manifest poor transcriptional rhythms, indicating a prominent contribution of post-transcriptional regulation to circadian mRNA expression. This analysis of rhythmic transcription also showed that the rhythmic DNA binding profile of the transcription factors CLOCK and BMAL1 does not determine the transcriptional phase of most target genes. This likely reflects gene-specific collaborations of CLK:BMAL1 with other transcription factors. These insights from Nascent-Seq indicate that it should have broad applicability to many other gene expression regulatory issues. Many biological processes oscillate with a period of roughly 24 hr, and the ability of organisms as diverse as bacteria and humans to maintain such circadian rhythms, even under conditions of continuous darkness, influences a range of phenomena, including sleep, migration and reproduction. One characteristic of circadian rhythms is that they can adjust to local time (with humans suffering from jet lag as they wait for this to happen). Experiments have shown that the circadian system in mammals relies on feedback loops that operate at the level of individual cells. These loops are controlled by two particular proteins, which comprise the transcription factor complex called BMAL1:CLK. Transcription factors cause particular sequences of bases in the DNA of cells to be transcribed into messenger RNA, thus starting the process by which target genes are expressed as proteins. In the case of BMAL1:CLK, these proteins are then modified, which inhibits any further transcription of the target genes. A reversal of these modifications is then followed by the synthesis of new proteins, which allows a new cycle of the transcription process to begin. The amounts of many messenger RNAs (mRNAs) in a cell also increases and decreases with a period of 24 hr, and it was generally assumed that this was due to the changes in the level of transcription. More recently, however, it was suggested that other processes, such as splicing and translation, might also contribute to rhythmic changes in the amount of mRNA associated with particular genes. Such post-transcriptional processes are known to have a role in other areas of cell biology, including aspects of the circadian system, but until very recently this had not been studied in detail for all genes. Now Menet et al. have directly assayed rhythmic transcription by measuring the amount of nascent mRNA being produced at a given time, six times a day, across all the genes in mouse liver cells using a high-throughput sequencing approach called Nascent-Seq. They compared this with the amount of liver mRNA expressed at six time points of the day. Although the authors found that many genes exhibit rhythmic mRNA expression in the mouse liver, about 70% of them did not show comparable transcriptional rhythms. Post-transcriptional regulation must, therefore, have a major role in the circadian system of mice and, presumably, other mammals. Menet et al. also found that the influence of CLK:BMAL1 differed from what was expected, which suggests that it collaborates with a number of other transcription factors to effect transcription of most target genes. In addition to showing that circadian systems of mammals are more complex than previously believed, the results also illustrate the potential of Nascent-Seq as a genome-wide assay technique for exploring a range of questions related to gene expression and gene regulation.

Bacterial messenger RNA (mRNA) synthesis by RNA polymerase (RNAP) and first-round translation by the ribosome are often coupled to regulate gene expression, yet how coupling is established and maintained is ill understood. Here, we develop biochemical and single-molecule fluorescence approaches to probe the dynamics of RNAP–ribosome interactions on an mRNA with a translational preQ₁-sensing riboswitch in its 5′ untranslated region. Binding of preQ₁ leads to the occlusion of the ribosome binding site (RBS), inhibiting translation initiation. We demonstrate that RNAP poised within the mRNA leader region promotes ribosomal 30S subunit binding, antagonizing preQ₁-induced RBS occlusion, and that the RNAP–30S bridging transcription factors NusG and RfaH distinctly enhance 30S recruitment and retention, respectively. We further find that, while 30S–mRNA interaction significantly impedes RNAP in the absence of translation, an actively translating ribosome promotes productive transcription. A model emerges wherein mRNA structure and transcription factors coordinate to dynamically modulate the efficiency of transcription–translation coupling.

The precise spatiotemporal gene expression is orchestrated by enhancers that lack general sequence features and thus are difficult to be computationally identified. By nascent RNA sequencing combined with epigenome profiling, we detect active transcription of enhancers from the complex bread wheat genome. We find that genes associated with transcriptional enhancers are expressed at significantly higher levels, and enhancer RNA is more precise and robust in predicting enhancer activity compared to chromatin features. We demonstrate that sub-genome-biased enhancer transcription could drive sub-genome-biased gene expression. This study highlights enhancer transcription as a hallmark in regulating gene expression in wheat.

Gene transcription is controlled and modulated by regulatory regions, including enhancers and promoters. These regions are abundant in non-coding bidirectional transcription that results in generally unstable RNA. Using nascent RNA transcription data across hundreds of human samples, we identified over 800,000 regions containing bidirectional transcription. We then identify tissue specific, highly correlated transcription between bidirectional and gene regions. The identified correlated pairs, a bidirectional region and a gene, are enriched for disease associated SNPs and often supported by independent 3D data. We present these resources as a database called DBNascent ( https://nascent.colorado.edu/ ) which serves as a resource for future studies into gene regulation, enhancer associated RNAs, and transcription factors.

Transcriptional regulation of gene expression is a major mechanism used by plants to confer phenotypic plasticity, and yet compared with other eukaryotes or bacteria, little is known about the design principles. We generated an extensive catalog of nascent and steady-state transcripts in Arabidopsis thaliana seedlings using global nuclear run-on sequencing (GRO-seq), 5′GRO-seq, and RNA-seq and reanalyzed published maize data to capture characteristics of plant transcription. De novo annotation of nascent transcripts accurately mapped start sites and unstable transcripts. Examining the promoters of coding and noncoding transcripts identified comparable chromatin signatures, a conserved “TGT” core promoter motif and unreported transcription factor-binding sites. Mapping of engaged RNA polymerases showed a lack of enhancer RNAs, promoter-proximal pausing, and divergent transcription in Arabidopsis seedlings and maize, which are commonly present in yeast and humans. In contrast, Arabidopsis and maize genes accumulate RNA polymerases in proximity of the polyadenylation site, a trend that coincided with longer genes and CpG hypomethylation. Lack of promoter-proximal pausing and a higher correlation of nascent and steady-state transcripts indicate Arabidopsis may regulate transcription predominantly at the level of initiation. Our findings provide insight into plant transcription and eukaryotic gene expression as a whole.

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker–induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.