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Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research. A study from the FANTOM consortium using single-molecule cDNA sequencing of transcription start sites and their usage in human and mouse primary cells, cell lines and tissues reveals insights into the specificity and diversity of transcription patterns across different mammalian cell types. Mapping the human transcription FANTOM5 (standing for functional annotation of the mammalian genome 5) is the fifth major stage of a major international collaboration that aims to dissect the transcriptional regulatory networks that define every human cell type. Two Articles in this issue of Nature present some of the project's latest results. The first paper uses the FANTOM5 panel of tissue and primary cell samples to define an atlas of active, in vivo bidirectionally transcribed enhancers across the human body. These authors show that bidirectional capped RNAs are a signature feature of active enhancers and identify more than 40,000 enhancer candidates from over 800 human cell and tissue samples. The enhancer atlas is used to compare regulatory programs between different cell types and identify disease-associated regulatory SNPs, and will be a resource for studies on cell-type-specific enhancers. In the second paper, single-molecule sequencing is used to map human and mouse transcription start sites and their usage in a panel of distinct human and mouse primary cells, cell lines and tissues to produce the most comprehensive mammalian gene expression atlas to date. The data provide a plethora of insights into open reading frames and promoters across different cell types in addition to valuable annotation of mammalian cell-type-specific transcriptomes.

RNA polymerase II (Pol II) core promoters are specialized DNA sequences at transcription start sites of protein-coding and non-coding genes that support the assembly of the transcription machinery and transcription initiation. They enable the highly regulated transcription of genes by selectively integrating regulatory cues from distal enhancers and their associated regulatory proteins. In this Review, we discuss the defining properties of gene core promoters, including their sequence features, chromatin architecture and transcription initiation patterns. We provide an overview of molecular mechanisms underlying the function and regulation of core promoters and their emerging functional diversity, which defines distinct transcription programmes. On the basis of the established properties of gene core promoters, we discuss transcription start sites within enhancers and integrate recent results obtained from dedicated functional assays to propose a functional model of transcription initiation. This model can explain the nature and function of transcription initiation at gene starts and at enhancers and can explain the different roles of core promoters, of Pol II and its associated factors and of the activating cues provided by enhancers and the transcription factors and cofactors they recruit.

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.

Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

Despite enormous progress in understanding the fundamentals of bacterial gene regulation, our knowledge remains limited when compared with the number of bacterial genomes and regulatory systems to be discovered. Derived from a small number of initial studies, classic definitions for concepts of gene regulation have evolved as the number of characterized promoters has increased. Together with discoveries made using new technologies, this knowledge has led to revised generalizations and principles. In this Expert Recommendation, we suggest precise, updated definitions that support a logical, consistent conceptual framework of bacterial gene regulation, focusing on transcription initiation. The resulting concepts can be formalized by ontologies for computational modelling, laying the foundation for improved bioinformatics tools, knowledge-based resources and scientific communication. Thus, this work will help researchers construct better predictive models, with different formalisms, that will be useful in engineering, synthetic biology, microbiology and genetics.In this Expert Recommendation, the authors review the definitions of classic concepts relating to bacterial gene regulation, with a focus on transcription initiation, and suggest up-to-date, precise definitions to provide a reference for knowledge representation, modelling and future research on bacterial gene regulation.

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. Transcription of Drosophila PIWI-interacting RNA (piRNA) clusters is enforced through RNA polymerase II pre-initiation complex formation within repressive heterochromatin, accomplished through the transcription factor IIA subunit paralogue Moonshiner. Initiating transcription in silent chromatin The PIWI-interacting RNA (piRNA) pathway is important for genome stability in the germline by establishing repressive heterochromatin at transposons. How piRNAs are transcribed from their loci within transposons that are transcriptionally silenced is not understood. Here Julius Brennecke and colleagues show that transcription initiation of Drosophila piRNA precursors involves a germline-specific TFIIA-L paralogue which they name Moonshiner. This protein is recruited to piRNA clusters in heterochromatin via an HP1 paralogue, Rhino, and couples to the core RNA polymerase II transcription machinery. Moonshiner therefore enables active piRNA transcription in a repressive heterochromatin environment.

The FANTOM5 project investigates transcription initiation activities in more than 1,000 human and mouse primary cells, cell lines and tissues using CAGE. Based on manual curation of sample information and development of an ontology for sample classification, we assemble the resulting data into a centralized data resource (http://fantom.gsc.riken.jp/5/). This resource contains web-based tools and data-access points for the research community to search and extract data related to samples, genes, promoter activities, transcription factors and enhancers across the FANTOM5 atlas.

Julia Zeitlinger and Wanqing Shao use ChIP-nexus to study RNA polymerase II (Pol II) promoter pausing and its relation to the formation of new initiation complexes in Drosophila cells. They find that pausing affects the initiation of new transcripts and propose that paused RNA Pol II helps to prevent new initiation between transcription bursts. 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.

During transcription initiation at promoters of protein-coding genes, RNA polymerase (Pol) II assembles with TBP, TFIIB and TFIIF into a conserved core initiation complex that recruits additional factors. The core complex stabilizes open DNA and initiates RNA synthesis, and it is conserved in the Pol I and Pol III transcription systems. Here, we derive the domain architecture of the yeast core pol II initiation complex during transcription initiation. The yeast complex resembles the human initiation complex and reveals that the TFIIF Tfg2 winged helix domain swings over promoter DNA. An ‘arm’ and a ‘charged helix’ in TFIIF function in transcription start site selection and initial RNA synthesis, respectively, and apparently extend into the active centre cleft. Our model provides the basis for further structure–function analysis of the entire transcription initiation complex. It was suggested that despite the conservation of their components, yeast and human pol II initiation complexes diverged in architecture. Mühlbacher et al. now demonstrate that the yeast and human core complexes are structurally conserved and provide insight into the conformations adopted by TFIIF during initiation.

Key Points Transcription initiation involves the interaction of DNA-dependent RNA polymerase with promoters. In bacteria, this is a highly regulated process. Many regulators interact directly with the bacterial DNA-dependent RNA polymerase, whereas other regulators interact directly with promoters. Regulation of transcription initiation occurs in the context of folding and compaction of bacterial chromosomes. A very wide range of different strategies are used to regulate transcription initiation in bacteria and these differ between species. In this Review, Browning and Busby describe the advances that have been made in recent years in understanding the molecular details of how transcription initiation is regulated to fine tune gene expression, highlighting factors that relate both to the RNA polymerase and to the promoter. Gene expression in bacteria relies on promoter recognition by the DNA-dependent RNA polymerase and subsequent transcription initiation. Bacterial cells are able to tune their transcriptional programmes to changing environments, through numerous mechanisms that regulate the activity of RNA polymerase, or change the set of promoters to which the RNA polymerase can bind. In this Review, we outline our current understanding of the different factors that direct the regulation of transcription initiation in bacteria, whether by interacting with promoters, with RNA polymerase or with both, and we discuss the diverse molecular mechanisms that are used by these factors to regulate gene expression.