Eukaryotic core promoters and the functional basis of transcription initiation.

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.

Coordinating the human looks.

Prescott et al. take a step forward in studying primate morphological evolution by a cellular anthropology approach. Through epigenomic profiling of in-vitro-derived cells, the authors identify and characterize candidate cis-regulatory elements underlying divergence in facial morphology between human and chimp, shedding new light on what makes us (look) human.

Differential cofactor dependencies define distinct types of human enhancers.

All multicellular organisms rely on differential gene transcription regulated by genomic enhancers, which function through cofactors that are recruited by transcription factors1,2. Emerging evidence suggests that not all cofactors are required at all enhancers3-5, yet whether these observations reflect more general principles or distinct types of enhancers remained unknown. Here we categorized human enhancers by their cofactor dependencies and show that these categories provide a framework to understand the sequence and chromatin diversity of enhancers and their roles in different gene-regulatory programmes. We quantified enhancer activities along the entire human genome using STARR-seq6 in HCT116 cells, following the rapid degradation of eight cofactors. This analysis identified different types of enhancers with distinct cofactor requirements, sequences and chromatin properties. Some enhancers were insensitive to the depletion of the core Mediator subunit MED14 or the bromodomain protein BRD4 and regulated distinct transcriptional programmes. In particular, canonical Mediator7 seemed dispensable for P53-responsive enhancers, and MED14-depleted cells induced endogenous P53 target genes. Similarly, BRD4 was not required for the transcription of genes that bear CCAAT boxes and a TATA box (including histone genes and LTR12 retrotransposons) or for the induction of heat-shock genes. This categorization of enhancers through cofactor dependencies reveals distinct enhancer types that can bypass broadly utilized cofactors, which illustrates how alternative ways to activate transcription separate gene expression programmes and provide a conceptual framework to understand enhancer function and regulatory specificity.

Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns.

Recruitment of RNA polymerase II (Pol II) to promoters is essential for transcription. Despite conflicting evidence, the Pol II preinitiation complex (PIC) is often thought to have a uniform composition and to assemble at all promoters via an identical mechanism. Here, using Drosophila melanogaster S2 cells as a model, we demonstrate that different promoter classes function via distinct PICs. Promoter DNA of developmentally regulated genes readily associates with the canonical Pol II PIC, whereas housekeeping promoters do not, and instead recruit other factors such as DREF. Consistently, TBP and DREF are differentially required by distinct promoter types. TBP and its paralog TRF2 also function at different promoter types in a partially redundant manner. In contrast, TFIIA is required at all promoters, and we identify factors that can recruit and/or stabilize TFIIA at housekeeping promoters and activate transcription. Promoter activation by tethering these factors is sufficient to induce the dispersed transcription initiation patterns characteristic of housekeeping promoters. Thus, different promoter classes utilize distinct mechanisms of transcription initiation, which translate into different focused versus dispersed initiation patterns.

Transcriptional cofactors display specificity for distinct types of core promoters.

Transcriptional cofactors (COFs) communicate regulatory cues from enhancers to promoters and are central effectors of transcription activation and gene expression1. Although some COFs have been shown to prefer certain promoter types2-5 over others (for example, see refs 6,7), the extent to which different COFs display intrinsic specificities for distinct promoters is unclear. Here we use a high-throughput promoter-activity assay in Drosophila melanogaster S2 cells to screen 23 COFs for their ability to activate 72,000 candidate core promoters (CPs). We observe differential activation of CPs, indicating distinct regulatory preferences or 'compatibilities'8,9 between COFs and specific types of CPs. These functionally distinct CP types are differentially enriched for known sequence elements2,4, such as the TATA box, downstream promoter element (DPE) or TCT motif, and display distinct chromatin properties at endogenous loci. Notably, the CP types differ in their relative abundance of H3K4me3 and H3K4me1 marks (see also refs 10-12), suggesting that these histone modifications might distinguish trans-regulatory factors rather than promoter- versus enhancer-type cis-regulatory elements. We confirm the existence of distinct COF-CP compatibilities in two additional Drosophila cell lines and in human cells, for which we find COFs that prefer TATA-box or CpG-island promoters, respectively. Distinct compatibilities between COFs and promoters can explain how different enhancers specifically activate distinct sets of genes9, alternative promoters within the same genes, and distinct transcription start sites within the same promoter13. Thus, COF-promoter compatibilities may underlie distinct transcriptional programs in species as divergent as flies and humans.

Epigenetic reprogramming enables the transition from primordial germ cell to gonocyte.

Gametes are highly specialized cells that can give rise to the next generation through their ability to generate a totipotent zygote. In mice, germ cells are first specified in the developing embryo around embryonic day (E) 6.25 as primordial germ cells (PGCs). Following subsequent migration into the developing gonad, PGCs undergo a wave of extensive epigenetic reprogramming around E10.5-E11.5, including genome-wide loss of 5-methylcytosine. The underlying molecular mechanisms of this process have remained unclear, leading to our inability to recapitulate this step of germline development in vitro. Here we show, using an integrative approach, that this complex reprogramming process involves coordinated interplay among promoter sequence characteristics, DNA (de)methylation, the polycomb (PRC1) complex and both DNA demethylation-dependent and -independent functions of TET1 to enable the activation of a critical set of germline reprogramming-responsive genes involved in gamete generation and meiosis. Our results also reveal an unexpected role for TET1 in maintaining but not driving DNA demethylation in gonadal PGCs. Collectively, our work uncovers a fundamental biological role for gonadal germline reprogramming and identifies the epigenetic principles of the PGC-to-gonocyte transition that will help to guide attempts to recapitulate complete gametogenesis in vitro.

Resolving systematic errors in widely used enhancer activity assays in human cells.

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfection into human cells: (i) the bacterial plasmid origin of replication (ORI) functions as a conflicting core promoter and (ii) a type I interferon (IFN-I) response is activated. These cause confounding false positives and negatives in luciferase assays and STARR-seq screens. We overcome both problems by employing the ORI as core promoter and by inhibiting two IFN-I-inducing kinases, enabling genome-wide STARR-seq screens in human cells. In HeLa-S3 cells, we uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells and are key to the characterization of human enhancers.

The aurora B kinase and the polycomb protein ring1B combine to regulate active promoters in quiescent lymphocytes.

Reversible cellular quiescence is critical for developmental processes in metazoan organisms and is characterized by a reduction in cell size and transcriptional activity. We show that the Aurora B kinase and the polycomb protein Ring1B have essential roles in regulating transcriptionally active genes in quiescent lymphocytes. Ring1B and Aurora B bind to a wide range of active promoters in resting B and T cells. Conditional knockout of either protein results in reduced transcription and binding of RNA Pol II to promoter regions and decreased cell viability. Aurora B phosphorylates histone H3S28 at active promoters in resting B cells as well as inhibiting Ring1B-mediated ubiquitination of histone H2A and enhancing binding and activity of the USP16 deubiquitinase at transcribed genes. Our results identify a mechanism for regulating transcription in quiescent cells that has implications for epigenetic regulation of the choice between proliferation and quiescence.

Two independent transcription initiation codes overlap on vertebrate core promoters.

A core promoter is a stretch of DNA surrounding the transcription start site (TSS) that integrates regulatory inputs and recruits general transcription factors to initiate transcription. The nature and causative relationship of the DNA sequence and chromatin signals that govern the selection of most TSSs by RNA polymerase II remain unresolved. Maternal to zygotic transition represents the most marked change of the transcriptome repertoire in the vertebrate life cycle. Early embryonic development in zebrafish is characterized by a series of transcriptionally silent cell cycles regulated by inherited maternal gene products: zygotic genome activation commences at the tenth cell cycle, marking the mid-blastula transition. This transition provides a unique opportunity to study the rules of TSS selection and the hierarchy of events linking transcription initiation with key chromatin modifications. We analysed TSS usage during zebrafish early embryonic development at high resolution using cap analysis of gene expression, and determined the positions of H3K4me3-marked promoter-associated nucleosomes. Here we show that the transition from the maternal to zygotic transcriptome is characterized by a switch between two fundamentally different modes of defining transcription initiation, which drive the dynamic change of TSS usage and promoter shape. A maternal-specific TSS selection, which requires an A/T-rich (W-box) motif, is replaced with a zygotic TSS selection grammar characterized by broader patterns of dinucleotide enrichments, precisely aligned with the first downstream (+1) nucleosome. The developmental dynamics of the H3K4me3-marked nucleosomes reveal their DNA-sequence-associated positioning at promoters before zygotic transcription and subsequent transcription-independent adjustment to the final position downstream of the zygotic TSS. The two TSS-defining grammars coexist, often physically overlapping, in core promoters of constitutively expressed genes to enable their expression in the two regulatory environments. The dissection of overlapping core promoter determinants represents a framework for future studies of promoter structure and function across different regulatory contexts.

A promoter-level mammalian expression atlas.

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.

Promoter architectures and developmental gene regulation.

Core promoters are minimal regions sufficient to direct accurate initiation of transcription and are crucial for regulation of gene expression. They are highly diverse in terms of associated core promoter motifs, underlying sequence composition and patterns of transcription initiation. Distinctive features of promoters are also seen at the chromatin level, including nucleosome positioning patterns and presence of specific histone modifications. Recent advances in identifying and characterizing promoters using next-generation sequencing-based technologies have provided the basis for their classification into functional groups and have shed light on their modes of regulation, with important implications for transcriptional regulation in development. This review discusses the methodology and the results of genome-wide studies that provided insight into the diversity of RNA polymerase II promoter architectures in vertebrates and other Metazoa, and the association of these architectures with distinct modes of regulation in embryonic development and differentiation.

CAGEr: precise TSS data retrieval and high-resolution promoterome mining for integrative analyses.

Cap analysis of gene expression (CAGE) is a high-throughput method for transcriptome analysis that provides a single base-pair resolution map of transcription start sites (TSS) and their relative usage. Despite their high resolution and functional significance, published CAGE data are still underused in promoter analysis due to the absence of tools that enable its efficient manipulation and integration with other genome data types. Here we present CAGEr, an R implementation of novel methods for the analysis of differential TSS usage and promoter dynamics, integrated with CAGE data processing and promoterome mining into a first comprehensive CAGE toolbox on a common analysis platform. Crucially, we provide collections of TSSs derived from most published CAGE datasets, as well as direct access to FANTOM5 resource of TSSs for numerous human and mouse cell/tissue types from within R, greatly increasing the accessibility of precise context-specific TSS data for integrative analyses. The CAGEr package is freely available from Bioconductor at http://www.bioconductor.org/packages/release/bioc/html/CAGEr.html.

Dynamic regulation of the transcription initiation landscape at single nucleotide resolution during vertebrate embryogenesis.

Spatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we provide a first and comprehensive description of the core promoter repertoire and its dynamic use during the development of a vertebrate embryo. By using cap analysis of gene expression (CAGE), we mapped transcription initiation events at single nucleotide resolution across 12 stages of zebrafish development. These CAGE-based transcriptome maps reveal genome-wide rules of core promoter usage, structure, and dynamics, key to understanding the control of gene regulation during vertebrate ontogeny. They revealed the existence of multiple classes of pervasive intra- and intergenic post-transcriptionally processed RNA products and their developmental dynamics. Among these RNAs, we report splice donor site-associated intronic RNA (sRNA) to be specific to genes of the splicing machinery. For the identification of conserved features, we compared the zebrafish data sets to the first CAGE promoter map of Tetraodon and the existing human CAGE data. We show that a number of features, such as promoter type, newly discovered promoter properties such as a specialized purine-rich initiator motif, as well as sRNAs and the genes in which they are detected, are conserved in mammalian and Tetraodon CAGE-defined promoter maps. The zebrafish developmental promoterome represents a powerful resource for studying developmental gene regulation and revealing promoter features shared across vertebrates.

Genomewide DNA methylation analysis identifies novel methylated genes in non-small-cell lung carcinomas.

INTRODUCTION: DNA methylation is part of the epigenetic regulatory mechanism present in all normal cells. It is tissue-specific and stably maintained throughout development, but often abnormally changed in cancer. Non-small-cell lung carcinoma (NSCLC) is the most deadly type of cancer, involving different tumor subtypes. This heterogeneity is a challenge for correct diagnosis and patient treatment. The stability and specificity make of DNA methylation a very suitable marker for epigenetic phenotyping of tumors. METHODS: To identify candidate markers for use in NSCLC diagnosis, we used genomewide DNA methylation maps that we had previously generated by MethylCap and next-generation sequencing and listed the most significant differentially methylated regions (DMRs). The 25 DMRs with highest significance in their methylation scores were selected. The methylation status of these DMRs was investigated in 61 tumors and matching control lung tissues by methylation-specific polymerase chain reaction. RESULTS: We found 12 novel DMRs that showed significant differences between tumor and control lung tissues. We also identified three novel DMRs for each of the two most common NSCLC subtypes, adenocarcinomas and squamous cell carcinomas. We propose a panel of five DMRs, composed of novel and known markers that exhibit high specificity and sensitivity to distinguish tumors from control lung tissues. CONCLUSION: Novel markers will aid the development of a highly specific epigenetic panel for accurate identification and subtyping of NSCLC tumors.

Genome-wide DNA methylation profiling of non-small cell lung carcinomas.

BACKGROUND: Non-small cell lung carcinoma (NSCLC) is a complex malignancy that owing to its heterogeneity and poor prognosis poses many challenges to diagnosis, prognosis and patient treatment. DNA methylation is an important mechanism of epigenetic regulation involved in normal development and cancer. It is a very stable and specific modification and therefore in principle a very suitable marker for epigenetic phenotyping of tumors. Here we present a genome-wide DNA methylation analysis of NSCLC samples and paired lung tissues, where we combine MethylCap and next generation sequencing (MethylCap-seq) to provide comprehensive DNA methylation maps of the tumor and paired lung samples. The MethylCap-seq data were validated by bisulfite sequencing and methyl-specific polymerase chain reaction of selected regions. RESULTS: Analysis of the MethylCap-seq data revealed a strong positive correlation between replicate experiments and between paired tumor/lung samples. We identified 57 differentially methylated regions (DMRs) present in all NSCLC tumors analyzed by MethylCap-seq. While hypomethylated DMRs did not correlate to any particular functional category of genes, the hypermethylated DMRs were strongly associated with genes encoding transcriptional regulators. Furthermore, subtelomeric regions and satellite repeats were hypomethylated in the NSCLC samples. We also identified DMRs that were specific to two of the major subtypes of NSCLC, adenocarcinomas and squamous cell carcinomas. CONCLUSIONS: Collectively, we provide a resource containing genome-wide DNA methylation maps of NSCLC and their paired lung tissues, and comprehensive lists of known and novel DMRs and associated genes in NSCLC.