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Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a precision nuclear run-on and sequencing (PRO-seq) assay to map the genome-wide distribution of transcriptionally engaged Pol II at base pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3′ polyadenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites, nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing. This \"complex interaction\" model was tested with insertional mutagenesis of the Drosophila Hsp70 core promoter.

RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of transcriptionally engaged RNA polymerases genome-wide. In this method, nuclear run-on RNA molecules are subjected to large-scale parallel sequencing and mapped to the genome. We show that peaks of promoter-proximal polymerase reside on ~30% of human genes, transcription extends beyond pre-messenger RNA 3' cleavage, and antisense transcription is prevalent. Additionally, most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription.

John Lis, Adam Siepel and colleagues map transcription start sites across the genome in two human cell lines using a nuclear run-on protocol called GRO-cap. They find a common architecture of initiation at both promoters and enhancers and that transcript elongation stability provides the strongest distinction between promoters and enhancers. Despite the conventional distinction between them, promoters and enhancers share many features in mammals, including divergent transcription and similar modes of transcription factor binding. Here we examine the architecture of transcription initiation through comprehensive mapping of transcription start sites (TSSs) in human lymphoblastoid B cell (GM12878) and chronic myelogenous leukemic (K562) ENCODE Tier 1 cell lines. Using a nuclear run-on protocol called GRO-cap, which captures TSSs for both stable and unstable transcripts, we conduct detailed comparisons of thousands of promoters and enhancers in human cells. These analyses identify a common architecture of initiation, including tightly spaced (110 bp apart) divergent initiation, similar frequencies of core promoter sequence elements, highly positioned flanking nucleosomes and two modes of transcription factor binding. Post-initiation transcript stability provides a more fundamental distinction between promoters and enhancers than patterns of histone modification and association of transcription factors or co-activators. These results support a unified model of transcription initiation at promoters and enhancers.

Mahat et al . describe how to map the genome-wide positions of active RNA polymerases using a modified nuclear run-on approach called PRO-seq. Details for PRO-cap, a modification that identifies transcription start sites, are also included. We provide a protocol for precision nuclear run-on sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under nuclear run-on conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3′ end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3′ end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4–5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.

Recent work has shown that the RNA polymerase II enzyme pauses at a promoter-proximal site of many genes in Drosophila and mammals. This rate-limiting step occurs after recruitment and initiation of RNA polymerase II at a gene promoter. This stage in early elongation appears to be an important and broadly used target of gene regulation.

The human genome encodes a variety of poorly understood RNA species that remain challenging to identify using existing genomic tools. We developed chromatin run-on and sequencing (ChRO-seq) to map the location of RNA polymerase for almost any input sample, including samples with degraded RNA that are intractable to RNA sequencing. We used ChRO-seq to map nascent transcription in primary human glioblastoma (GBM) brain tumors. Enhancers identified in primary GBMs resemble open chromatin in the normal human brain. Rare enhancers that are activated in malignant tissue drive regulatory programs similar to the developing nervous system. We identified enhancers that regulate groups of genes that are characteristic of each known GBM subtype and transcription factors that drive them. Finally we discovered a core group of transcription factors that control the expression of genes associated with clinical outcomes. This study characterizes the transcriptional landscape of GBM and introduces ChRO-seq as a method to map regulatory programs that contribute to complex diseases. Chromatin run-on and sequencing (ChRO-seq) is a new method that maps the location of RNA polymerase using virtually any input sample. Here, ChRO-seq is used to study nascent transcription in human glioblastoma, and to identify regulators of tumor subtype.

Mammalian genomes are pervasively transcribed, yielding a complex transcriptome with high variability in composition and cellular abundance. Although recent efforts have identified thousands of new long non-coding (lnc) RNAs and demonstrated a complex transcriptional repertoire produced by protein-coding (pc) genes, limited progress has been made in distinguishing functional RNA from spurious transcription events. This is partly due to present RNA classification, which is typically based on technical rather than biochemical criteria. Here we devise a strategy to systematically categorize human RNAs by their sensitivity to the ribonucleolytic RNA exosome complex and by the nature of their transcription initiation. These measures are surprisingly effective at correctly classifying annotated transcripts, including lncRNAs of known function. The approach also identifies uncharacterized stable lncRNAs, hidden among a vast majority of unstable transcripts. The predictive power of the approach promises to streamline the functional analysis of known and novel RNAs. Despite our growing understanding of their complexity, different types of RNA are still classified using technical rather than functional criteria. Andersson et al. show that categorization of RNAs based on stability and direction of transcription is an effective means of functional classification.

X chromosome expression in the balance Different organisms use a variety of mechanisms to compensate for X chromosome dosage imbalance between the sexes. In Drosophila , the MSL (Male-specific lethal) complex increases transcription on the single X chromosome of males and is thought to regulate transcription elongation, although mechanistic details have been unclear. A global run-on sequencing technique has now been used to reveal that the MSL complex seems to enhance transcription by facilitating the progression of RNA polymerase II across the bodies of active X-linked genes. In this way, MSL can impose dosage compensation on diverse genes with a wide range of transcription levels along the X chromosome. Different organisms use a variety of mechanisms to compensate for X chromosome dosage imbalance between the sexes. In Drosophila , the MSL complex increases transcription on the single X chromosome of males and is thought to regulate transcription elongation, although mechanistic details have been unclear. Here, a global run-on sequencing technique is used to reveal that the MSL complex seems to enhance transcription by facilitating the progression of RNA polymerase II across the bodies of active X linked genes. In this way, MSL can impose dosage compensation on diverse genes with a wide range of transcription levels along the X chromosome. The evolution of sex chromosomes has resulted in numerous species in which females inherit two X chromosomes but males have a single X, thus requiring dosage compensation. MSL (Male-specific lethal) complex increases transcription on the single X chromosome of Drosophila males to equalize expression of X-linked genes between the sexes 1 . The biochemical mechanisms used for dosage compensation must function over a wide dynamic range of transcription levels and differential expression patterns. It has been proposed 2 that the MSL complex regulates transcriptional elongation to control dosage compensation, a model subsequently supported by mapping of the MSL complex and MSL-dependent histone 4 lysine 16 acetylation to the bodies of X-linked genes in males, with a bias towards 3′ ends 3 , 4 , 5 , 6 , 7 . However, experimental analysis of MSL function at the mechanistic level has been challenging owing to the small magnitude of the chromosome-wide effect and the lack of an in vitro system for biochemical analysis. Here we use global run-on sequencing (GRO-seq) 8 to examine the specific effect of the MSL complex on RNA Polymerase II (RNAP II) on a genome-wide level. Results indicate that the MSL complex enhances transcription by facilitating the progression of RNAP II across the bodies of active X-linked genes. Improving transcriptional output downstream of typical gene-specific controls may explain how dosage compensation can be imposed on the diverse set of genes along an entire chromosome.

DNA sequence variation has been associated with quantitative changes in molecular phenotypes such as gene expression, but its impact on chromatin states is poorly characterized. To understand the interplay between chromatin and genetic control of gene regulation, we quantified allelic variability in transcription factor binding, histone modifications, and gene expression within humans. We found abundant allelic specificity in chromatin and extensive local, short-range, and long-range allelic coordination among the studied molecular phenotypes. We observed genetic influence on most of these phenotypes, with histone modifications exhibiting strong context-dependent behavior. Our results implicate transcription factors as primary mediators of sequence-specific regulation of gene expression programs, with histone modifications frequently reflecting the primary regulatory event.

Key Points Many RNA polymerase II (Pol II)-transcribed genes are regulated at the level of transcription elongation, which has three main stages: promoter escape, promoter-proximal pausing, and productive elongation. Immediately following transcription initiation, the transcription complex is unstable. Promoter escape is the earliest step in transcription elongation during which the transcription complex breaks its contacts with promoter-bound factors and simultaneously tightens its grip on the nascent RNA. The combined results from several recent studies have identified potential protein–nucleic acid interactions, as well as mechanisms, that are instrumental in the completion of promoter escape by Pol II. Promoter-proximal pausing might provide a checkpoint before committing to productive elongation, and provides a highly dynamic regulatory step in Pol II transcription. There are several key factors (including DSIF, NELF and P-TEFb) that are thought to be important for transcription regulation during early Pol II elongation. Several elongation factors also facilitate productive elongation by Pol II through the gene by directly altering its elongation properties or by affecting chromatin structure. The main constituent of chromatin is the nucleosome, and the obstacle that nucleosomes pose to transcription elongation must be overcome. The histone components of nucleosomes can be modified covalently, or the nucleosome can be altered structurally, to facilitate transcription. There are several elongation factors known to be important for the covalent and structural modification of nucleosomes. Recent studies have challenged the view that transcription is predominantly regulated at the level of RNA polymerase II recruitment to promoters. Transcription is also regulated at the level of elongation by factors that act directly upon RNA polymerase II or that manipulate the chromatin environment. Hundreds of protein factors participate in transcription and its regulation in eukaryotes. Many of these proteins regulate specific genes by targeting upstream promoter regions, whereas a smaller but mechanistically diverse set of factors functions at most genes during RNA polymerase II (Pol II) elongation. These elongation factors can affect mRNA production at particular stages and in different ways during transcription. Some factors act directly on Pol II, whereas others manipulate the chromatin environment.