Unknown,Transcriptomics,Genomics,Proteomics

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Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters

ABSTRACT: RNA polymerases are highly regulated molecular machines. We present a method (GRO-seq) that maps the position, amount, and orientation of transcriptionally-engaged RNA polymerases genome-wide. In this method, nuclear run-on RNAs are subjected to large-scale parallel sequencing and mapped to the genome. Here, we show that peaks of promoter-proximal polymerase reside on ~30% of human genes, transcription extends beyond pre-mRNA 3M-bM-^@M-^Y 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. Two biological replicates of nascent RNA sequencing

ORGANISM(S): Homo sapiens

SUBMITTER: Leighton Core

PROVIDER: E-GEOD-13518 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters.

Core Leighton J LJ Waterfall Joshua J JJ Lis John T JT

Science (New York, N.Y.) 20081204 5909

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 approximately 30% of human genes, transcription extends beyond pre-messenger RNA 3' cleavage, and an ...[more]

PMID: 19056941

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Project description:Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genomeM-bM-^@M-^Ys primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide, nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally-engaged RNA polymerases in mouse embryonic stem cells (ESCs) and embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ~40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol IIM-bM-^@M-^Ys entry into elongation. Furthermore, M-bM-^@M-^\bivalentM-bM-^@M-^] ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb Group Complexes PRC 1 and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5M-bM-^@M-^Y proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation. Mapping engaged RNA polymerase density in two cell types by sequencing run-on transcripts. SUPPLEMENTARY FILES: All fastq files have sanger-fastq format q values. Alignments were generated with eland and the mm9 mouse genome assembly. Reads aligning to regions annotated as similar to rRNA by RepeatMasker were then removed. Wiggle files are in units of RPKM (reads per kilobase per million aligned reads) and are broken up by cell type and chromosome to aid in uploading to UCSC. Each file furthermore contains two tracks - one for each strand. As in the published paper, plus strand RPKM densities are in red with positive values and minus strand RPKM densities are in blue with negative values.

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Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters

Publications

Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters.

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