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Massively‐Parallel Sequencing Of Nascent RNA Sequencing Reveals Widespread Pausing And Divergent Initiation From Mammalian Promoters
Author(s) -
Lis John T,
Core Leighton J,
Min Irene,
Waterfall Joshua J
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.316.1
Subject(s) - biology , rna polymerase ii , promoter , polymerase , transcription (linguistics) , gene , transcription factor ii d , genetics , rna , rna polymerase ii holoenzyme , microbiology and biotechnology , gene expression , linguistics , philosophy
RNA polymerases are highly‐modulated molecular machines. We have developed a highly‐sensitive method (GRO‐seq) that maps the position, amount, and orientation of transcriptionally‐engaged RNA polymerases across the entire genome, and have applied it to a primary human fibroblast cell line, and to mouse embryonic stem (ES) cells and isogenic embryonic fibroblasts (MEFs). In this method, nuclear run‐on reactions allow RNA polymerase to incorporate BrU affinity‐tags into nascent RNA that is fragmented, purified 10,000 fold, and subjected to large‐scale parallel sequencing. Mapping these sequences to the genome in these different cell types shows that 30% of all genes have promoter‐proximal paused polymerase, that transcription continues kilobases beyond the 3' cleavage for many genes, and that antisense transcription is prevelant. Surprisingly, in addition promoter‐proximal paused polymerase, most promoters also 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. This research was supported by NIH.