Development And Application Of A Genomic Method To Map The Position, Amount, And Orientation Of Transcriptionally Engaged Rna Polymerases

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RNA polymerases are highly-regulated molecular machines that can be modulated at the level of recruitment to a gene promoter, pre-initiation complex formation, initiation, elongation, and termination. Studies using the chromatin immunoprecipitation assay coupled to genomic DNA microarrays (ChIP-chip) or to high throughput sequencing (ChIP-seq) indicate that RNA polymerase II (Pol II) is present at disproportionately higher levels near the 5? end of many eukaryotic genes relative to downstream regions. This pattern is consistent with Pol II being either in a pre-initiation complex, or transcriptionally engaged and paused proximal to the promoter. Promoterproximal pausing is proposed to be an important post-initiation, rate-limiting target for gene regulation, and usually occurs within the first 20-50 bases of a transcription unit. However, the ChIP assay cannot determine whether Pol II is simply promoter-bound or engaged in transcription. The goal of this dissertation project was to develop a method that would assess the generality of promoter-proximal pausing, genome-wide. To that end, I have developed a highly-sensitive method, Global RunOn sequencing (GRO-seq), that maps the position, amount, and orientation of transcriptionally-engaged RNA polymerases across the entire genome. We have applied GRO-seq to a primary human fibroblast cell line (IMR90). In this method, nuclear run-on reactions allow RNA polymerase to incorporate BrU affinity-tags into nascent RNA. The RNA is fragmented, purified at least 10,000 fold, and subjected to large-scale parallel sequencing. Mapping these sequences to the genome in this 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 to 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.

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