A Unified View of the Sequence and Functional Organization of the Human RNA Polymerase II Promoter

To expand our understanding of the interplay of human RNA polymerase II (Pol II) promoter elements, promoter-proximal pausing, local chromatin structure, and co-transcriptional RNA processing, the 5′ and 3′ ends of nascent, capped transcripts and the locations of nearby nucleosomes were accurately identified. High-quality visualization tools revealed sequence elements defining the core promoter, sites of pausing, and nucleosome positioning across over 177,000 transcription start regions (TSRs). We found that cap methylation begins as transcripts attain a length of about 30 nt. A nuclear run-off assay was developed that utilizes the unique properties of the DNA fragmentation factor (DFF) to demonstrate the relative location of nucleosomes and paused Pol II. Our data support the conclusion that human Pol II promoters are functional TFIID binding sites with built-in downstream information directing ubiquitous promoter proximal pausing and the downstream nucleosome location. NasCap design: PRO-Seq protocol with a modified front end to rapidly isolate nuclei followed by selection of capped transcripts containing m7G or non methylated capped transcripts (NasCap). DFF-Seq design: The level of nucleosome occupancy across the human genome has been estimated using Micrococcal Nuclease (MNase) coupled with DNA sequencing. However, the nucleosome occupancy levels generated from the more selective nuclease, DNA Fragmentation Factor (DFF), has not been investigated. We generated our own measure of nucleosome occupancy using DFF and subsequent DNA sequencing. HeLa cells were treated with the P-TEFb inhibitor flavopiridol prior to nuclei isolation and were then treated with DFF. Here, we have sequenced the undigested DNA from HeLa cells treated with 1 µM flavopiridol for 1 h and DFF for 30 minutes.