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Single-molecule nascent RNA sequencing identifies regulatory domain architecture at promoters and enhancers

Abstract

Eukaryotic RNA polymerase II (Pol II) has been found at both promoters and distal enhancers, suggesting additional functions beyond mRNA production. To understand this role, we sequenced nascent RNAs at single-molecule resolution to unravel the interplay between Pol II initiation, capping and pausing genome-wide. Our analyses identify two pause classes that are associated with different RNA capping profiles. More proximal pausing is associated with less complete capping, less elongation and a more enhancer-like complement of transcription factors than later pausing. Unexpectedly, transcription start sites (TSSs) are predominantly found in constellations composed of multiple divergent pairs. TSS clusters are intimately associated with precise arrays of nucleosomes and correspond with boundaries of transcription factor binding and chromatin modification at promoters and enhancers. TSS architecture is largely unchanged during the dramatic transcriptional changes induced by heat shock. Together, our results suggest that promoter- and enhancer-associated Pol II is a regulatory nexus for integrating information across TSS ensembles.

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Fig. 1: CoPRO simultaneously measures initiation and the active site of Pol II genome-wide.
Fig. 2: Sequence determinants of pause position choice.
Fig. 3: Late-pause TSNs are capped more efficiently, but later than early-pause TSNs.
Fig. 4: A global view of initiation shows rules for divergent pairing and widespread complex organization.
Fig. 5: Massive regulatory changes in heat shock occur by modulating the activity of pre-established TSSs.
Fig. 6: TID organization is linked to chromatin environment.

Data availability

All sequencing data and processed bigwig and Rdata files have been deposited at GEO under accession GSE116472.

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Acknowledgements

We thank members of the Lis, Kwak and Danko laboratories for helpful discussions throughout the experimental design and analysis phases. J. Mahat assisted in designing CoPRO libraries for sequencing without PCR amplification and the heat shock protocol. The Cornell Sequencing core and especially P. Schweitzer were extremely helpful in designing libraries and eminently patient in accommodating our technical requests. We acknowledge funding from National Institutes of Health (NIH) grant nos. HG009393 and GM025232 (to J.T.L.). N.D.T. was supported by NIH training grant no. T32HD057854.

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J.M.T. and N.D.T. conceived of CoPRO, carried out experiments and analyzed data. N.D.T. developed the computational framework for analysis. J.M.T., N.D.T. and J.T.L. interpreted results and prepared the manuscript.

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Correspondence to John T. Lis.

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Tome, J.M., Tippens, N.D. & Lis, J.T. Single-molecule nascent RNA sequencing identifies regulatory domain architecture at promoters and enhancers. Nat Genet 50, 1533–1541 (2018). https://doi.org/10.1038/s41588-018-0234-5

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