Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing
RNA polymerase II (Pol II) pausing is a general regulatory step in transcription, yet the stability of paused Pol II varies widely between genes. Although paused Pol II stability correlates with core promoter elements, the contribution of individual sequences remains unclear, in part because no rapi...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2019-04-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/41461 |
| _version_ | 1811253131949899776 |
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| author | Wanqing Shao Sergio G-M Alcantara Julia Zeitlinger |
| author_facet | Wanqing Shao Sergio G-M Alcantara Julia Zeitlinger |
| author_sort | Wanqing Shao |
| collection | DOAJ |
| description | RNA polymerase II (Pol II) pausing is a general regulatory step in transcription, yet the stability of paused Pol II varies widely between genes. Although paused Pol II stability correlates with core promoter elements, the contribution of individual sequences remains unclear, in part because no rapid assay is available for measuring the changes in Pol II pausing as a result of altered promoter sequences. Here, we overcome this hurdle by showing that ChIP-nexus captures the endogenous Pol II pausing on transfected plasmids. Using this reporter-ChIP-nexus assay in Drosophila cells, we show that the pausing stability is influenced by downstream promoter sequences, but that the strongest contribution to Pol II pausing comes from the initiator sequence, in which a single nucleotide, a G at the +2 position, is critical for stable Pol II pausing. These results establish reporter-ChIP-nexus as a valuable tool to analyze Pol II pausing. |
| first_indexed | 2024-04-12T16:45:11Z |
| format | Article |
| id | doaj.art-54c070d8bf684432a6d02e69e51ca644 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T16:45:11Z |
| publishDate | 2019-04-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-54c070d8bf684432a6d02e69e51ca6442022-12-22T03:24:36ZengeLife Sciences Publications LtdeLife2050-084X2019-04-01810.7554/eLife.41461Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausingWanqing Shao0https://orcid.org/0000-0003-4234-2095Sergio G-M Alcantara1https://orcid.org/0000-0003-3695-4677Julia Zeitlinger2https://orcid.org/0000-0002-5172-3335Stowers Institute for Medical Research, Kansas City, United StatesStowers Institute for Medical Research, Kansas City, United StatesStowers Institute for Medical Research, Kansas City, United States; Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, United StatesRNA polymerase II (Pol II) pausing is a general regulatory step in transcription, yet the stability of paused Pol II varies widely between genes. Although paused Pol II stability correlates with core promoter elements, the contribution of individual sequences remains unclear, in part because no rapid assay is available for measuring the changes in Pol II pausing as a result of altered promoter sequences. Here, we overcome this hurdle by showing that ChIP-nexus captures the endogenous Pol II pausing on transfected plasmids. Using this reporter-ChIP-nexus assay in Drosophila cells, we show that the pausing stability is influenced by downstream promoter sequences, but that the strongest contribution to Pol II pausing comes from the initiator sequence, in which a single nucleotide, a G at the +2 position, is critical for stable Pol II pausing. These results establish reporter-ChIP-nexus as a valuable tool to analyze Pol II pausing.https://elifesciences.org/articles/41461RNA polymerase II pausingcore promoter elementreporter-ChIP-nexusKc167 cellsTFIID |
| spellingShingle | Wanqing Shao Sergio G-M Alcantara Julia Zeitlinger Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing eLife RNA polymerase II pausing core promoter element reporter-ChIP-nexus Kc167 cells TFIID |
| title | Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing |
| title_full | Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing |
| title_fullStr | Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing |
| title_full_unstemmed | Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing |
| title_short | Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing |
| title_sort | reporter chip nexus reveals strong contribution of the drosophila initiator sequence to rna polymerase pausing |
| topic | RNA polymerase II pausing core promoter element reporter-ChIP-nexus Kc167 cells TFIID |
| url | https://elifesciences.org/articles/41461 |
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