Genome binding/occupancy profiling by high throughput sequencing Other
Summary
RNA polymerase II (Pol II) is generally paused at promoter-proximal regions in most metazoans, and based on in vitro studies, this function has been attributed to the negative elongation factor (NELF). Here, we show that upon rapid depletion of NELF, Pol II fails to be released into gene bodies, stopping instead around the +1 nucleosomal dyad-associated region. The transition to the 2nd pause region is independent of positive transcription elongation factor P-TEFb. During the heat shock response, Pol II is rapidly released from pausing at heat shock induced genes, while most genes are paused and transcriptionally downregulated during the heat shock response. We find that both aspects of the heat shock response remain intact upon NELF loss. We find that NELF depletion results in global loss of cap-binding complex from chromatin without global reduction of nascent transcript 5’ cap stability. Thus, our studies implicate NELF functioning in early elongation complexes distinct from Pol II pause-release.
Overall design
Examination of NELF functions in human cells using the auxin-inducible degron system and high-throughput sequencing (PRO-seq, ChIP-seq, PRO-cap).
Less...
More...