Expression profiling by high throughput sequencing Other
Summary
Cdk9 is an essential transcriptional kinase that is conserved across distant eukaryotes, but we previously found that acute inhibition of Cdk9 causes different transcriptional phenotypes in NELF-containing higher eukaryotes (like mammals and Drosophila) vs. the NELF-lacking fission yeast S. pombe. NELF is known to participate in promoter-proximal pausing of RNA Polymerase II, and using NELF-depleted Drosophila cells, we find that this NELF-mediated pause is necessary to prevent gene body entry by Pol II in the absence of Cdk9. Without NELF, the abnormal gene body entry that occurs following Cdk9 inhibition is strikingly similar to that seen in S. pombe, and in either case, these Pol II complexes have elongation defects and are unable to complete gene transcription. These results show that NELF enforces an early checkpoint for Cdk9 and efficiently shuts down gene transcription in its absence. We propose this would have been critical for the emergence of gene regulatory strategies acting via Cdk9 to modulate transcription of specific genes.
Overall design
Drosophila S2 cells were treated with dsRNA to knockdown either NELF-E or LacZ (an unexpressed control). Knockdowns were performed in 2 independent experiments (each with 2 biological replicates) in which cells were treated with Flavopiridol or DMSO (control) before harvesting for PRO-seq and RNA-seq. PRO-cap libraries are derived from a third independent experiment without replicates or drug treatments. In all cases, cells were combined with a small quantity of mouse embryonic fibroblasts as an exogenous spike-in control for normalization purposes.
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