Abstract
Transcription progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps from enhancer activation to termination. Here we utilize a rapid degradation system and reveal crucial functions of SPT5 in maintaining cellular and chromatin RNA polymerase II (Pol II) levels. Rapid SPT5 depletion causes a pronounced reduction of paused Pol II at promoters and enhancers, distinct from negative elongation factor (NELF) degradation resulting in short-distance paused Pol II redistribution. Most genes exhibit downregulation, but not upregulation, accompanied by greatly impaired transcription activation, altered chromatin landscape at enhancers, and severe Pol II processivity defects at gene bodies. Phosphorylation of an SPT5 linker at serine 666 potentiates pause release and is antagonized by Integrator-PP2A (INTAC) targeting SPT5 and Pol II, while phosphorylation of the SPT5 C-terminal region links to 3' end termination. Our findings position SPT5 as an essential positive regulator of global transcription.
Highlights
Transcription by RNA polymerase II (Pol II) in mammals is a highly orchestrated process involving complex transitions from initiation, Pol II pausing, elongation, to termination (Chen et al, 2018; Core and Adelman, 2019; Cramer, 2019; Jonkers and Lis, 2015)
SPT5 maintains cellular and chromatin levels of Pol II protein To explore the direct role of SPT5 in transcriptional regulation, we utilized the degradation tag technology by integrating the FLAG-FKBP12F36V tag at the N terminus of the endogenous SPT5 locus (SUPT5H) in DLD-1 cells (Figures 1A and S1A; Jaeger and Winter, 2021; Nabet et al, 2018)
SPT5 knockdown resulted in a reduction in RPB1 levels (Figure S1D), ruling out the possible artifacts caused by the acute protein degradation system
Summary
Transcription progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps from enhancer activation to termination. We utilize a rapid degradation system and reveal crucial functions of SPT5 in maintaining cellular and chromatin RNA polymerase II (Pol II) levels. Rapid SPT5 depletion causes a pronounced reduction of paused Pol II at promoters and enhancers, distinct from negative elongation factor (NELF) degradation resulting in short-distance paused Pol II redistribution. But not upregulation, accompanied by greatly impaired transcription activation, altered chromatin landscape at enhancers, and severe Pol II processivity defects at gene bodies. Phosphorylation of an SPT5 linker at serine 666 potentiates pause release and is antagonized by Integrator-PP2A (INTAC) targeting SPT5 and Pol II, while phosphorylation of the SPT5 C-terminal region links to 30 end termination. Our findings position SPT5 as an essential positive regulator of global transcription
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