Coordinated regulation of RNA polymerase II pausing and elongation progression by PAF1

Abstract

Pleiotropic transcription regulator RNA polymerase II (Pol II)–associated factor 1 (PAF1) governs multiple transcriptional steps and the deposition of several epigenetic marks. However, it remains unclear how ultimate transcriptional outcome is determined by PAF1 and whether it relates to PAF1-controlled epigenetic marks. We use rapid degradation systems and reveal direct PAF1 functions in governing pausing partially by recruiting Integrator-PP2A (INTAC), in addition to ensuring elongation. Following acute PAF1 degradation, most destabilized polymerase undergoes effective release, which presumably relies on skewed balance between INTAC and P-TEFb, resulting in hyperphosphorylated substrates including SPT5. Impaired Pol II progression during elongation, along with altered pause release frequency, determines the final transcriptional outputs. Moreover, PAF1 degradation causes a cumulative decline in histone modifications. These epigenetic alterations in chromatin likely further influence the production of transcripts from PAF1 target genes.

The pleiotropic roles of SPT5 in transcription

“…With the help of DSIF, NELF contributes to the stabilization of the first but not the second pausing [ 52 , 53 ], with its depletion resulting in the downstream shift of Pol II to the second pausing sites around the +1 nucleosome dyad. The stabilization of Pol II at the second pausing sites is aided by the pausing and elongation regulator PAF1 complex (PAF1C) and probably chromatin remodelers [ 54–56 ], whereas Pol II release from the second pausing sites requires factors such as P-TEFb and MYC [ 52 , 53 , 57 ].…”

“…Structural comparison of NELF- and PAF1C-containing Pol II complexes reveals steric clashes between NELF and PAF1C, thus eliciting a plausible model that the shift of Pol II from the first to the second pausing sites is accompanied by a substantial change in the composition of Pol II complex, such as the replacement of NELF by PAF1C [ 15 , 54 , 58 ]. Different from the eviction of NELF during this transition, DSIF remains with Pol II at the second pausing site ( Figure 2a ).…”

“…Therefore, loss of INTAC phosphatase activity confers cellular resistance to P-TEFb inhibition ( Figure 3b ) [ 77 ]. The balance between P-TEFb and INTAC in modulating pausing and pause release could be harnessed by other transcription regulators such as PAF1C, which can directly interact with INTAC [ 54 , 58 ]. Through this interaction, PAF1 can recruit INTAC to genomic regions, including promoters, to suppress SPT5 phosphorylation, which could potentially explain the broad function of PAF1 in restraining the release of Pol II from the second pausing sites [ 54–56 ].…”

“…The balance between P-TEFb and INTAC in modulating pausing and pause release could be harnessed by other transcription regulators such as PAF1C, which can directly interact with INTAC [ 54 , 58 ]. Through this interaction, PAF1 can recruit INTAC to genomic regions, including promoters, to suppress SPT5 phosphorylation, which could potentially explain the broad function of PAF1 in restraining the release of Pol II from the second pausing sites [ 54–56 ]. However, it is important to highlight that SPT5 is not the only player in the antagonism between P-TEFb and INTAC, since disruption of the INTAC phosphatase module in SPT5-null cells still stimulates transcriptional activation [ 19 ].…”

Spt6 directly interacts with Cdc73 and is required for Paf1C recruitment to active genes

“…In vitro transcription reactions with purified factors showed that human Paf1C (hPaf1C) can stimulate transcription elongation on chromatin templates in cooperation with TFIIS (11). More recently, depletion of individual Paf1C subunits, including Paf1 and Rtf1, from human or mouse cell lines has revealed positive effects of the complex on elongation rate and Pol II processivity as well as context-dependent effects on promoter proximal Pol II pausing (12)(13)(14)(15)(16)(17). The ability of hRtf1 to stimulate Pol II elongation rate in vitro involves an intimate contact between the hRtf1 latch domain and the Pol II catalytic center (5).…”