Abstract
Reversible phosphorylation of Pol II and accessory factors helps order the transcription cycle. Here, we define two kinase-phosphatase switches that operate at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3′ and 5′ ends of genes, respectively, and have overlapping but distinct specificities for Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between Kyrpides-Ouzounis-Woese (KOW) motifs 4 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation decreases as transcription complexes pass the cleavage and polyadenylation signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in termination first uncovered in yeast. Depletion of PP4-complex subunits increases phosphorylation of both Ser666 and the CTR, and promotes redistribution of promoter-proximally paused Pol II into gene bodies. These results suggest that switches comprising Cdk9 and either PP4 or PP1 govern pause release and the elongation-termination transition, respectively.
Highlights
Reversible phosphorylation of polymerase II (Pol II) and accessory factors helps order the transcription cycle
Two phosphatase 1 (PP1) catalytic-subunit isoforms and two residues of Spt[5] were among targets of human positive transcription elongation factor b (P-TEFb) we identified in a chemical-genetic screen[9]
We identify a second site of Cdk9-mediated inhibitory phosphorylation in PP4R2, a regulatory subunit of the protein phosphatase 4 (PP4) complex
Summary
Reversible phosphorylation of Pol II and accessory factors helps order the transcription cycle. Depletion of PP4-complex subunits increases phosphorylation of both Ser[666] and the CTR, and promotes redistribution of promoter-proximally paused Pol II into gene bodies These results suggest that switches comprising Cdk[9] and either PP4 or PP1 govern pause release and the elongation-termination transition, respectively. A promoter–proximal pause soon after the transition from initiation to elongation is a rate-limiting step in transcription of many Pol II-dependent genes in metazoans[10,11] This pause is established within the first ~100 nucleotides (nt) downstream of the transcription start site (TSS) by recruitment of the DRBsensitivity inducing factor (DSIF)—a heterodimer of Spt[4] and Spt[5] subunits conserved in all eukaryotes—and a metazoanspecific negative elongation factor (NELF)[12]. Pol II transcription, phosphorylating Spt[5] to enable its function as a processivity factor[20], and stimulating elongation rate by three to fourfold[21,22]
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