Abstract
Using an improved chromatin immunoprecipitation assay designed to increase immunoprecipitation efficiency, we investigated changes in RNA polymerase II (Pol II) density and carboxyl-terminal domain (CTD) phosphorylation during transcription of the cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and several androgen-responsive genes in LNCaP cells. As generally observed in higher eukaryotes, promoter proximal pausing of Pol II appeared to occur on the PPIA and GAPDH genes, but apparently not on the androgen-responsive genes PSA and NKX3-1. Unlike some mammalian studies, we found that the CTD of Pol II in promoter regions contains little phosphorylation at Ser-2 of the heptad repeat, suggesting that Ser-2 phosphorylation is not involved in polymerase exit from the promoter region. In contrast, Pol II near the promoter displayed high levels of Ser-5 phosphorylation, which decreased as polymerase transcribed beyond the promoter region of the PPIA and GAPDH genes. However, total Pol II levels appear to decrease as much or more, suggesting that Ser-5 phosphorylation is maintained. In support of this conclusion, a phosphoserine 5-specific antibody quantitatively immunoprecipitates native hyperphosphorylated Pol II, suggesting that all polymerase with phosphoserine 2 also contains phosphoserine 5. Given reports indicating that phosphoserine 5 is present during elongation in yeast, our data suggest that gross changes in CTD phosphorylation patterns during transcription may be more conserved in yeast and humans than recognized previously.
Highlights
Phosphorylation of the carboxyl-terminal domain (CTD)1 of the largest subunit of RNA polymerase II (Pol II) plays an important role in regulating transcription and mRNA processing [1,2,3,4]
Using an improved chromatin immunoprecipitation assay designed to increase immunoprecipitation efficiency, we investigated changes in RNA polymerase II (Pol II) density and carboxyl-terminal domain (CTD) phosphorylation during transcription of the cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and several androgen-responsive genes in LNCaP cells
We found that the CTD of Pol II in promoter regions contains little phosphorylation at Ser-2 of the heptad repeat, suggesting that Ser-2 phosphorylation is not involved in polymerase exit from the promoter region
Summary
Materials—RPMI 1640 medium and penicillin/streptomycin (15140-122) were from Invitrogen, and heat-inactivated fetal calf serum (SH30071.03) was from Hyclone. Preparation of Antibody Beads—Protein G-agarose was bound to IgG antibodies by incubation of each antibody with a 5% matrix slurry for 1 h at 25 °C with rotation in IP dilution buffer (10 mM Tris, pH 7.5, at 25 °C, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, protease inhibitors (1 mM PMSF and 1 ϫ Complete PIC), phosphatase inhibitors (1 mM Na3VO4, 10 mM Na4P2O7, and 10 mM NaF)). Plates were placed on ice, and 2 ml of chilled (2 min on ice) lysis buffer (10 mM Tris, pH 7.5, at 25 °C, 0.5% SDS, protease inhibitors (1 mM PMSF and 5 ϫ Complete PIC), phosphatase inhibitors as above) was added.
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