Extended Data Fig. 5: Regulation of pSpt5 by Cdk9 and Dis2 occurs independently of CPF recruitment and upstream of CPF function.
From: A Cdk9–PP1 switch regulates the elongation–termination transition of RNA polymerase II
a, Core CPF recruitment to chromatin is unaffected by Cdk9 inhibition. Cells with different GFP-tagged CPF subunits expressed from their respective chromosomal loci (cdk9as pfs2–GFP–HA, cdk9as pla1–GFP–HA or cdk9as cft1–GFP) were grown at 30 °C and treated with 10 μM 3-MB-PP1 or DMSO for 10 min. ChIP–qPCR analysis of GFP:Pol II signal ratios was performed at the hxk2+ gene. Data are mean + s.d. from three biological replicates. b, Wild-type (dis2+) and mutant (dis2-11) cells were grown at 30 °C and shifted to 18 °C (or not shifted) for 10 min. ChIP–qPCR analysis of Pol II-pTyr1:Pol II (top), pSpt5:Spt5 (middle) and Pol II-pSer2:Pol II (bottom) signal ratios was performed at the psu1+ gene. c, Loss of Dis2 activity does not affect chromatin recruitment of a core CPF subunit, Pfs2. Cells of dis2+ and dis2-11 strains with Pfs2–GFP–HA expressed from the chromosomal pfs2+ locus were grown at 30 °C and shifted to 18 °C (or not shifted) for 10 min before formaldehyde crosslinking and chromatin isolation. ChIP–qPCR analysis of GFP:Pol II signal ratios was performed at the psu1+ gene at 30 °C (top) and 18 °C (bottom). d, Spt5 phosphorylation is not affected by thermal inactivation of an essential CPF subunit. Cells of pfs2+ and pfs2-11 (temperature-sensitive) strains were grown at 30 °C, shifted to 37 °C (or not shifted) and incubated for various times as indicated. ChIP–qPCR analysis of pSpt5, total Spt5, pSpt5:Spt5 signal ratios, Pol II and Pol II signal ratios (pfs2-11 over pfs2+), was performed at the hxk2+ locus. b–d, Data are mean + s.d. from two biological replicates.
