Extended Data Fig. 3: Chromatin-associated Spt5 is dephosphorylated rapidly upon Cdk9 inhibition and stabilized in dis2-11 cells.

a, Rapid pSpt5 turnover on chromatin. ChIP–qPCR analysis of crosslinking of pSpt5 versus total Spt5 at the eng1⁺ gene after 3-MB-PP1 treatment for various lengths of time. Left, absolute ChIP signals for anti-Myc; middle, absolute signals for anti-pSpt5; right, ratio of pSpt5 to total Spt5, expressed as a percentage of the ratio in the absence of the inhibitor. b, Loss of Dis2 function stabilizes pSpt5 on chromatin. Left and middle, either cdk9^as spt5-13Myc dis2⁺ or cdk9^as spt5-13Myc dis2-11 cells were shifted to 18 °C and treated with 10 μM 3-MB-PP1 or mock-treated with DMSO for 2 min and subjected to ChIP–qPCR analysis at the eng1⁺ locus for Spt5–Myc (left) or pSpt5 (middle). Right, the pSpt5:Spt5 signal ratios of 3-MB-PP1-treated samples versus DMSO-treated samples. The pSpt5:Spt5 signal ratios between treatments (DMSO and 3-MB-PP1) were plotted for each condition (dis2⁺ or dis2-11). Note, higher residual levels of pSpt5 in cdk9^as dis2⁺ cells, compared to those analysed in a, may reflect less efficient dephosphorylation at 18 °C, relative to 30 °C. c, As in b, except measuring at the aro1⁺ gene. d, As in b, but measuring at the hxk2⁺ gene (raw data for pSpt5 and total Spt5 from which ratios in Fig. 2c were calculated). a–d, Data are mean + s.d. from three biological replicates. b–d, P values (Student’s t-test) are indicated between wild-type (dis2⁺) and mutant (dis2-11) cells.

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