Fig. 4: Degradation of CTDP1, but not other phosphatases, leads to the destabilization of RPB1.
From: CTDP1 and RPB7 stabilize Pol II and permit reinitiation
a Western blot analyses of the indicated protein in CTDP1 degron cells, CTDSP1 degron cells, SSU72 degron cells, and RAP74 degron cells after a time-course auxin treatment. b Volcano plot showing the protein enrichment changes identified by label-free MS of whole-cell lysates upon CTDP1 degradation. The red dot indicates CTDP1, blue dots indicate subunits of Pol II. The differentially enriched proteins were identified by DEP (|log2FC|> 1, p value < 0.05, analyzed with two-sided t-test with standard deviation moderated by empirical Bayes method in Limma software). c Western blot analyses of the indicated proteins in two subcellular fractions of CTDP1 degron cells in the presence and absence of auxin treatment. Right: The fold changes in hypophosphorylated Pol II (IIA) and hyperphosphorylated Pol II (IIO) upon depletion of CTDP1 are shown (n = 2, biological replicates). d Meta plots showing CTDP1 (up) and RPB1 (down) ChIP-Seq signal upon RPB7 degradation, as well as an input and IgG ChIP-Seq signal. Right shows the quantification of the meta plots at TSS region (−0.5 to +0.5 kb around TSS). The bounds of box indicate 25% (Q1) to 75% (Q3) of the data. The center of the box indicates 50% of the data. The upper and lower whiskers indicate Q3 + 1.5IQR and Q1 − 1.5IQR, respectively (IQR = Q3 − Q1). Statistics in two biological replicates were identified using two-sided Wilcoxon test. e Schematic of exogenous expression of wild-type or four mutated CTDP1 (D302K, Δ2–133, Δ619–729, and D188E/D190E) in CTDP1 degron cells. f Western blot analyses in exogenous CTDP1-expressing cells treated with or without 500 μM auxin for 3 h. GFP signal served as the degradation control, FLAG signal served as exogenous CTDP1-expressing control, and β-Actin served as the loading control. Right: Fold changes of total RPB1 signals in the left panel (n = 3, biological replicates) are shown as means ± SD. Statistical significance, two-sided unpaired t-test; ***p < 0.001; **p < 0.01; n.s., not significant; error bars, SD. L-R p value: 0.0005, 0.0007, 0.1174, and 0.0021.
