Fig. 4. PCIF1 regulates ETS1 expression.
a The mRNA expressions of ETS1 was enhanced by PCIF1. The mRNA levels of the ETS1 were analyzed using RT-qPCR in control and PCIF1 KO Jurkat cells, or PCIF1 overexpressed MT4 cells. **p = 0.001, ***p = 0.0001. b PCIF1 regulates ETS1 through its methyltransferase domain. ETS1 mRNA was quantified by RT-qPCR in PCIF1 KO cells rescued with wild-type or methyltransferase inactivated PCIF1 mutant as in Fig. 2c. **p = 0.001, **p = 0.0012, **p = 0.0076, ns not significant. c ETS1 mRNA decay is accelerated in PCIF1 KO cells. Control or PCIF1 KO Jurkat cells were treated with Actinomycin D (ActD) for the indicated time points and cells were collected for RNA quantification. *p = 0.019, *p = 0.012. d CLIP-qPCR showing the association of ETS1 transcript with PCIF1 in PCIF1 overexpressing MT4 cells. RNA and protein in control or Flag-PCIF1 MT4 cells were UV-crosslinked and sonicated. PCIF1 bound RNA was pulled down using Flag antibody. Bound protein and chromatin were enzyme digested and ETS1 and GAPDH RNA were quantified by RT-qPCR. RNA levels were normalized to Input RNA. *p = 0.025. e Protein levels of ETS1 are decreased in PCIF1 KO cells. PCIF1 and ETS1 protein expression was detected in control and PCIF1 KO cells by western blotting. f ETS1 protein is decreased by HIV infection. PCIF1 and ETS1 protein expressions were analyzed in MT4 cells infected with HIVLAI at an MOI of 0.4 or 2 for 3 days. g Kinetics of ETS1 mRNA during HIVLAI-∆Vpr infection. ETS1 mRNA expression was quantified in MT4 cells infected with HIVLAI-∆Vpr at MOI 0.4 for 2, 3, or 5 days. All data are represented as mean ± SD and analyzed by a two-sided t-test in a–d, g. n = 3 (a, c, d, g), or 4 (b) independent experiments. Similar results were obtained from three independent experiments in e and f.