Prolyl isomerase Pin1 shares functional similarity with phosphorylated CTD interacting factor PCIF1 in vertebrate cells.

The carboxy-terminal domain (CTD) of the RNA polymerase II (Pol II) largest subunit undergoes reversible phosphorylation during transcription cycle. The phosphorylated CTD plays critical roles in coordinating transcription with chromatin modification and RNA processing by serving as a scaffold to recruit various proteins. Recently, we identified a novel human WW domain-containing protein PCIF1 as a phosphorylated CTD-interacting factor and demonstrated that PCIF1 negatively modulates Pol II activity in vivo. In the present study, to explore cellular functions of PCIF1, we generated PCIF1-deficient chicken DT40 cell lines. We observed significant up-regulation of WW domain-containing prolyl isomerase Pin1 in two independently established PCIF1-deficient mutant clones. As reconstitution of PCIF1 in the mutants did not reduce Pin1 expression, PCIF1 may not be a negative regulator of Pin1 expression. We assume that Pin1 over-expression might suppress defects caused by PCIF1 deficiency in DT40 cells. We furthermore compared PCIF1 and Pin1 for their functional properties and found that these two proteins exhibit most similar target specificity among other CTD-binding WW proteins, overlapping subcellular localization and comparative inhibitory effects on transcriptional activation by Pol II in human cultured cells. These results suggest that Pin1 may have overlapping cellular function with PCIF1 in vertebrate cells.

Human phosphorylated CTD-interacting protein, PCIF1, negatively modulates gene expression by RNA polymerase II.

Phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) regulates transcription cycle and coordinates recruitment of RNA processing factors and chromatin regulators. Recently, we reported the identification of human PCIF1 as a novel protein that directly binds to the phosphorylated CTD via its WW domain, which is highly homologous to the WW domain of human peptidylprolyl isomerase Pin1. Although PCIF1 has been shown to associate with phosphorylated Pol II, functional consequence of the interaction remains unclear. Here we further characterized the cytological, structural, and functional properties of human PCIF1. Immunofluorescence microscopy revealed that endogenous PCIF1 was colocalized with the phosphorylated Pol II and the transcription elongation factor DSIF in the cell nucleus. We also found that PCIF1 WW domain inhibits the CTD phosphatase activity of SCP1 in vitro. By examining the effect of either PCIF1 overexpression or knockdown on the transactivation of reporter gene expression by various transcriptional activation domains, we found that PCIF1 significantly repressed the transactivation depend on its CTD binding ability. These data suggest that PCIF1 modulates phosphorylation status of the CTD and negatively regulates gene expression by Pol II.