DPF2 regulates OCT4 protein level and nuclear distribution

• DPF2 is upregulated during H9 cell differentiation induced by retinoic acid.
• DPF2 interacts with OCT4 and promotes OCT4 ubiquitination in vitro.
• DPF2 interacts with OCT4 in vivo and redistributes OCT4 in nuclei.
• DPF2 enhances the ubiquitination and degradation of OCT4 in a PHD finger-dependent manner.
• Ubiquitination of OCT4 by DPF2 is mainly through lysine (K) 48 linkages.

The amount of transcription factor OCT4 is strictly regulated. A tight regulation of OCT4 levels is crucial for mammalian embryonic development and oncogenesis. However, the mechanisms underlying regulation of OCT4 protein expression and nuclear distribution are largely unknown. Here, we report that DPF2, a plant homeodomain (PHD) finger protein, is upregulated during H9 cell differentiation induced by retinoic acid. Endogenous interaction between DPF2 and OCT4 in P19 cells was revealed by an immunoprecipitation assay. GST-pull down assay proved that OCT4 protein in H9 cells and recombinant OCT4 can precipitate with DPF2 in vitro. In vitro ubiquitination assay demonstrated DPF2 might serve as an E3 ligase. Knock down of dpf2 using siRNA increased OCT4 protein level and stability in P19 cells. DPF2 siRNAs also up-regulates OCT4 but not NANOG in H9 cells. However, RA fails to downregulates OCT4 protein level in cells infected by lenitviruses containing DPF2 siRNA. Moreover, overexpression of both DPF2 and OCT4 in 293 cells proved the DPF2–OCT4 interaction. DPF2 but not PHD2 mutant DPF2 enhanced ubiquitination and degradation of OCT4 in 293 cells co-expressed DPF2 and OCT4. Both wild type DPF2 and PHD2 mutant DPF2 redistributes nuclear OCT4 without affecting DPF2–OCT4 interaction. Further analysis indicated that DPF2 decreases monomeric and mono-ubiquitinated OCT4, assembles poly-ubiquitin chains on OCT4 mainly through Ub–K48 linkage. These findings contribute to an understanding of how OCT4 protein level and nuclear distribution is regulated by its associated protein.