Oct4 pseudogene lncRNA: dissecting its role in embryonic stem cell differentiation and epigenetic silencing of its ancestral gene in trans

Pseudogenes are defined as non-functional genomic sequences originally derived from functional genes to which they are similar but with important defects, such as mutations, insertions and deletions, that make them unable to produce a functional protein. For a long time, pseudogenes have been considered as non-functional RNA relics, however, recently, some pseudogenes-derived lncRNA have been shown to be functional and play an important role in various physiological and pathological conditions. We recently discovered a murine Oct4 pseudogene (mOct4P4) which encodes a nuclear restricted lncRNA able to silence the transcription of the ancestral Oct4 gene. mOct4P4 is upregulated during mESC differentiation and recruits SUV39H1 to the Oct4 promoter to impose repressive heterochromatin in trans, leading to Oct4 gene silencing to promote cell differentiation. In this study, we obtained novel insights into the molecular mechanism of mOct4P4 pseudogene-dependent gene silencing and its conservation in human cells. CRISPR/Cas9 mediated mOct4P4 loss-of-function experiments showed an essential role for mOct4P4 in orchestrating an effective mESC differentiation. In addition, mOct4P4 deletion analysis identified the minimal functional region in mOct4P4 lncRNA (base pairs 984-1183) required for SUV39H1 binding and Oct4 ancestral gene targeting. Moreover, mass spectrometry analysis on mOct4P4 lncRNA pull-down identified a novel mOct4P4 interactor, the RNA binding protein FUS, which is required for mOct4P4 dependent silencing of Oct4 expression, suggesting that a mOct4P4 lncRNA/FUS/SUV39H1 complex has an important role in the silencing of Oct4 during mESC differentiation. Finally, we characterized the human homolog of mOct4P4 (hOCT4P3) and demonstrated the functional conservation between the two pseudogene-derived lncRNAs. In fact, we show that hOCT4P3 is able to form a complex with SUV39H1 and FUS that is able to impose repressive heterochromatin marks on ancestral gene promoter leading to silencing of OCT4 expression in ovarian cancer cells. In conclusion, we provided novel insights into the epigenetic mechanism that the murine Oct4P4 lncRNA uses to control ancestral Oct4 expression in trans to regulate self-renewal and differentiation and further demonstrated that this mechanism is also conserved in humans.