Down-Regulation Of OCT4 By DPF2 Promotes Neuroectoderm Differentiation Of Embryonic Stem Cells

Backgroud: Embryonic stem cells(ESCs)can form neuroectoderm(NE),providing a platform for in vitro dissection of NE formation.Because differentiation of human ESCs into NE is prerequisite for differentiation of human ESCs into region-specific neural cells,it is important to understand roles of extrinsic or intrinsic factors involved in NE differentiation.OCT4 plays crucial roles in maintaining stem cell pluripotency and self-renewal.OCT4 protein level is important for its multilineage differentiation capacity.DPF2 contains double plant homeodomain(PHD)fingers,which is considered as one subgroup of RING-related E3 s,suggesting DPF2 might have shared an E3 activity.It has been reported that DPF2 interacts with OCT4 or a protein complex containing OCT4 in vivo.However,if DPF2 regulates OCT4 protein levels is largely unknown.Objective: Our study here aim to: 1)study the interaction of DPF2 and OCT4;2)investigate whether down-regulation of OCT4 by DPF2 affects differentiation of human ESC line,H9 cell,and P19 cells into neuroectoderm;3)investigate the effect of the cell density on inducing differentiation of ESCs into neuroectoderm;4)explore the expression of DPF2 in spinal cord and hippocampus of C57/BL6 mice.Methods:1)Human ESC cell line,H9 cell,was plated at different cell densities and subjected to neuroectoderm induction with RA.Then IF and IB assay were used to detect the protein levels of PAX6 in the induced cells.2)P19 cells were subjected to Co-IP and IF assay to verify the combination of DPF2 and OCT4;3)P19 cells were transfected with DPF2 si RNA and subjected to NE differentiation.H9 cells were infected with lentivirus carring DPF2 si RNA and subjected to differentiation induced by RA.Then IB assay was performed to detect the protein levels of OCT4,DPF2 and PAX6;4)The C57/BL6 E13.5 embryos and adult mice(10-12 weeks,weight 25-30g)were employed to detect DPF2 expression in embryonic spinal cord and adult mouse spinal cord and hippocampus using immunohistochemical assay.Results:1)High plating cell density,with the supplement of SB431542 and NOGGIN,increased significantly PAX6-positvie H9-derived NE cells and decreased OCT4-positive H9-derived cells;2)DPF2 interacts with OCT4 in P19 cells;3)During the H9 cell differentiation induced by RA,DPF2 was upregulated and accompanied by downregulation of OCT4 protein level.When DPF2 was knockdown,RA was not able to induce the downregulation of OCT4;4)In P19 cells,upregulation of DPF2 and PAX6 was accompanied by downregulation of OCT4 induced by RA.Upon knockdown of DPF2,downregulation of OCT4 and upregulation of PAX6 was not observed;5)The expression of DPF2 is in both the embryonic spinal cord at E13.5 and the adult mouse spinal cord and hippocampus,with its localization is mainly in nuclei.The expression of DPF2 in CA1 region is higher than the other regions in the hippocampus.Robust expression of DPF2 was found in the ependymal cells of the mouse spinal cord and the DG cells of the adult mouse hippocampus.Conclusion: 1)High plating cell density promotes NE differentiation,and further differentiated into neurons;2)After DPF2 combined with OCT4,downregulation of OCT4 protein levels can promote neural differentiation of embryonic stem cells;3)DPF2may be related to the development of new neural cells.