EID3 directly associates with DNMT3A during transdifferentiation of human umbilical cord mesenchymal stem cells to NPC-like cells

Abstract

There has been recently been increased interest in the plasticity of human umbilical cord mesenchymal stem cells (UMSCs) and their potential in the treatment of neurological disorders. In this study, UMSCs were transdifferentiated into neural stem-like cells (uNSCL), these cells grow in neurosphere-like structures and express high levels of NSCs markers. Epigenetics-related gene screening was here used to assess the relationship between E1A-like inhibitor of differentiation 3 (EID3), a p300 inhibitor, and DNA methyltransferase 3 A (DNMT3A) during the transdifferentiation of UMSCs into uNSCL in vitro. Before transdifferentiation of UMSCs into uNSCLs, high levels of EID3 and low levels of DNMT3A were detected; after transdifferentiation, low levels of EID3 and high levels of DNMT3A were detected. The current work showed that EID3 and DNMT3A co-localized in cell nuclei and EID3 interacted directly with DNMT3A in uNSCL. In summary, these results suggest that DNMT3A is probably directly regulated by EID3 during UMSC transdifferentiation into uNSCLs. These findings indicated a novel mechanism by which EID3, a p300 acetyltransferase inhibitor, could directly affect DNMT3A, this enzyme possesses dual methylation and demethylation abilities. These studies may be helpful for understanding a complex regulation mode of DNMT3A, which is a unique member of the methyltransferase family.

Figure 1. Immunophenotype of human umbilical mesenchymal stem cells.

Cell surface markers of human umbilical mesenchymal stem cells (UMSCs) were detected by flow cytometric analysis at passage 3. (A) The related isotype control was used as a negative control. UMSCs did not express (B) CD34 and CD45, but expressed (C) CD29, CD44, (D) CD90, and CD105.

Figure 2

Characteristics of human UMSCs-derived neural stem cell-like cells (uNSCLs) (A) Morphology of human UMSCs and uNSCL. (B) NESTIN and PAX6 expression of UMSCs (upper panels) and uNSCLs (below panels). (C) GFAP and SOX2 expression of UMSCs (upper panels) and uNSCLs (below panels). Scale bars represent 100 μm. (D) Quantitative real-time RT-PCR analyses of NSC marker gene expression (Nestin, Pax6, VIM, MSI1, GFAP, NeuroD1, SOX1). Expression levels are expressed relative to the housekeeping gene GAPDH. Data were analyzed by one-way ANOVA, the experiments were performed at least three times. Abbreviations: UMSCs, umbilical cord mesenchymal stromal cells; uNSCL, UMSCs-derived neural stem cell-like cells; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

Figure 3. The expression of EID3 and DNMT3A in UMSC, uNSCL and NSC cells.

(A) Screen of relative epigenetic gene expression (DNMT1, DNMT3A, DNMT3B, EID1, EID3, p300, HDAC1) by qRT-PCR. Expression levels are expressed relative to the housekeeping gene GAPDH. (B) Western blotting protein analysis of EID3 and DNMT3A in UMSCs, uNSCL and NSCs. GAPDH expression was used as a protein loading control. (C) Histogram of the densitometric quantification of data presented in the left panel. Data were analyzed by one-way ANOVA, the experiments were performed at least three times. (D) Intracellular localization of EID3 and DNMT3A; EID3 and DNMT3A were expressed in UMSCs, uNSCL, and NSCs as detected by immunofluorescence using EID3 (green) and DNMT3A (red) antibodies. Nuclei were stained with DAPI (blue). More than 30 cells were studied. Scale bars represent 10 μm. Abbreviations: DNMT 3 A/3B, DNA methyltransferases 3 A/3B; EID3, E1A-like inhibitor of differentiation 3; p300, E1A binding protein p300; HDAC1, Histone Deacetylase 1; UMSCs, umbilical cord mesenchymal stromal cells; uNSCL, UMSCs-derived neural stem cell-like cells; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

Figure 4. EID3 interacts with DNMT3A.

(A)EID3 co-immunoprecipitates with endogenous DNMT3A in uNSCL. Endogenous EID3 is associated with DNMT3A. (B) EID3 co-immunoprecipitates with DNMT3A in HEK293 cells. pcDNA3.1-EID3, pEGFP-DNMT3A, or their control vectors were co-transfected into HEK293 cells, cells were harvested and lysed with Co-IP lysis buffer after 48 h transfection. Cells lysates were incubated with anti-EID3 antibody and protein A/G beads at 4 °C overnight followed by immunoblotting. (C) EID3 and DNMT3A were expressed in HEK293. Scale bars represent 10 μm.