The distributions of 5-MeC, DNMT1, DNMT3a, MBD1, and Ac-H4 changed upon differentiation (summarized in Table 2). DNA methyltransferase demonstrated a dramatic translocation during differentiation. 5-MeC was present throughout the nucleus of undifferentiated NSCs, evacuated from nucleoli of NSC nucleus in the core of differentiating neurospheres, and further translocated into the perinuclear compartment in migrated and differentiated NSCs (Figure 4A, B). DNMT1 was found in both the nucleus and the cytoplasm in undifferentiated neurospheres, but translocated entirely into the cytoplasm in differentiating core and in migrated NSCs (Figure 4C, D). DNMT3a was distributed exclusively in the nucleus of undifferentiated NSCs, but nearly all expression translocated into the cytoplasm at the beginning of differentiation, as seen in the core and periphery of differentiated and migrated neurospheres. Interestingly, MBD1 was not expressed (or expressed at very low levels) in undifferentiated NSCs (Figure 1C), but was seen in both the nucleus and cytoplasm of differentiating core and migrated NSCs (Figure 1F, arrow). It is worthwhile to note that DNMT1, DNMT3a, and MBD1 were the only marks to be found in the cytoplasm.
