Neural Differentiation of Mouse Embryonic Stem Cells-An in vitro Approach to Profile DNA Methylation of Reprogramming Factor Sox2-SRR2

Abstract

Sox2 is one of the core transcription factors maintaining the embryonic stem cells (ES) pluripotency and, also indispensable for cellular reprogramming. However, limited data is available about the DNA methylation of pluripotency genes during lineage-specific differentiations. This study investigated the DNA methylation of Sox2 regulatory region 2 (SRR2) during directed differentiation of mouse ES into neural lineage. ES cells were first grown to form embryoid bodies in suspension which were then dissociated, and cultured in defined medium to promote neural differentiation. Typical neuronal morphology together with the up-regulation of Pax6, neuroepithelial stem cell intermediate filament and β-tubulin III and, down-regulation of pluripotency genes Oct4, Nanog and Sox2 showed the existence of neural phenotype in cells undergoing differentiation. Three CpGs in the core enhancer region of neural-specific SRR2 were individually investigated by direct DNA sequencing post-bisulfite treatment and, found to be unmethylated in differentiated cells at time-points chosen for analysis. This analysis does not limit the possibility of methylation at other CpG sites than those profiled here and/or transient methylation. Hence, similar analyses exploring the DNA methylation at other regions of the Sox2 gene could unravel the onset and transitions of epigenetic signatures influencing the outcome of differentiation pathways and neural development. The data presented here shows that in vitro neural differentiation of embryonic stem cells can be employed to study and characterize molecular regulatory mechanisms governing neurogenesis by applying diverse pharmacological and toxicological agents.

Keywords: DNA methylation; SOX2; SRR2; embryonic stem cells; epigenetic regulation; mouse; neural differentiation; pluripotency.

FIGURE 1

Some representative pictures of mES cells undergoing neural differentiation. Mouse embryonic stem cells (mES) undergoing differentiation. E14Tg2a cells were photographed at different time-points during in vitro neural differentiation at 0.1 mm (10X) and 0.2 mm (20X) magnification. Day-0 refers to undifferentiated mES cells before starting differentiation and Day-8 are embryoid bodies (EBs) grown in suspension culture in non-adherent petri plates. Cells having specific neuronal morphology can be seen at day-12 and by day-21 and day-28 majority of cells in culture were differentiated. These are the results from three independent differentiation experiments.

FIGURE 2

Gene expression analysis of mES during neural differentiation time-course. Gene expression analysis of mES subjected to neural differentiation. RNA extracted from E14Tg2a cells grown in neural differentiation medium at day-0, day-12, day-21, and day-28 was reverse transcribed using random primers. Pluripotency markers, i.e., Oct4, Nanog and Sox2 were found to be down-regulated after day-8 (Supplementary Figure 4). Neural-markers, i.e., Sox9, Nestin, Pax6, and beta-tubulin III were up-regulated in the cells undergoing differentiation after day-8. Gapdh was used as endogenous control. Each PCR was performed in triplicates from three independent differentiation experiments.

FIGURE 3

Immunocytochemistry with Anti-Sox2 Antibody. Almost all cells of undifferentiated ES cell colony were found positive with nuclear localization (Figure 4 with higher resolution at day-0) and no expression was detected at day-12, day-21, and day-28. Dapi (4′,6-diamidino-2-phenylindole) was used to stain the nucleus and anti-rabbit IgG secondary antibody (FITC-conjugated) against primary anti-Sox2 antibody was used (scale bar is 32 μm) for visualization.

FIGURE 4

Immunocytochemistry with Anti-Sox2 Antibody (Day-0). The nuclear localization of Sox2 antibody has been shown for clarity in undifferentiated ES cells. All the cells can be seen clearly expressing Sox2.

FIGURE 5

DNA methylation analysis of Sox2-SRR2 by MS-PCR. Top panel shows multiplex PCR for control primers at all time-points, i.e., day-0, day-8, day-12, day-21, and day-28 indicating complete digestion of the genomic DNA. Lower panel is Sox2-SRR2 amplified product in H lane indicative of DNA methylation at all the sites in the fragment as no product was observed in M lane. Any product in the M lane would have indicated the presence of undigested genomic DNA thus giving false positive results. This analysis was performed in triplicates from three independent differentiation experiments.

FIGURE 6

Sequencing chromatogram of Sox2-SRR2. SRR2 spans from +3641 to +4023 downstream of the gene relative to transcription start site (GeneBank ID: NG_051227.1). Part of the sequence which was analyzed by direct DNA sequencing after bisulfite conversion and contained 81bp core enhancer sequence (+3931 to +4011) highlighted in gray is shown here at the bottom. Sequencing chromatogram shows the three unmethylayted CpGs of which two are in core enhancer region, i.e., +3961 and +3967.

FIGURE 7

Lollipop diagram of CpGs analyzed by direct DNA sequencing. Lollipop diagram generated from BiQ Analyzer after bisulfite sequencing. The three CpGs in the enhancer region of SRR2 appear to be unmethylated in undifferentiated ES cells and after differentiation at day-8, day-12, day-21, and day-28. This was done in duplicates from two independent differentiation experiments.