Research Article

TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing

Figure 5

(a) Expression of methylated reporter plasmids is identical in differentiated and undifferentiated ES cells. A Dual Luciferase Reporter Assay (Promega) was modified to determine if E14 murine ES cells undergoing differentiation were more able to demethylate (unsilenced) a reporter gene plasmid. Methylation of the pRL reporter by SssI methylase (Promega M0226S) showed optimal gene silencing, and relative expression of the Renilla Luciferase was compared to the unmodified pGL3 plasmid expression of Firefly Luciferase via a luminometer. E14 cells were plated at cells per well in a 24-well plate in triplicate experiments and induced to differentiation on day 1 posttransfection by removal of leukemia inhibitory factor (LIF), and transient transfection of both the pGL3 and methylated pRL was conducted using the Lipofectamine LTX (Invitrogen) reagent as per the manufacturer’s instructions on day 5 after plating. Analysis of Renilla and Firefly expression was conducted on day 6. Transient transfection of unmodified pRL and pGL3 was used as a control. (a) shows that although methylation clearly had a significant silencing effect on pRL, there was no significant difference seen between the −LIF (differentiating) or +LIF (undifferentiated) conditions on relative expression of pRL. (b) TET2 is unable to overcome silenced expression of a cotransfected methylated Renilla plasmid. Human embryonic kidney (HEK) 293T cells were plated at cells per well in a 24-well plate in triplicate and transiently transfected using the Lipofectamine LTX (Invitrogen) reagent as per the manufacturer’s instruction on day 1 after plating with both Dual Luciferase reporter plasmids and a TET2-pCDNA3 construct plasmid. Control experiments were run in parallel with either no pRL methylation, no pCDNA-TET2 cotransfect, or a pCDNA3 (empty vector) cotransfect. Dual Luciferase experiments were modified by methylation of the pRL reporter by SssI methylase (Promega M0226S), which showed optimal gene silencing, and relative expression of the Renilla Luciferase was compared to the unmodified pGL3 plasmid expression of Firefly Luciferase via a luminometer. Experiments were conducted in triplicate and averaged. Cotransfection of a TET2-pCDNA3 plasmid vector at varying concentrations with the Dual Luciferase plasmids does not lead to changes in the unsilencing (demethylation) of the methylated reporter. Control 1 shows again that unmethylated pRL plasmid has much higher expression levels than the methylated form. There was no dose response, even when 2 μg of TET2-pCDNA3 plasmid DNA was added to each well, and there was no difference in Renilla expression between controls 2 and 3 and between those cotransfections that contained the TET2-pCDNA3 plasmid.
986571.fig.005a
(a) Effect of differentiation on ES cell ability to demethylate reporter plasmid
986571.fig.005b
(b) Effect of differing concentrations of TET2 plasmid on methylated Renilla expression