TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing
Figure 1
Hydroxymethylation status in ES cells under differentiation and TET2 knockdown and in patient samples. HPLC mass spectrometry was used to determine the levels of methylcytosine (C) and hydroxymethylcytosine (C) expressed as a percentage of total guanosine bases. All experiments were in triplicate and averaged, unless otherwise stated. (a) 5′-hydroxymethylcytosine is 0.15% of wild-type ES cells. Levels of C were compared to levels of C in undifferentiated E14 ES cells, grown in the presence of LIF to maintain an undifferentiated state; levels were 0.15% and 7.5%, respectively. (b) 5′-hydroxymethylcytosine levels fall in differentiated ES cells. C levels dropped by approximately 50% from 0.15% to 0.076% relative to guanosine bases upon differentiation via the removal of LIF from the growth media. C levels were determined on day 5 after removal of LIF from the growth medium. (c) Effect of TET2 knockdown on 5′-hydroxymethylcytosine levels. Small inhibitory RNA (siRNA) knockdown of TET2 using a control scrambled oligonucleotide (wild-type ES), and two specific TET2 oligonucleotides (Oligo 1 and Oligo 2). Oligo 1 showed no significant knockdown of C levels compared to the control, whereas Oligo 2 showed reduced levels of C upon HPLC mass spectrometric analysis of around 18% compared to control. (d) Bone marrow samples from patients with myelodysplasia. Marrow samples from myelodysplastic syndrome (MDS) patients were analysed with the mass spectrometer for C status. Patients were genotyped for mutations in the TET2 gene locus and any mutants were found to be heterozygous for the TET2 gene.
(a) 5-Hydroxymethylcytosine is 0.15% of guanosine in wild-type ES cells
(b) 5-Hydroxymethylcytosine levels fall in differentiated ES cells
(c) Effect of TET2 knockdown on 5-hydroxymethylcytosine levels
