Abstract
The TET family of 5-methylcytosine (5mC) dioxygenases plays critical roles in development by modifying DNA methylation. Using CRISPR, we inactivated the TET1 gene in H9 human embryonic stem cells (hESCs). Mutant H9 hESCs remained pluripotent, even though the level of hydroxymethylcytosine (5hmC) decreased to 30% of that in wild-type cells. Neural differentiation induced by dual SMAD inhibitors was not significantly affected by loss of TET1 activity. However, in a morphogen-free condition, TET1 deficiency significantly reduced the generation of NESTIN+SOX1+ neuroectoderm cells from 70% in wild-type cells to 20% in mutant cells. This was accompanied by a 20-fold reduction in the expression level of PAX6 and a significant decrease in the amount of 5hmC on the PAX6 promoter. Overexpression of the TET1 catalytic domain in TET1-deficient hESCs significantly increased 5hmC levels and elevated PAX6 expression during differentiation. Consistent with these in vitro data, PAX6 expression was significantly decreased in teratomas formed by TET1-deficient hESCs. However, TET1 deficiency did not prevent the formation of neural tube-like structures in teratomas. Our results suggest that TET1 deficiency impairs the intrinsic ability of hESCs to differentiate to neuroectoderm, presumably by decreasing the expression of PAX6, a key regulator in the development of human neuroectoderm.
Highlights
Among the three members of the TET family, TET1 is the most abundant in ESCs22,23
After CRISPR/Cas9-mediated gene targeting in H9 human embryonic stem cells (hESCs), two independent clones with a frame shift mutation in each allele of TET1 were obtained
In total RNA isolated from these teratomas, we found that the expression levels of PAX6 and SOX1 were significantly decreased in the teratomas formed by the two TET1-deficient hESCs (Fig. 8a)
Summary
Several studies have generated Tet[1] loss-of-function mouse ESCs22,24 or Tet[1] knockout mice[25,26]. The conflicting phenotypes in mouse and the important role of human TET1 in epigenetic reprogramming of human fibroblasts to neurons[28] motivated us to study the consequence of TET1 loss in human. TET1-deficient hESCs maintained pluripotency but exhibited impaired differentiation to neuroectoderm and neurons in a morphogen-free condition. The 5hmC level on PAX6 promoter was significantly decreased, as was the expression of PAX6, a critical regulator of neuroectoderm development in human[30]. Overexpression of TET1 catalytic domain in TET1-deficient hESCs rescued the defects in 5hmC levels in hESCs and PAX6 expression during differentiation. The study reveals a critical function of TET1 in the differentiation of hESCs to neuroectoderm and neurons
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