Abstract
The chromatin remodeler CHD8, which belongs to the ATP-dependent chromatin remodelers CHD family, is one of the most high-risk mutated genes in autism spectrum disorders. However, the role of CHD8 in neural differentiation and the mechanism of CHD8 in autism remains unclear, despite there are a few studies based on the CHD8 haploinsufficient models. Here, we generate the CHD8 knockout human ESCs by CRISPR/Cas9 technology and characterize the effect of loss-of-function of CHD8 on pluripotency maintenance and lineage determination by utilizing efficient directed differentiation protocols. The results show loss-of-function of CHD8 does not affect human ESC maintenance although having slight effect on proliferation and cell cycle. Interestingly, CHD8 depletion results in defective neuroectoderm differentiation, along with severe cell death in neural progenitor stage. Transcriptome analysis also indicates CHD8 does not alter the expression of pluripotent genes in ESC stage, but in neural progenitor cells depletion of CHD8 induces the abnormal expression of the apoptosis genes and suppresses neuroectoderm-related genes. These results provide the evidence that CHD8 plays an essential role in the pluripotency exit and neuroectoderm differentiation as well as the regulation of apoptosis during neurogenesis.
Highlights
Human embryonic stem cells (ESCs), derived from the inner cell mass of blastocysts, possess indefinite proliferative capacity and can differentiate into all three germ layers cell type
CHD8 knockout resulted in severe defect of activation of neural lineage genes that explained the blocked neuroectoderm differentiation, and caused significant derepression of apoptosis-related genes during neurogenesis, which led to massive cell death in neural progenitor cell (NPC) stage (Fig. 7E)
Using the CHD8 knockout human ESCs, we found the complete depletion of CHD8 has no significant effect on the expression of pluripotent markers, the ESCs with CHD8 knockout exhibit altered cell cycle progress with a shortened G1 phase, eventually resulting in modestly promoted cell proliferation (Fig. 2)
Summary
Human embryonic stem cells (ESCs), derived from the inner cell mass of blastocysts, possess indefinite proliferative capacity and can differentiate into all three germ layers cell type. As such, they can serve as a model for development and disease studies, and as the ideal resource for regenerative medicine and drug screening [1]. During neuronal differentiation from mouse neural stem cells, CHD8 was reported to be recruited by SMAD3 together with histone demethylase KDM6B to create appropriate chromatin environment to activate posterior gene transcription [16]. Scientists have utilized human ESCs/induced pluripotent stem cells (iPSCs) and the derived neural lineage model as well as neural progenitor cell
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