The Functional Role Of KDM6 In Human Early Neuroectodermal Differentiation

Epigenetic trait is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence,including DNA modification(methylation),histone modifications(methylation,acetylation,ubiquitination,etc.),chromatin remodeling,histone variants,non-coding RNA.Epigenetic modifications have been shown to be important for development and cell reprogramming,but how it contributes to the differentiation process of human early neuroectoderm is still largely unclear.This study mainly focuses on the role of enzymes for histone methylations in early neuroectoderm development.The main contents of this study are as follows.Firstly,we stablish and optimize the neuroectoderm differentiation protocol by titrating the small molecules,and then perform RNA-seq analysis to identify histone H3 demethylase KDM6 B as the most significantly upregulated gene during human neuroectoderm differentiation.Next,we construct the KDM6 A and KDM6 B single knockout and double knockout human embryonic stem cell lines by CRISPR/Cas9 and determine that the KDM6-knockout cells exhibit higher efficiency of neuroectoderm differentiation by marker examination and RNA-seq analysis.Then we identify WNT/beta-catenin signaling pathway as the downstream of KDM6 through KEGG(Kyoto Encyclopedia of Genes and Genomes;KEGG)analysis based on transcriptome and then experimentally confirm this notion by examining the amount of activated beta catenin protein and the expression levels of WNT target genes WNT3 and FZD5.More importantly,demonstrate that WNT agonist(CHIR99021)can inhibit neuroectoderm differentiation and the inhibitor(IWR-1)can improve the efficiency of early neural differentiation,while both CHIR99021 and IWR-1 can replenish the phenotype that KDM6 knockout enhances neuroectoderm differentiation to some extent.Continuly,we construct murine-derived Kdm6 b demethylase domain Jmj C mutant and WT expressing invasion viruses.Adding corresponding viruses during the differentiation of human early neural progenitor cells and the results of neuroectodermal differentiation on day 7 were detected.The mutant Kdm6 b cannot replenish the phenotype,while the WT Kdm6 b can replenish the phenotype.This indicated that the enzymatic activity of KDM6 is required for the loss of KDM6 family could promote neuroectoderm differentiation.At last,we apply the KDM6 inhibitor GSK J1 in neuroectoderm differentiation from both human embryonic stem cells and induced pluripotent stem cells to improve neuroectoderm differentiation.In summary,we found that the KDM6 family could inhibit human early neuroectodermal differentiation through the WNT/beta-catenin signaling pathway,which depended on the demethylase activity.Additionally,the chemical inhibitor of KDM6,GSK J1,can improve human early neural differentiation,which should facilitate the following translational study for degenerative neurological diseases.