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RNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library in haploid human embryonic stem cells (hESCs) differentiated into three germ layers

ABSTRACT: Pluripotent stem cells can differentiate into all embryonic germ layers, a process that is orchestrated by the dissolution of pluripotency network and activation of pro-differentiation pathways. Yet, the genes essential for these cell fate transitions in human are still elusive. Here, we used a genome-wide loss-of-function library established in haploid hPSCs and differentiated these cells into into the three germ layers. We analyzed the transcriptome of the differentiated mutant library cells to assess their differentiation status, as well as to determine lineage-specific expression of essential genes for the differentiation of hESCs into each germ layer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE143373 | GEO | 2020/07/30

REPOSITORIES: GEO

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Project description:Embryogenesis is tightly regulated by multiple levels of epigenetic systems such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent reprogramming occurs by de novo methylation and demethylation. Variance of DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analysed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions in the three germ layers and in the three adult somatic tissues are shared in common. This commonly methylated gene set is enriched in germ cell associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns with global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Taken together, our findings indicate that differentiation from ES cells to the three germ layers is accompanied by an increase in the number of commonly methylated DNA regions and that these tissue-specific alterations are present for only a small number of genes. Our findings indicate that DNA methylation at the proximal promoter regions of commonly methylated genes act as an irreversible mark which fixes somatic lineage by repressing transcription of germ cell specific genes. Using the MeDIP on chip protocol, we immunoprecipitated methylated DNA from R1 ES cell, SK7 cell, as well as SK7 derived-Ectoderm, - Endoderm, -Paraxial mesoderm, brain, liver, skeletal muscle, and sperm, and hybridized to a genome tiling array. The three germ layer lineages from ES cells were confirmed by the expression of specific marker genes in each germ layer (see related expression analysis).

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RNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library in haploid human embryonic stem cells (hESCs) differentiated into three germ layers

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