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Transcriptome analysis of human embryonic stem cells (hESCs) upon siRNA-mediated knockdown of hESC-essential genes

ABSTRACT: The maintenance of pluripotency requires the coordinated expression of a set of essential genes. Thorough identification of these genes is vital for gaining a better understanding of the pluripotent state. To this end, we have recently generated a genome-wide loss-of-function library in haploid human pluripotent stem cells utilizing the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology using nearly 180,000 single guide RNAs (sgRNAs) and targeting 18,166 protein-coding genes. By combining the results of our high-throughput genetic screening with the analysis of publicly available RNAseq data of pluripotent stem cells and several somatic cells, we have determined the essential genes which have an enriched expression in hESCs and termed these genes as hESC-essential genes. Here, we have individually knocked down four of these hESC-essential genes with siRNAs, namely SALL4, DSCC1, SEPHS1 and VRTN, in human embryonic stem cells and analyzed the transcriptomes of the cells upon downregulation of these transcripts in order to unravel part of the pathways affected by hESC-essential genes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE103846 | GEO | 2018/04/30

REPOSITORIES: GEO

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Project description:Embryonic stem cells have potential utility in regenerative medicine due to their pluripotent characteristics. Sall4, a zinc-finger transcription factor, is expressed very early in embryonic development with Oct4 and Nanog, two well characterized pluripotency regulators. Sall4 plays an important role in governing the fate of stem cells through transcriptional regulation of both Oct4 and Nanog. Using chromatin immunoprecipitation coupled to microarray hybridization (ChIP on Chip), we have mapped global gene targets of Sall4 unveiling possible regulation of broad ES cell functions. Approximately 5,000 genes were identified that were bound by the Sall4 protein and many of these have major functions in developmental and regulatory pathways. Sall4 bound more than six times as many annotated genes within promoter regions as Oct4 and twice as many as Nanog. Immunoprecipitation revealed a heterotrimeric protein complex between Sall4, Oct4, and Nanog, consistent with binding site co-occupancies. Further, Sall4 bound many genes that are regulated in part by the chromatin-based epigenetic events mediated by polycomb-repressive complexes and bivalent domains. This suggests that Sall4 plays a central and diverse role in regulating stem cell pluripotency during early embryonic development that involves integration of transcriptional and epigenetic control processes. Keywords: ChIP-chip We used ChIP-chip to map global promoter bound by Sall4, a zinc finger transcription factor. Growing evidence has suggested that Sall4 plays a vital role in ES cell pluripotency maintenance. Evidence for this includes its recent use as a marker for somatic cell reprogramming, in a genetic signature for ES cells, and evidence that Sall4 enhances reprogramming. These recent findings and two previously described interactions with Oct4 and Nanog strongly support the role of Sall4 in ES cells. This hypothesis was furthered here as we show that Sall4 plays important roles through transcriptional regulation of vital ES cell genes.

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Transcriptome analysis of human embryonic stem cells (hESCs) upon siRNA-mediated knockdown of hESC-essential genes

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