Abstract
It is essential to understand the network of transcription factors controlling self-renewal of human embryonic stem cells (ESCs) and human embryonal carcinoma cells (ECs) if we are to exploit these cells in regenerative medicine regimes. Correlating gene expression levels after RNAi-based ablation of OCT4 function with its downstream targets enables a better prediction of motif-specific driven expression modules pertinent for self-renewal and differentiation of embryonic stem cells and induced pluripotent stem cells.We initially identified putative direct downstream targets of OCT4 by employing CHIP-on-chip analysis. A comparison of three peak analysis programs revealed a refined list of OCT4 targets in the human EC cell line NCCIT, this list was then compared to previously published OCT4 CHIP-on-chip datasets derived from both ES and EC cells. We have verified an enriched POU-motif, discovered by a de novo approach, thus enabling us to define six distinct modules of OCT4 binding and regulation of its target genes.A selection of these targets has been validated, like NANOG, which harbours the evolutionarily conserved OCT4-SOX2 binding motif within its proximal promoter. Other validated targets, which do not harbour the classical HMG motif are USP44 and GADD45G, a key regulator of the cell cycle. Over-expression of GADD45G in NCCIT cells resulted in an enrichment and up-regulation of genes associated with the cell cycle (CDKN1B, CDKN1C, CDK6 and MAPK4) and developmental processes (BMP4, HAND1, EOMES, ID2, GATA4, GATA5, ISL1 and MSX1). A comparison of positively regulated OCT4 targets common to EC and ES cells identified genes such as NANOG, PHC1, USP44, SOX2, PHF17 and OCT4, thus further confirming their universal role in maintaining self-renewal in both cell types. Finally we have created a user-friendly database (http://biit.cs.ut.ee/escd/), integrating all OCT4 and stem cell related datasets in both human and mouse ES and EC cells.In the current era of systems biology driven research, we envisage that our integrated embryonic stem cell database will prove beneficial to the booming field of ES, iPS and cancer research.
Highlights
Human embryonic stem cells, derived from the inner cell mass (ICM) of the blastocyst, have the ability to differentiate into all cell types and hold great potential for regenerative medicine and studying early development [1]
TAMALPAIS was used for the promoter analysis as it assumes that only a small fraction (,5%) of probes on an array harbours binding sites of transcription factors
As similar approaches have recently been adopted in constructing the HaemAtlas which serves as a reference library for gene expression in human blood cells and as a resource for identifying key genes with roles in blood cell function [41], we have developed a specialized database, which enables rapid and convenient access and comparisons between published datasets related to embryonic stem cell biology to help overcome this shortfall
Summary
Human embryonic stem cells (hESCs), derived from the inner cell mass (ICM) of the blastocyst, have the ability to differentiate into all cell types and hold great potential for regenerative medicine and studying early development [1]. The stem cell phenotype of hESCs cells has recently been shown to be maintained by a selfstabilizing network of transcription factors, NANOG, OCT4, and SOX2 [3]. These factors maintain their own and each other’s transcriptional level, through combinatorial interactions. They positively regulate genes responsible for the ES cell phenotype whilst repressing transcription of genes required for inducing differentiation
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