Silencing of core transcription factors in human EC cells highlights the importance of autocrine FGF signaling for self-renewal.
BACKGROUND
Despite their distinct origins, human embryonic stem (hES) and embryonic carcinoma (hEC) cells share a number of similarities such as surface antigen expression, growth characteristics, the ability to either self-renew or differentiate, and control of the undifferentiated state by the same core transcription factors. To obtain further insights into the regulation of self-renewal, we have silenced hES/hEC cell-specific genes in NCCIT hEC cells and analysed the downstream effects by means of microarrays.


RESULTS
RNAi-mediated silencing of OCT4 and SOX2 induced differentiation with mesodermal characteristics. Markers of trophoblast induction were only transiently up-regulated in the OCT4 knock-down. Independent knock-downs of NANOG produced a proliferation rather than a differentiation phenotype, which may be due to high NANOG expression levels in the cell line used. Published ChIP-chip data from hES cells were used to identify putative direct targets. RNAi-mediated differentiation was accompanied by direct down-regulation of known hES/hEC cell markers. This included all three core transcription factors in the case of the OCT4 and SOX2 knock-downs, confirming previous findings of reciprocal activation in ES cells. Furthermore, large numbers of histone genes as well as epigenetic regulators were differentially expressed, pointing at chromatin remodeling as an additional regulatory level in the differentiation process. Moreover, loss of self-renewal was accompanied by the down-regulation of genes involved in FGF signaling. FGF receptor inhibition for short and prolonged periods of time revealed that the ERK/MAPK cascade is activated by endogenously expressed fibroblast growth factors and that FGF signaling is cruicial for maintaining the undifferentiated state of hEC cells, like in hES cells.


CONCLUSIONS
Control of self-renewal appears to be very similar in hEC and hES cells. This is supported by large numbers of common transcription factor targets and the requirement for autocrine FGF signaling.
Greber
Boris
B
Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Berlin, Germany. greber@molgen.mpg.de <greber@molgen.mpg.de>
Lehrach
Hans
H
Adjaye
James
J
D019898:Autocrine Communication / Q000235:genetics*; D015153:Blotting, Western; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics; D015536:Down-Regulation; D054278:Embryonal Carcinoma Stem Cells; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D020868:Gene Silencing; D024002:HMGB Proteins / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D000071317:Nanog Homeobox Protein; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors; D015398:Signal Transduction / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*
D004268:DNA-Binding Proteins; D024002:HMGB Proteins; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D034741:RNA, Small Interfering; D017468:Receptors, Fibroblast Growth Factor; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D014157:Transcription Factors