Germ cell nuclear factor is a repressor of CRIPTO-1 and CRIPTO-3.
The pluripotency of embryonic stem and embryonic carcinoma cells is maintained by the expression of a set of "stemness" genes. Whereas these genes are down-regulated upon induction of differentiation, the germ cell nuclear factor (GCNF) is transiently up-regulated and represses several pluripotency genes. CRIPTO-1, a co-receptor for the morphogen nodal, is strongly expressed in undifferentiated cells and is rapidly down-regulated during retinoic acid-induced differentiation. Although CRIPTO-1 is expressed at very low levels in adult tissues under normal conditions, it is found highly expressed in a broad range of tumors, where it acts as a potent oncogene. We show that expression of CRIPTO-1 is directly repressed by GCNF during differentiation of the human teratocarcinoma cell line, NT2. GCNF bound to a DR0 element of the CRIPTO-1 promoter in vitro, as shown by electrophoretic mobility shift assays, and in vivo, as demonstrated by chromatin immunoprecipitation. Reporter gene assays demonstrated that GCNF-mediated repression of the CRIPTO-1 promoter is dependent upon the DR0 site. Overexpression of GCNF in NT2 cells resulted in repression of CRIPTO-1 transcription, whereas expression of the transcription-activating fusion construct GCNF-VP16 led to an induction of the CRIPTO-1 gene and prevented its retinoic acid-induced down-regulation. Furthermore, we demonstrated that CRIPTO-3, a processed pseudogene of CRIPTO-1 on the X chromosome, is expressed in undifferentiated NT2 cells and is regulated by GCNF in parallel to CRIPTO-1. Thus, our study supports the hypothesis of GCNF playing a central role during differentiation of stem cells by repression of stem cell-specific genes.
Hentschke
Moritz
M
Institute of Medical Microbiology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. m.hentschke@uke.uni-hamburg.de
Kurth
Ingo
I
Borgmeyer
Uwe
U
Hübner
Christian A
CA
D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D058851:GPI-Linked Proteins; D005786:Gene Expression Regulation; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D057136:Nuclear Receptor Subfamily 6, Group A, Member 1; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011544:Pseudogenes / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014212:Tretinoin
D004268:DNA-Binding Proteins; D058851:GPI-Linked Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C539950:NR6A1 protein, human; D009363:Neoplasm Proteins; D057136:Nuclear Receptor Subfamily 6, Group A, Member 1; D018160:Receptors, Cytoplasmic and Nuclear; C070636:TDGF1 protein, human; D014212:Tretinoin; D004815:Epidermal Growth Factor