Characterization of a negative retinoic acid response element in the murine Oct4 promoter.
Expression of Oct4 in embryonic stem cells is controlled by a distal upstream stem cell-specific enhancer that is deactivated during retinoic acid (RA)-induced differentiation by an indirect mechanism not involving binding of RA receptors (H. Okazawa, K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, and H. Hamada, EMBO J. 10:2997-3005, 1991). Here we report that in RA-treated P19 embryonal carcinoma cells the Oct4 promoter is also subject to negative regulation by RA. The minimal Oct4 promoter sequence mediating repression consists of a promoter-proximal sequence containing a GC-rich SP1 consensus-like sequence and several hormone response element half-sites that can be arranged into direct repeats with different spacing. The GC box binds a nuclear factor that is invariably present in undifferentiated and RA-treated differentiated P19 cells. By contrast, the hormone response element-containing sequence binds factors that are induced following RA treatment. Mutational analysis and competition experiments show that the functional entity binding the RA-induced factor is a direct repeat sequence with a spacing of one nucleotide, previously shown to be a binding site for COUP transcription factors (COUP-TFs). Cotransfected orphan receptors COUP-TF1, ARP-1, and EAR-2 were able to repress the activity of Oct4 promoter-driven reporters in P19 EC cells, albeit with different efficiencies. Furthermore, the negative transcriptional effect of COUP-TFs is dominant over the activating effect of the Oct4 embryonic stem cell-specific enhancer. These results show that negative regulation of Oct4 expression during RA-induced differentiation of embryonic stem cells is controlled by two different mechanisms, including deactivation of the embryonic stem cell-specific enhancer and promoter silencing by orphan nuclear hormone receptors.
Schoorlemmer
J
J
Hubrecht Laboratorium, Netherlands Institute for Developmental Biology, Utrecht.
van Puijenbroek
A
A
van Den Eijnden
M
M
Jonk
L
L
Pals
C
C
Kruijer
W
W
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004622:Embryo, Mammalian; D005809:Genes, Regulator / Q000187:drug effects; D015698:Genomic Library; D051379:Mice; D008808:Mice, Inbred CBA; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D050814:Octamer Transcription Factor-3; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013154:Spleen / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D050814:Octamer Transcription Factor-3; D009838:Oligodeoxyribonucleotides; C494174:Pou5f1 protein, mouse; D014157:Transcription Factors; D014212:Tretinoin