The stem cell-specific factor Oct-4 is expressed in undifferentiated e
mbryonal carcinoma and embryonic stem cells and is quickly downregulat
ed upon RA-induced differentiation. Irrespective of the direction of d
ifferentiation, Oct-4 repression in P19 EC cells requires treatment wi
th high doses of either all-trans or 9-cis RA. Unlike in P19 cells, no
RA-induced downregulation of Oct-4 expression is observed in the P19-
derived RA-resistant RAC65 cells. However, in these cells Oct-4 promot
er repression can be rescued in a RA-dependent manner by cotransfectio
n of RAR alpha 2 or RAR beta 2 but not RARr gamma 1, matching previous
ly reported transactivation properties of these receptor types. In the
vicinity of the transcription initiation site of the Oct-4 gene, thre
e Hormone Response Element (HRE) half sites are present which are arra
nged as direct repeats with different spacing. In vitro translated RAR
and RXR proteins bind to this HRE as heterodimers with low affinity,
in such a way that all three I-IRE half sites contribute to complex fo
rmation. Although P19 EC cells contain weak binding activity interacti
ng with the Oct-4 promoter HRE, strong binding activity is observed in
nuclear extracts from RA-treated P19 cells. This binding activity was
shown to correspond to COUP-TFs but not nuclear RA receptors. Moreove
r, the presence of these binding factors in nuclear extracts correspon
ds to silencing of Oct-4 expression. These results implicate RA and th
e action of its nuclear receptors in silencing Oct-4 expression upon d
ifferentiation of EC cells. The observed silencing is most likely not
exerted by direct binding of RARs to the Oct-4 proximal promoter HRE.
Our results support models in which different nuclear receptor complex
es sequentially occupy different sites in the Oct-4 promoter HRE to si
lence Oct-4 expression during RA-induced differentiation.