Comparative analysis of human, bovine, and murine Oct-4 upstream
* Karin Hu¨bner,
Hans R. Scho¨ler
The Center for Animal Transgenesis and Germ Cell Research, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, 382
West Street Road, Kennett Square, Pennsylvania 19348, USA
EMBL, Gene Expression Programme, Meyerhofstrasse 1, D-69126, Heidelberg, Germany
Received: 12 September 2000 / Accepted: 5 December 2000
Abstract. The Oct-4 gene encodes a transcription factor that is
specifically expressed in embryonic stem cells and germ cells of
the mouse embryo. Cells that differentiate into somatic tissues lose
Oct-4 expression. Regulation of Oct-4 gene transcription involves
a TATA-less minimal promoter and two upstream elements: the
proximal (PE) and distal enhancers (DE). We report here the
nucleotide sequence of the 5Ј upstream regulatory regions of the
human and murine Oct-4 genes. A comparative alignment analysis
between these regions and those of the bovine Oct-4 ortholog
reveals four conserved regions of homology (CR 1 to 4) between
these species (66–94% conservation). The 1A sequence within the
mouse PE is located approximately half-way between CR 2 and
CR 3. A putative Sp1/Sp3 binding site and the overlapping hor-
mone responsive element (HRE) in CR 1 are identical in all three
species. A high number of CCC(A/T)CCC motifs exhibit various
levels of homology in these upstream regions. We discuss the
importance of these and other sequences and present candidate
factors that may bind and regulate Oct-4 gene expression.
The Oct-4 gene encodes a transcription factor that is expressed in
embryonic stem cells and germ cells of the mouse embryo. Oct-4
is crucial for the maintenance of embryonic cell totipotency during
early developmental stages as demonstrated recently by homolo-
gous recombination (Nichols et al. 1998; Niwa et al. 2000). The
Oct-4 transcription factor is postulated to function as a transcrip-
tional activator of genes required in maintaining an undifferenti-
ated totipotent state and may prevent the expression of genes that
are activated during stem cell differentiation (Pesce et al., 1998,
1999; Pesce and Scho¨ler 2000; Tomilin et al. 2000).
As a member of the POU (Pit, Oct, Unc) transcription factor
family, Oct-4 contains a bipartite DNA binding domain, the POU
domain (Scho¨ler 1991; Herr and Cleary 1995), with which it binds
to an octamer sequence motif present in the promoter and enhancer
of candidate genes, thereby regulating their expression. Oct-4, also
designated Oct-3, or POU5F1, is a maternally inherited transcript
that is developmentally regulated in mice. It is expressed at low
levels in all blastomeres until the four-cell stage (Palmieri et al.
1994), at which time it undergoes zygotic activation (Yeom et al.
1991) resulting in high Oct-4 protein levels in the nuclei of all
blastomeres until the compacted morula stage (Palmieri et al.
1994). After cavitation, Oct-4 is maintained only in cells of the
inner cell mass (ICM) in the blastocyst and is downregulated in
outer cells of the morula that have differentiated into trophecto-
derm (Okamoto et al. 1990; Rosner et al. 1990; Scho¨ler et al.
1990b). Following implantation, Oct-4 expression is restricted to
the primitive ectoderm (epiblast) although it is transiently ex-
pressed at high levels in cells of the hypoblast (primitive endo-
derm) before they differentiate into parietal/visceral endoderms,
which do not express Oct-4 (Scho¨ler 1991; Palmieri et al. 1994).
During gastrulation, Oct-4 expression becomes downregulated in
the epiblast in an anterior-posterior manner (Yeom et al. 1996).
Following gastrulation from 8.5 dpc on, Oct-4 expression becomes
restricted to the germline as evidenced by strict Oct-4 confinement
to primordial germ cells (PGCs), precursors of the gametes (Yeom
et al. 1996). Embryonic germ (EG) cells derived from PGC pre-
cursors (Labosky et al. 1994) express Oct-4 (Yeom et al. 1996).
Oct-4 is also expressed in undifferentiated embryonic stem (ES)
and embryonic carcinoma (EC) cell lines, derived from the ICM
and epiblast, respectively, and is not expressed in somatic lineages.
Differentiation of ES and EC cells with retinoic acid (RA) or other
differentiating agents results in rapid Oct-4 downregulation (Scho¨-
ler et al. 1990a, 1990b; Minucci et al. 1996).
The expression pattern is reflected by a complex arrangement
of regulatory elements. Oct-4 gene expression is driven by a
TATA-less minimal promoter (Okazawa et al. 1991) and at least
two enhancer elements (Yeom et al. 1996). The Oct-4 minimal
promoter is located within the first 250 base pairs of the transcrip-
tion initiation sites and contains a cluster of overlapping binding
sites (Pikarsky et al. 1994; Schoorlemmer et al. 1994; Sylvester
and Scho¨ler 1994). Among these is a GC box representing a pu-
tative high-affinity binding site for Sp1 and Sp3 transcription fac-
tors that mediates Oct-4 minimal promoter activity (Sylvester and
Scho¨ler 1994; Pesce et al., 1999). This site partially overlaps with
the hormone-responsive element (HRE) which resembles the ca-
nonical RA responsive element (RARE; Sylvester and Scho¨ler
1994). The HRE is arranged in three direct repeats (R1, R2, R3) of
an AGGTCA-like sequence with R1 and R2 separated by one base
pair (DR1) and R2 and R3 directly adjacent to each other (DR0)
(Ovitt and Scho¨ler 1998; Fuhrman et al. 1999). HRE is recognized
by a number of receptors belonging to the steroid-thyroid receptor
family including RA and retinoid X-receptors and to the orphan
nuclear receptor superfamily (specifically COUP-TFI, ARP-1, and
EAR-2). All these factors specifically bind to DR1 and are be-
lieved to be important in Oct-4 downregulation (Okazawa et al.
1991; Pikarsky et al. 1994; Schoorlemmer et al. 1994; Sylvester
and Scho¨ler 1994; Ben-Shushan et al. 1995; Barnea and Bergman
The use of Oct-4/Lac Z transgenes has allowed the identifica-
tion of two distinct enhancer elements that reciprocally drive the
cell-specific expression of Oct-4 (Yeom et al. 1996). Located in a
* Present address: Institute of Pharmacology and Toxicology, University
of Mu¨nster, Domagkstr. 12, 48149 Mu¨nster, Germany
Correspondence to: H.R. Scho¨ler; E-mail: firstname.lastname@example.org
The human and mouse nucleotide sequence data reported in this paper have
been submitted to EMBL Genbank and have been assigned accession num-
bers AJ297527 and AJ297528, respectively.
Mammalian Genome 12, 309–317 (2001).
© Springer-Verlag New York Inc. 2001