ABBS 2005,38(03): Second Intron of Mouse Nestin Gene Directs its Expression in Pluripotent Embryonic Carcinoma Cells through POU Factor Binding Site

Abstract        Nestin, an intermediate filament protein,
is expressed in the neural stem cells of the developing central nervous system.
This tissue-specific expression is driven by the neural stem cell-specific
enhancer in the second intron of the nestin gene. In this study, we showed that
the mouse nestin gene was expressed in pluripotent embryonic carcinoma (EC) P19
and F9 cells, not in the differentiated cell types. This cell type-specific
expression was conferred by the enhancer in the second intron. Mutation of the
conserved POU factor-binding site in the enhancer abolished the reporter gene
expression in EC cells. Oct4, a Class V POU factor, was found to be coexpressed
with nestin in EC cells. Electrophoretic mobility-shift assays and supershift
assays showed that a unique protein-DNA complex was formed specifically with
nuclear extracts of EC cells, and Oct4 protein was included. Together, these
results suggest the functional relevance between the conserved POU
factor-binding site and the expression of the nestin gene in pluripotent EC
cells.

Key words        nestin; embryonic carcinoma cells;
enhancer; POU factor

nestin, an
intermediate filament protein, is expressed in the neural stem cells (NSCs) of
the developing central nervous system (CNS), and is used as an NSC marker [1–4]. In situ hybridization results showed that nestin mRNA was
found predominantly in the neuroepithelial and radial glia cells of the neural
tube of mouse embryos at embryonic day 10.5 [4]. During CNS development, NSCs
differentiate into mature neurons and glial cells, concomitantly nestin
expression is downregulated and replaced by neurofilament and glial fibrillary
acidic protein [1,4]. Nestin expression shows a sharp decline in the motor
neurons of the spinal cord and marginal layer neurons of the telencephalon when
NSCs become post-mitotic young neurons [4].

The
nestin gene is well conserved in its genomic structure in human, rat and mouse,
and it contains four exons and three introns [2,3,5]. Transgenic mice showed
that the rat nestin gene had two tissue-specific enhancers in the first and
second introns, which could drive nestin gene expression in muscle progenitor
cells and NSCs, respectively [6]. The NSC-specific enhancer resides in the 3‘
region of the second intron, and a cis-element for the POU transcription
factors is responsible for nestin expression in the developing CNS [7–9]. It has been shown that group B1/C Sox and Class III POU factors
might interact synergistically to determine nestin expression in neural
primordial cells [10]. We previously described the cloning of the mouse nestin
gene, and found that nestin protein existed in the developing eye, the growth
cones of P19 neurons and the mouse cerebellar granule cells [11–13]. We further characterized the promoter of the mouse nestin gene,
and showed that transcription factors Sp1 and Sp3 were involved in the
regulation of nestin expression [14].

The
onset of nestin expression occurred in the neural plate of mouse embryos at
approximately embryonic day 7.5 [4,7]. Using
more sensitive methods, the expression of nestin was detected in embryonic day
7.0 mouse embryos [15,16]. Recently, however, weak expression of nestin was
found in the inner cell mass of mouse blastocysts as well as in human and mouse
embryonic stem (ES) cells, and the expression was upregulated upon
differentiation from ES cells into NSCs [17,18]. We previously reported nestin
expression in undifferentiated mouse embryonic carcinoma (EC) P19 cells and
retinoic acid-induced P19 NSCs at both mRNA and protein levels [13,19]. In
contrast to the well-known mechanisms of expression regulation of the nestin
gene in NSCs, little is known of the molecular basis of earlier nestin
expression in pluripotent stem cells. In this study, we showed the nestin gene
expression in pluripotent EC cells, and found that the POU factor binding site
in the second intron of the mouse nestin gene was probably involved in this
cell type-specific gene expression.

Materials and Methods

The luciferase reporter constructs
pNH92 and pNH94 were generated in our laboratory previously using the vector
pGL3-Px’ which contained the SV40 promoter [5]. We also prepared the luciferase
construct, in which the mini-enhancer was placed in front of the nestin
endogenous promoter (pNH142). To prepare enhanced green fluorescent protein
(EGFP) reporter constructs, the vector ptkEGFP (kind gift from Dr. H. Kondoh, Cell
lines, including mouse embryonic carcinoma P19, F9, mouse fibroblast NIH3T3, Chinese
hamster ovary CHO, African green monkey kidney Cos7 and human neuroblastoma
SH-SY5Y, were maintained in DMEM/F12 (1:1) medium (Gibco, Carlsbad, USA)
supplemented with 10% fetal bovine serum (HyClone, Logan, USA) at 37 ºC in 5% CO2. Cells were seeded in
24-well plates for 24 h, then 0.4 mg of firefly
luciferase reporter constructs and 0.1 mg of pRL-TK plasmid (Promega, Madison, USA) were cotransfected into
cells with Fugene6 reagent
(Roche, Basel, Switzerland) according to the manufacturer’s protocol. Forty
hours later, cells were washed with phosphate-buffered saline and lysed with passive lysis buffer (Promega). The
supernatant was analyzed for luciferase reporter activity on a 20/20n luminometer
(Turner Biosystems, Total
RNA was extracted from cells using Trizol reagent (Invitrogen, Nuclear
extracts were prepared from cells as described previously [14]. Nuclear
proteins (3–6 mg) were
preincubated with 2 mg poly(dG-dC)·poly(dG-dC)
(Amersham, Uppsala, Sweden) on ice for 30 min in 5´binding buffer
containing 50% glycerol,  

Results and Discussion

To
survey nestin gene expression in different cell lines, we analyzed nestin
transcript with RT-PCR. As shown in Fig. 1(A), nestin mRNA could be
detected in mouse EC P19 and F9 cells, though the expression level is
relatively low in F9 cells. However, the expression of the nestin gene was even
low or undetectable in differentiated cell lines, such as mouse fibroblast
NIH3T3 cells, Chinese hamster ovary CHO cells, human neuroblastoma SH-SY5Y
cells and The POU
factor binding site in the second intron of the nestin gene has been shown to
be critical for its expression in NSCs of developing CNS [8]. To examine
whether this binding site is also essential to nestin expression in EC cells,
we transfected the EGFP reporter gene under the control of the mini-enhancer of
the second intron (pNH161) and its POU site-mutated counterpart (pNH166) into
P19 cells. As shown in Fig. 2(A), this mini-enhancer could induce
reporter gene expression in P19 cells, but the expression of EGFP was severely
reduced in the cells transfected with the mutant construct. To further validate
the importance of this POU factor binding site in EC cells, we transfected, in
different cell lines, the mini-enhancer construct (pNH94) and POU site-mutated
construct (pNH135) linked with the luciferase reporter gene. It was found that
the mutation of this binding site caused a sharp reduction of luciferase
activity in P19 and F9 EC cells, but it did not induce significant changes in
other cell lines [Fig. 2(B)]. Taken together, these results show that
the POU factor binding site is required for the enhancer activity in
pluripotent EC cells.

To
search for the possible trans-factors binding to the POU site, we
examined the expression patterns of members of the POU transcription factor
family by RT-PCR. Oct1, the Class II POU factor, was expressed in each cell
line, and showed no cell type specificity. In contrast, Oct4, the Class V POU
factor, was only expressed in the EC cells [Fig. 3(A)]. This is
consistent with the notion that Oct4 is a very important transcription factor
for the self-renewal and pluripotency of ES cells [20–22]. To test whether there are nuclear proteins actually binding to
the POU factor binding site, we used nuclear extracts from different cells and
performed electrophoretic mobility-shift assay with a 32P-labeled
oligonucleotide probe containing the POU site. As shown in Fig. 3(B), a
low mobility band (band 1) could be detected in SY5Y and CHO cells as well as
F9 and P19 cells, and a fast migrating band (band 2) existed only in F9 and P19
cells. The protein-binding specificity of the POU site was confirmed because
these two bands (band 1 and band 2) were effectively competed by a non-radiolabeled
probe, but not by an irrelevant competitor, probe SIE [Fig. 3(C)].

To
identify the proteins binding to the POU site, supershift assays were
performed. Results showed that band 1 was supershifted by antibody against
Oct1, and band 2 was specifically diminished by antibody against Oct4 [Fig.
3(C)]. Thus, POU factors Oct1 and Oct4 did bind to the POU site in P19 EC
cells. Interestingly, the Sox2 gene was expressed in pluripotent ES
cells and a core enhancer in the 5‘ flanking region activated transcription
in ES cells depending on POU factor binding sites [23,24]. In the Sox2 5‘
enhancer, two bands of protein-DNA complexes were formed between the POU
binding sites and nuclear extracts from ES cells, in which the fast migrating
band was the complex with Oct4 protein and the slow band was with Oct1 [23].
The binding of Oct4 to POU sites in the Sox2 5‘ enhancer might impart
its specific expression in ES cells. Based on these observations, we speculate
that Oct4 and Oct1 proteins might directly bind to the POU factor binding site
and regulate nestin expression in EC cells.

In
summary, we showed that the nestin gene was expressed specifically in
pluripotent EC cells, and that this cell type-specific expression was regulated
by the NSC-specific enhancer in the second intron of the mouse nestin gene. The
conserved POU factor binding site in this enhancer mediated nestin gene
expression in EC cells. These results shed light on the molecular mechanisms of
regulation of nestin gene expression in pluripotent stem cells.

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