Oncogene (1997) 15, 1123 ± 1131
 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
MAZ, a Myc-associated zinc ®nger protein, is essential for the ME1a1mediated expression of the c-myc gene during neuroectodermal
di€erentiation of P19 cells
Masaaki Komatsu1,3, Hai-Ou Li1, Hatsumi Tsutsui1, Keiichi Itakura2, Masatoshi Matsumura3,
and Kazunari K Yokoyama1
1
Tsukuba Life Science Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan; 2Department of Molecular Genetics,
Beckman Research Institute of the City of Hope, 1450 East Duarte, Duarte, California 91010, USA; 3Institute of Applied
Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan
To investigate whether MAZ (Myc-associated zinc ®nger
protein) a€ects the expression of the c-myc gene during
the retinoic acid-induced (RA-induced) neuroectodermal
di€erentiation of P19 embryonal carcinoma (EC) cells,
we introduced a CAT reporter construct, human c-myc
promoter/CAT (pMyc2CAT), and a mutant CAT
derivative that lacked an ME1a1 site (pMyc1CAT) into
P19EC cells to monitor the promoter activity of the cmyc gene. The expression of CAT in pMyc2CATtransformed cells declined ®vefold after 24 h in the
presence of RA, returned to the normal level within 48 h,
and decreased again to below 20% of the normal level
after 96 h. By contrast, the expression of CAT in
pMyc1CAT-transformed cells did not return to the
normal level after 48 h in the presence of RA. In
addition, an electrophoretic mobility shift assay (EMSA)
with ME1a1 DNA as probe demonstrated that the
kinetics of the DNA-binding activity of MAZ were
closely correlated with the changes in the expression of
CAT from the c-myc promoter/CAT gene during the
di€erentiation of P19EC cells. Taken together, these
results suggest that MAZ plays a key role in the
transient increase in the expression of the c-myc gene
after 48 h of exposure to RA during the neuroectodermal
di€erentiation of P19EC cells.
Keywords: MAZ; c-myc; ME1a1 site; gene expression
Introduction
The c-myc proto-oncogene encodes a nuclear phosphoprotein, namely a DNA sequence-speci®c transcriptional factor, which forms dimers with a related
polypeptide, Max, to optimize the binding to DNA
(Cole, 1986; Spencer and Groudine, 1991; Blackwood
and Eisenman, 1991; Marcu et al., 1992). Appropriate
regulation of expression of c-myc is necessary for
normal cellular proliferation, di€erentiation and
progression of the cell cycle, and deregulation of the
expression of c-myc is associated with tumorigenesis
and apoptosis (Cole, 1986; Marcu et al., 1992).
Regulation of c-myc occurs at multiple levels, which
include the initiation of transcription, the termination
of transcription, and the attenuation of transcription
Correspondence: K Yokoyama
Received 3 February 1997; revised 14 May 1997; accepted
14 May 1997
(Spencer and Groudine, 1991; Marcu et al., 1992). In
proliferating cells, the initiation of transcription of the
c-myc gene is regulated by P1 and P2 promoters and
the RNA initiated from the P2 promoter accounts for
80 ± 90% of the total RNA initiated from the P0, P1
and P2 promoters (Marcu et al., 1992; DesJardins and
Hey, 1993). Optimal initiation of transcription from
the P2 promoter requires the ME1a2, E2F and ME1a1
elements in the P2 promoter (Moberg et al., 1991,
1992). Several transcriptional factors, including Sp1
(DesJardins and Hey, 1993; Ash®eld et al., 1994), and
the zinc-®nger protein ZF87/MAZ (a zinc-®nger
protein of 87 kDa; Pyrc et al., 1992; Bossone et al.,
1992; DesJardins and Hey, 1993) or a mouse homolog
Pur-1 (purine-binding factor; Kennedy and Rutter,
1992) and E2F (Kovesdi et al., 1986) bind to these
elements in vitro and in vivo. The Myc-associated zinc
®nger protein (MAZ) was identi®ed as a transcriptional
factor that bind to a GA box (GGGAGGG) at the
ME1a1 site, to the attenuator region of P2 within the
®rst exon of the c-myc gene, and to a related sequence
that is involved in the termination of transcription of
the gene for complement 2 (C2) (DesJardins and Hey,
1993; Bossone et al., 1992). Kennedy and Rutter (1992)
found that the Pur-1 protein bound to the GAGA
boxes of rat genes for insulin I and II and of the
human gene for islet amyloid polypeptide. Recently, we
reported isolation of a cDNA clone for a member of
the family of MAZ proteins in human islet cells that
had a similar DNA-binding speci®city (Tsutsui et al.,
1996). A MAZ plays a role in the control of the
initiation of transcription of genes for CD4 (Duncan et
al., 1995) and serotonin (Parks and Shenk, 1996) and
in the termination of transcription between closely
spaced human genes for complement (Ash®eld et al.,
1994) and also between the introns of the mouse gene
for IgM-D (Ash®eld et al., 1994). Therefore, MAZ
appears to be a transcriptional factor with a dual role
in the initiation and termination of transcription.
In this study we examined the role of MAZ protein
in the expression of the c-myc gene during the
neuroectodermal di€erentiation of mouse P19 embryonal carcinoma (EC) cells. P19 cells can be induced to
di€erentiate into neurons, astrocytes and ®broblasts by
exposure to retinoic acid (RA). By contrast, P19 cells
treated with dimethyl sulfoxide (DMSO) di€erentiate
into cardiac and skeletal muscle cells (Jones-Villeneuve
et al., 1982; McBurney et al., 1982; Edwards et al.,
1983). In the presence of RA, transcription of the cmyc gene during the di€erentiation of P19 cells
MAZ regulates the expression of c-myc
M Komatsu et al
1124
increases transiently and biphasically: ®rst after 3 h
and then from 48 h to 96 h. However, the latter
increase does not occur in the case of induction with
DMSO (St-Arnaud et al., 1988). The ®rst peak in the
level of c-myc mRNA at 3 h is associated with an
increase in the stability of the mRNA without any
change in either the extent of transcription or the
utilization of promoters, as assessed by nuclear run-on
assays (St-Arnaud et al., 1988). Thus, the increase in
the stability of c-myc mRNA appears to be responsible
for this early increase in the level of c-myc mRNA. By
contrast, the stability of c-myc mRNA at 48 and 96 h
is similar to that in untreated cells. However, a ®vefold
increase has been demonstrated in the rate of
transcription of exons 1, 2 and 3 of the c-myc gene
after 48 h of treatment with RA (St-Arnaud et al.,
1988). Thus, the second peak of expression of c-myc
mRNA seems to be due to the increased rate of
transcription of the c-myc gene. However, the
transcriptional factors required for the regulation of
the rate of transcription of the c-myc gene have not yet
been identi®ed.
In the present study, we found that at least the
ME1a1 element in the P2 promoter of the c-myc gene
is necessary for the second increase in the expression of
c-myc mRNA that occurs during the neuroectodermal
di€erentiation of P19 cells and, moreover, that the
MAZ protein, bound to the ME1a1 element, seems to
be associated with this aspect of the regulation of
transcription of the c-myc gene.
Results
The ME1a1 element is essential for the transcription of
the c-myc gene during RA-induced di€erentiation
To determine whether the ME1a1 element in the P2
promoter a€ects the expression of the c-myc gene
during the di€erentiation of P19 cells, we cotransfected
P19 cells with a DNA fragment that consisted of the 5'region of the c-myc gene fused to a CAT reporter gene
(Figure 1a) or to a mutant CAT derivative that lacked
the ME1a1 site, together with pSV2neo. After selection
with G418 for 3 weeks, we obtained several hundred
stable transformed clones. Representative transformed
clones such as pMyc1 and pMyc2, respectively, were
treated with RA at 1076 M for di€erent lengths of time,
and then the level of expression of the CAT reporter
gene was measured (Figure 1b). The expression of
pMyc2CAT fell ®vefold within 24 h upon exposure of
cells to RA; it returned to the normal level within 48 h
and it decreased again to below 20% of the normal
level after 96 h (Figure 1b, lanes 9 ± 16). The expression
of pMyc1CAT did not return to the normal level
within 48 h in the presence of RA (Figure 1b, lanes 1 ±
8). We did not detect an initial increase in CAT activity
1 ± 6 h after the start of treatment with RA (Figure 1b,
lanes 10 and 11). These results indicate that the ME1a1
element is necessary for the transient increase in
transcription of the c-myc gene that occurs 48 h after
the addition of RA during the neuroectodermal
di€erentiation of P19 cells.
We next analysed the expression of the c-myc gene
during the RA-induced di€erentiation of P19 cells by
Northern blotting (Figure 1c). As expected, the level of
the transcript of the c-myc gene was depressed 7 ± 8-fold
at 18 to 24 h, it returned to the initial level at 48 h and it
decreased again 5 ± 6-fold at 96 h in the presence of RA,
as compared to the level of c-myc mRNA in undifferentiated P19 cells (Figure 1c, lanes 1 ± 5). By contrast, the
level of expression of the gene for b-actin remained
unchanged (Figure 1c, lanes 6 ± 10).
We also examined the e€ect of the MAZ protein on
the promoter activity of the c-myc gene by transforming cells with a c-myc promoter/CAT reporter
construct, pMyc2CAT; a mutant variant construct,
pMyc1CAT; and pICAT, as the control plasmid
(Kitabayashi et al., 1991). As shown in Figure 1d,
the CAT activity of cells transformed with the c-myc
promoter/CAT construct with the ME1a1 site was
increased 2 ± 3-fold by MAZ protein (lanes 6 and 7),
while the CAT activity of cells transformed with the cmyc promoter/CAT construct without the ME1a1 site
was not (lanes 4 and 5). These results indicate that the
MAZ protein can a€ect the ME1a1 site-mediated
regulation of expression of the c-myc gene in P19 cells.
No changes were detected in the expression of MAZ
mRNA and protein during RA-induced di€erentiation
As shown in Figure 1, the ME1a1 element in the P2
promoter of the c-myc gene was essential for the
second increase in the level of expression of the c-myc
gene during the di€erentiation of P19 cells. To
determine whether the expression of c-myc is regulated
by MAZ, we prepared total RNAs from P19 cells that
had been treated with RA at 1076 M for di€erent
lengths of time and analysed it by Northern blotting
with MAZ cDNA as probe (Figure 2a). Transcripts of
the gene for MAZ of approximately 1.6, 2.6 and 4.5 kb
were detected and the levels of these mRNAs did not
change during the di€erentiation of P19 cells (Figure
2a, lanes 1 ± 8). Furthermore, to examine the changes in
expression of MAZ protein during the di€erentiation,
we prepared lysates of P19 cells that had been treated
with RA at 1076 M for di€erent lengths of time, and
analysed the proteins by Western blotting with
antibodies speci®c for MAZ (Figure 2c). Two major
proteins of 58 ± 60 kD and 38 kD were detected with
antibodies against MAZ protein. The 38 kDa protein
appeared to be generated from the internal methionine
codon that starts at nt 857 in the coding region, as
described previously (Tsutsui et al., 1996). No changes
in the levels of these proteins were seen during the
di€erentiation of P19 cells (Figure 2c, lanes 2 ± 5).
Moreover, the preimmune serum did not generate any
bands of proteins on Western blots (Figure 2c, lanes
6 ± 9).
MAZ binds to the ME1a1 element
We showed that the ME1a1 element in the promoter of
the c-myc gene was necessary for the second increase in
level of the transcript of the c-myc gene during the
neuroectodermal di€erentiation of P19 cells (Figure
1b). By contrast, the levels of both the MAZ protein
and MAZ mRNA did not change to any signi®cant
extent (Figure 2a and c). Therefore, we next examine
the DNA-binding activity of MAZ to the ME1a1
element of the c-myc gene. Nuclear extracts of
undi€erentiated P19 cells were prepared and analysed
MAZ regulates the expression of c-myc
M Komatsu et al
ME1a1 element (lanes 3 and 4). However, the shifted
bands were unchanged after addition of the mutant
oligodeoxynucleotide as competitor (lanes 6 and 7).
Unrelated antibodies, raised against c-jun/AP-1, did
not alter the mobility of the shifted bands of DNAprotein complexes (lane 8). It has been reported that
in an electrophoretic mobility shift assay (EMSA) with
the double-stranded ME1a1 sequence as probe (Figure
3a). As shown in lane 2 of Figure 3b, three shifted
bands were detected. The three bands disappeared after
the addition of the unlabeled competitor oligodeoxynucleotide that corresponded to the sequence of the
a
b
Lane 1
2
3
4
5
6
7
8
Conv.(%) 2.5 3.8 2.1 1.0 0.8 0.8 0.7 0.5
+RA – 1 6 12 24 48 72 96
9
10
11 12
13 14 15
5.2 4.0 3.2 2.4 1.2 4.4 2.8 0.8
– 1 6 12 24 48 72 96
16
(h)
c
RA
–
18
24
48
96 (h)
d
Lane
Lane
1
2
6
7
3
8
4
9
–
+
+
pCMV-MAZ
+
5
10
18S
28S
Lane 11
12
13
14
Lane 1
Conv.(%) 1.0
2
1.6
3
2.0
4
5
6
1.8 2.1 6.3
7
8
4.5 0.6
9
0.8
15
Figure 1 The ME1a1 element was necessary for the transient second increase in expression of the c-myc gene during RA-induced
di€erentiation of P19 cells. (a) Outline of the c-myc promoter-CAT reporter constructs. The numbers indicate the positions of the
DNA fragments relative to the P1 promoter. ME1a2, E2F, ME1a1 and TATA boxes are indicated. A large X represents the
deletion of the ME1a1 element. (b) CAT activity in P19 cells that had been stably transfected with the CAT reporter constructs
during RA-induced di€erentiation. P19 cells were transfected with the CAT reporter (10 mg) and pSV2neo (2 mg). After selection
with G418 for 3 weeks, stably transformed cells (pMyc1 and pMyc2 clones, respectively) were treated with RA at 1076 M for
di€erent lengths of time and assayed for their CAT activities. The percent conversion of chloramphenicol to its acetylated form and
the duration of incubation with RA are indicated. (c) Expression of the c-myc gene in undi€erentiated and di€erentiated P19 cells.
Total RNA (10 mg) from cells treated with RA at 161076 M for di€erent lengths of time, as indicated, was transferred after
electrophoresis to a nylon membrane and allowed to hybridize to a fragment of c-myc cDNA or of b-actin cDNA. Lanes 1 ± 5,
mRNA transcribed from the c-myc gene (arrowhead); lanes 6 ± 10, mRNA for b-actin (arrowhead). To demonstrate loading of
equivalent samples, bands of 28S and 18S ribosomal RNA are shown for all Northern analyses (lanes 11 ± 15). Lanes 1, 6 and 11,
control P19 cells; lanes 2, 7 and 12, P19 cells after 18 h of treatment with RA; lanes 3, 8 and 13, P19 cells after 24 h of treatment
with RA; lanes 4, 9 and 14, P19 cells after 48 h of treatment with RA; lanes 5, 10 and 15, P19 cells after 96 h of treatment with RA.
(d) Enhancement of the expression of the c-myc promoter/CAT reporter gene by MAZ protein. The percent conversion of
chloramphenicol to its acetylated form is indicated. Five mg of pICAT (lanes 1, 8 and 9), pMyc1CAT (lanes 2, 4 and 5) or
pMyc2CAT (lanes 3, 6 and 7), 2 mg of pSV2neo and 3 mg of pCH110 were used to transfect P19 cells together with (lanes 4 ± 9) or
without (lanes 1 ± 3) 5 mg of the pCMV-MAZ expression vector. After selection with G418 for 3 weeks, stably transformed
independent clones were isolated. Lysates were prepared and analysed for CAT activity as described in Materials and methods
1125
MAZ regulates the expression of c-myc
M Komatsu et al
1126
the ME1a1 element is recognized by MAZ and by the
transcriptional factor Sp1 (DesJardins and Hey, 1993;
Ash®eld et al., 1994). In an attempt to characterize the
proteins that bound to the ME1a1 element, we added
monoclonal antibodies against MAZ and polyclonal
antibodies against Sp1 to reaction mixtures for the
EMSA. The monoclonal antibodies against MAZ
shifted the retarded band still further (Figure 3b, lane
5). However, the antibodies against Sp1 did not a€ect
the mobility of the DNA-protein complexes (Figure 3b,
lane 8; Figure 3c, lane 4). Moreover, we used puri®ed
a
Lane
1 2 3 4 5 6 7 8
4.5 kb
— 2.6 kb
1.3 kb
+RA
Sp1 protein from Hela cells for an EMSA with the
double-stranded ME1a1 as probe and no retarded
bands were detected on the gel (Figure 3c, lane 5).
To investigate the binding of the MAZ protein to
the ME1a1 element during the neuroectodermal
di€erentiation of P19 cells, we prepared nuclear
extracts of P19 cells that had been treated with RA
at 1076 M for di€erent lengths of time. We incubated
the extracts with a 32P-labeled double-stranded
oligodeoxynucleotide probe that corresponded to the
ME1a1 site and analysed the mixture by EMSA
(Figure 4a). The anity of the MAZ protein for the
ME1a1 site declined after 24 h in the presence of RA,
as compared to the binding anity of MAZ from
undi€erentiated P19 cells (compared complex C in
lanes 2 and 3). The level of the DNA-protein complex
that corresponded to band C returned to the normal
level after 48 h (lane 4), and it fell again from 72 h to
96 h (lanes 5 and 6). We determined the relative
– 1 6 12 24 48 72 96 (h)
a
b
Lane
1 2 3 4 5 6 7 8
b
+RA
A
B
– 1 6 12 24 48 72 96 (h)
C
c
Anti-MAZ
Preimmune serum
F
Lane 1 2 3 4 5
6 7 8 9
Lane
1
2
3
4
5
6
7
8
190 kDa —
c
120 kDa —
87 kDa —
A
B
C
58-60 kDa
49.1 kDa —
38 kDa
F
Lane
+RA
0 24 48 96
(h)
0 24 48 96 (h)
Figure 2 Expression of MAZ mRNA and protein during the
RA-induced di€erentiation of P19 cells. (a) Northern blotting
analysis of MAZ transcripts. The size of each mRNA was
determined by reference to mobilities of RNA markers and is
indicated on the right. (b) Total RNA (10 mg) was fractionated
and allowed to hybridize with a fragment of MAZ cDNA. The
duration of incubation with RA is indicated. To demonstrate
loading of equivalent samples, bands of ribosomal RNA, 28S and
18S, are depicted, as revealed by Northern analysis. (c)
Expression of MAZ protein was unchanged during RA-induced
di€erentiation of P19 cells. Whole-cell lysates were prepared from
P19 cells that had been treated with RA at 1076 M for di€erent
lengths of time. The proteins were subjected to SDS-gradient gel
(2 ± 15%) polyacrylamide electrophoresis and then transferred to
a PVDF membrane. Immunoblot analysis was performed with
MAZ-speci®c polyclonal antibodies (lanes 2 ± 5) and with control
pre-immune serum (lanes 6 ± 9). The duration of incubation with
RA is indicated
1
2
3
4
5
Figure 3 Electrophoretic mobility shift assay (EMSA) with the
ME1a1 element as DNA probe. (a) Double-stranded oligodeoxynucleotides corresponding to the ME1a1 element and the
mutated oligodeoxynucleotide are shown. (b) EMSA of DNAprotein complexes with nuclear extracts of undi€erentiated P19
cells (5 mg of protein). Lane 1, free ME1a1 DNA probe; lane 2,
nuclear extract of P19 cells; lanes 3 and 4, nuclear extracts plus a
100-fold and 300-fold molar excess of the speci®c ME1a1 doublestranded oligodeoxynucleotide, respectively, as a competitor; lane
5, monoclonal antibodies against MAZ; lanes 6 and 7, nuclear
extracts and a 100- and 300-fold molar excess of mutant ME1a1
double-stranded oligodeoxynucleotide, respectively, as a competitor; lane 8, control unrelated antibodies against c-jun/AP-1. (c)
The transcriptional factor Sp1 did not bind to the doublestranded oligodeoxynucleotide that corresponded to ME1a1. Lane
1, the DNA probe; lane 2, nuclear extract of P19EC cells; lane 3
nuclear extract plus a 300-fold molar excess of the speci®c ME1a1
double-stranded oligodeoxynucleotide as a competitor; lane 4,
Sp1-speci®c polyclonal antibodies; lane 5, puri®ed Sp1 protein
(10 ng). The DNA-protein complexes designated A, B and C and
the free probe (F) are indicated. See text for full details
MAZ regulates the expression of c-myc
M Komatsu et al
a
+RA
–
24
48
72
96
(h)
A
B
C
F
Lane
1
2
3
4
5
6
b
Figure 4 EMSA with nuclear extracts of P19 cells that had been
treated with RA at 1076 M for di€erent lengths of time. (a) The
DNA probe was the double-stranded oligodeoxynucleotide
corresponding to the ME1a1 site. The duration of incubation
with RA is indicated. Lane 1, the DNA probe; lane 2, nuclear
extract of P19 cells; lanes 3 ± 6, nuclear extracts from cells treated
with RA at 1076 M for the indicated times. The DNA-protein
complexes A, B and C and the free DNA probe (F) are indicated.
See text for full details. (b) The amounts of DNA-protein complex
(band C) shifted on the gel relative to the total radioactivity of
the DNA probe. The radioactivity of shifted band C was
measured and the relative values was calculated. The average of
results from at least four experiments and the standard deviation
for each value are indicated
amounts of the shifted DNA-protein complex obtained
from the extracts of undi€erentiated and di€erentiated
P19 cells on the basis of the radioactivity of complex C
as a function of the total radioactivity of the
[32P]ME1a1 oligodeoxynucleotide probe used for
EMSA (Figure 4b). The level of complex C decreased
to approximately 60% of the initial level after 24 h in
the presence of RA and then it returned to the original
level at 48 h. Finally, it decreased again to 35 ± 40% of
the initial amount of the DNA-protein complex
(Figure 4b). These results indicate that the binding of
MAZ protein to the ME1a1 element was altered during
the di€erentiation of P19 cells and the change in the
binding of MAZ to the ME1a1 element, in particular
the change in the binding that generated complex C,
corresponded to the changes in the regulated expression of the c-myc gene during the neuroectodermal
di€erentiation of the P19 cells.
Discussion
The RA-mediated di€erentiation of P19 cells into
various types of cells derived from the neuroectoderm
is associated with a biphasic increase in the steadystate levels of c-myc mRNA and protein (St-Arnaud et
al., 1988). The ®rst peak in the expression of the cmyc gene at 3 h might be associated with the
induction of di€erentiation of P19 cells by RA
(Jones-Villeneuve et al., 1982). Di€erentiated neurons
appear in RA-treated cultures between 3 and 5 days
after the start of treatment with RA (McBurney et al.,
1982). The second peak in c-myc expression, from 2 ± 4
days, appears slightly to precede this event, suggesting
that elevated levels of c-myc mRNA and protein might
occur in neuroblast cells just prior to their differentiation into post-mitotic neurons. However, it is not yet
known whether the late increase and decrease in levels
of c-myc mRNA and protein occur in only these cells
that are destined to di€erentiate into neurons. The
period from 48 h to 96 h is equivalent to the so-called
the commitment phase of di€erentiation. That is, a
large number of cells progress to a stable di€erentiated
phenotype after this phase, even in the absence of any
di€erentiation-inducing agents (McBurney et al.,
1982). The temporal correlation between these late
changes in the chromatin, the initiation of transcription and the reversibility of the down-regulation of the
c-myc gene is intriguing. The down-regulation of the cmyc gene in an essential component of di€erentiation,
as reported in other systems (Lachman et al., 1986;
Prochownik and Kukowska, 1986; Yokoyama and
Imamoto, 1987; WickstroÈm et al., 1988; Holt et al.,
1988). The constitutive expression of a transfected cmyc gene prevents the di€erentiation of mouse
erythroleukemia cells (Coppola and Cole 1986;
Dmitrovsky et al., 1986; Lachman et al., 1986;
Prochownik and Kukowska, 1986; Kume et al.,
1988), but its critical function has not been well
documented. Moreover, the biphasic transient accumulation of c-myc mRNA in P19 cells that have been
treated with 161076 M RA appears to be well
correlated with di€erentiation in the neuroectodermal
lineage (McBurney et al., 1982; Edwards and
McBurney, 1983). Neither treatment with a lower
concentration of RA nor induction with DMSO
induce such a biphasic increase in the accumulation
of c-myc mRNA and the appearance of the neuronlike phenotypes. As shown in Figure 5, 20 ± 30% of
P19 cells treated with RA developed neural ®laments
that could be stained with antibodies speci®c for a
160 kDa neural ®lament protein. A systematic study
of the phases of di€erentiation of P19 cells using a
lower concentration of RA indicated that the second
increase in the level of c-myc mRNA is well correlated
with neural di€erentiation (Edwards and McBurney,
1983; St-Arnaud et al., 1988). These results prompted
us to attempt to de®ne the role of the second increase
in expression of the c-myc gene in the di€erentiation
of P19 cells.
1127
MAZ regulates the expression of c-myc
M Komatsu et al
1128
We found here that the second increase in the level
of c-myc mRNA was associated with the activation of
transcription of the c-myc gene via the ME1a1 site in
the c-myc promoter after 48 h of treatment with RA
(Figure 1). Moreover the DNA-binding activity of
MAZ protein to ME1a1 was well correlated with the
ME1a1-mediated promoter activity of the c-myc gene
(Figures 1b and 3b). However, we did not detect an
initial peak of CAT activity under control of the c-myc
promoter between 1 and 6 h after the start of exposure
of RA, a result that does not re¯ect the changes in the
level of c-myc mRNA as reported previously (Figure
1b; St-Arnaud et al., 1988). This result indicates that
the ®rst increase in level of c-myc mRNA was due not
only to the ME1a1-mediated transcription but also to
the post-transcriptional regulation of the expression of
the c-myc gene.
Three distinct elements, ME1a2, E2F and ME1a1,
within the P2 promoter of the c-myc gene are located
at positions +65 ± 88, +98 ± 105 and +112 ± 121
respectively, relative to the P1 promoter (Asselin et
al., 1989; Hall, 1990) and they required for optimal
transcription of the c-myc gene (Moberg et al., 1991,
1992), in addition to the consensus TATA box. It was
reported that the ME1a1 element had a small positive
a
e€ect on the initiation of transcription in a study in
vitro (Hall, 1990) and was necessary for P2 promoterdependent transcription in vivo (Asselin et al., 1989).
Moberg et al. (1992) reported that, individually, the
three elements ME1a2, E2F and ME1a1 are weak, but
together they contribute to full promoter activity. As
shown in Figure 3b (lanes 6 and 7), the formation of
the DNA-protein complex was not subject to any
competition when the oligonucleotide added as a
potential competitor was changed from CGGAGGG,
the ME1a1 element, to CGGATTG, which is not
recognized by MAZ (Wong et al., 1995). Moreover,
ectopic expression of MAZ protein in P19 cells resulted
in a small but signi®cant increase in the CAT activity
due to the pMyc2CAT reporter gene, while such was
not the case with the pMyc1CAT deletion construct
(Figure 1d, lanes 4 and 5). These results imply that the
ME1a1 element, at least, is critical for the transient
second increase in expression of the c-myc gene that
occurs during the RA-induced di€erentiation of P19
cells. However a direct association between the increase
in c-myc mRNA and the induction of the differentiation of P19 cells remains to be proven.
Let us consider now the transcription factor(s) that
bind to the ME1a1 element of the c-myc promoter
b
Figure 5 Neural di€erentiation of P19 cells. (a) The morphology of P19 cells after RA-induced di€erentiation. (b) Cells were
stained with monoclonal antibodies against neuro®lament 160 kDa protein (NF 160K), biotinylated second antibodies raised in
rabbits against mouse IgG and 3,3'-diaminobenzidine. The neurites and neurons with immunostaining of NF 160K are indicated by
arrows (magni®cation6200)
MAZ regulates the expression of c-myc
M Komatsu et al
during the di€erentiation of P19 cells. We detected
three speci®c DNA-protein complexes, namely, A, B
and C (Figure 3b, lane 2). Previous reports showed
that the ME1a1 element is recognized not only by
MAZ but also by the transcriptional factor Sp1
(DesJardins and Hey, 1993; Ash®eld et al., 1994).
With the exception of a single base within the ME1a1
element, this element is conserved in the mouse, rat
and human homologs of the c-myc gene (Moberg et al.,
1991). The same or a very closely related protein in the
extracts of Hela cells bound to both the ME1a1 and
ME1a2 elements (Moberg et al., 1992). The regulation
of the c-myc gene is complex since a number of
transcriptional factors, as well as both transcriptional
and post-transcriptional regulation, are involved
(Marcu et al., 1992). However, we found here that
antibodies speci®c for MAZ but not for Sp1 caused
shifted bands on a gel in EMSA (Figure 3b).
Moreover, puri®ed Sp1 protein from Hela cells did
not bind to the probe that corresponded to ME1a1
(Figure 3c). These results indicate that MAZ protein
might be important for ME1a1-mediated expression of
c-myc gene after 48 h of treatment of P19 cells with
RA. The DNA complexes A, B and C observed with
the ME1a1 probe were all super-shifted after addition
of antibodies against MAZ protein (Figure 3b, lane 5).
In turn, MAZ activated the initiation of transcription
of the c-myc gene in P19 cells (Figure 1d). It was
reported that the forced expression of MAZ was not
associated with a signi®cant increase in the level of cmyc mRNA in NIH3T3 cells (Pyrc et al., 1992).
However, we demonstrated a weak but signi®cant
enhancement of the activity of the promoter of the cmyc gene by MAZ protein (Figure 1d). The levels of
MAZ mRNA and protein did not change signi®cantly
during the di€erentiation of P19 cells (Figure 2). By
contrast, the kinetics of changes in the DNA-binding
anity of MAZ for the ME1a1 element (band C) were
closely correlated with the kinetics of the changes in
level of expression of c-myc mRNA and in the
promoter activity of the c-myc gene during the
di€erentiation of P19 cells (Figures 1b and c).
Therefore, it seems likely that some modi®cation of
MAZ, such as phosphorylation, might be responsible
for the change in the DNA-binding activity of MAZ. A
previous study demonstrated that the MAZ protein is
phosphorylated in COS cells, possibly on serine and
threonine residues (Pyrc et al., 1994). Similarly, in our
preliminary study of DNA binding in vitro, phosphorylation by casein kinase (CKII) signi®cantly enhanced
the DNA-binding activity to the NHE (nucleasehypersensitive element)-III2, as compared with that of
unphosphorylated MAZ protein (data not shown).
Treatment of neuroblastoma cells with an antisense
oligodeoxynucleotide speci®c for the gene for CKII
blocked neuritogenesis (Ullow et al., 1993). Changes in
the extent of phosphorylation by CKII during
di€erentiation of neuroblastoma cells might a€ect the
DNA-binding activity of certain transcription factors,
including MAZ (Ullow et al., 1993). However, we
cannot rule out the possibility that other factors might
associate with MAZ and a€ect the regulation of
transcription of the c-myc gene. Preliminary studies
with u.v.-crosslinking, treatment of the DNA-protein
complex with deoxycholate (DOC) and the `reversed'
combination of immunoprecipitation with antibodies
speci®c for MAZ protein and a subsequent gel shift
assay tend to support the presence of the other
associated factors with MAZ (data not shown).
The ME1a1-mediated transcription of the c-myc
gene that involves the MAZ protein is associated with
a transient second increase in the level of c-myc mRNA
after a 48 h incubation of P19 cells in the presence of
RA, which seems to be well correlated with the
di€erentiation of these cells into neuroectoderm. The
molecular mechanism of the interaction of MAZ
protein with other factors, including E2F or other
factors that bind to the ME1a2 site, and the way in
which these factors function to stabilize the interaction
of MAZ with E2F, with or without other e€ective
proteins, are totally unknown. An understanding of the
mechanism of the binding of MAZ protein to these
proximal and distal sites and the subsequent regulation
of transcription of the c-myc gene after 48 h of
exposure of P19 cells to RA should shed more light
on the mechanisms that control the di€erentiation of
P19 cells to neuroectoderm.
Materials and methods
Plasmids
pCMV-MAZ was constructed as described previously
(Tsutsui et al., 1996). The pMyc2CAT reporter was
generated by insertion of the DNA fragment from 7187
to +412 relative to the P1 site of initiation of transcription
into the SpeI ± HindIII site of pICAT (Kitabayashi et al.,
1991). The pMyc1CAT was generated by deletion from
pMyc2CAT of the DNA fragment from +112 to +121
relative to the P1 initiation site.
Cell culture, transfection and CAT assay
P19EC (P19) cells were grown in a-MEM (GIBCO BRL,
Gaithersburg, MD, USA) supplemented with 10% fetal
bovine serum (GIBCO BRL) and 60 mg/ml kanamycin
monosulfate (Sigma Chemical Co., St Louis, MO, USA).
Retinoic acid (Sigma Chemical Co.) for induction of
di€erentiation was added to cultured cells in monolayers
at 161076 M. For induction of neural di€erentiation of
P19 cells, cells were cultured in bacteriological-grade dishes
(Becton Dickinson, Franklin Lakes, NJ, USA) in the
presence of RA at 161076 M for 2 days, transferred to
tissue culture-grade dishes (Corning, Corning, NY, USA),
and cultured in the absence of RA for another 2 days as
described elsewhere (Jones-Villeneuve et al., 1982; McBurney et al., 1982; Edward et al., 1983). Transient and stable
transfections and assays of chloramphenicol acetyltransferase (CAT) activity were performed as described elsewhere
(Kitabayashi et al., 1992). The extent of conversion of
chloramphenicol to its acetylated form was determined
with a Bio-Imaging analyzer (model BAS 2000; Fuji,
Tokyo, Japan). b-Galactosidase activity was assayed as
described by Kitabayashi et al. (1992). The ratio of CAT
activity to the activity of b-galactosidase was used for
normalization of results.
Isolation of RNA and Northern hybridization
Total RNA (10 mg) from P19 cells that had been cultured
in the presence or absence of RA at 161076 M for di€erent
lengths of time was isolated and fractionated on a
formaldehyde-1% agarose gel and transferred to a nylon
membrane as described elsewhere (Tsutsui et al., 1996;
Sambrook et al., 1989). The blot was allowed to hybridize
1129
MAZ regulates the expression of c-myc
M Komatsu et al
1130
with a 1.8 kb EcoRI DNA fragment derived from pCMVMAZ (Tsutsui et al., 1996) or a 2.3 kb BamHI DNA
fragment derived from pC5-8 c-myc expression vector
(Yokoyama and Imamoto, 1987). The DNA probe was
labeled with a-32P-dCTP by the random-priming method
(Feinberg and Vogelstein 1983).
Immunoblot analysis
Whole-cell lysates were prepared as described elsewhere
(Kawasaki et al., 1996). Immunoblotting was performed by
the standard protocol as described elsewhere (Kaufmann et
al., 1987). In brief, proteins were fractionated on an SDSpolyacrylamide gradient (2 ± 15%) multigel (Daiichi Pure
Chemicals Co., Tokyo, Japan) and then transferred to
Immunobilon PVDF-nylon membrane (Daiichi Pure Chemicals). The membrane was blocked with 5% nonfat milk in
phosphate-bu€ered saline (PBS-T; 0.2% Tween 20 in
phosphate-bu€ered saline, PBS(7)) for 1 h at 258C and then
washed four times with PBS-T. It was then incubated with
1000-fold diluted polyclonal antibodies against MAZ in 1%
BSA in PBS-T overnight at 48C and then washed four times
with PBS-T. Antibodies bound to the membranes were
detected with horseradish peroxidase (HPR)-conjugated
antibodies against rabbit immunoglobulin G (Zymed
Laboratories, San Francisco, CA, USA) and a chemiluminescence kit (Dupont NEN, Boston, MA, USA).
Preparation of nuclear extracts and the electrophoretic mobility
shift assay (EMSA)
Nuclear extracts were prepared as described elsewhere
(Schmitt-Ney et al., 1991; Ye et al., 1996). P19 cells were
treated with 300 ml of lysis bu€er (50 mM KCl, 0.5%
Nonidet P-40 (NP-40), 25 mM HEPES (pH 7.8), 1 mM
phenylmethanesulfonyl ¯uoride (PMSF), 10 mg/ml leupeptin, 10 mg/ml pepstatin, 20 mg/ml aprotinin, 100 mM dithiothreitol (DTT) on ice for 4 min. The supernatant was saved
after centrifugation at 40 000 g for 1 min at 48C. The nuclei
were washed once with the same volume of bu€er without
NP-40 and then placed in 100 ml of extraction bu€er
(500 mM KCl and 10% glycerol with the same concentrations of HEPES (pH 7.8), PMSF, leupeptin, aprotinin and
DTT as those in the lysis bu€er). After centrifugation at
40 000 g for 5 min at 48C, the supernatant was harvested as
the nuclear extract. Protein concentrations were determined
with bicinchoninic acid (BCA; Pierce, Rockford, III.). For
EMSA, the reaction was conducted in 25 ml of a reaction
mixture that included 3 mg of poly dI.dC (Sigma Chemical
Co.), 5 mg of nuclear protein, 16104 c.p.m. of 32P-labeled
double-stranded oligodeoxynucleotide that corresponded to
the ME1a1 site, and 5 ml of 56binding bu€er (50 mM
HEPES (pH 7.8), 250 mM KCl, 5 mM EDTA (pH 8.0),
25 mM MgCl2, 50% glycerol, 25 mM DTT, 3.5 mM PMSF,
10 mg/ml aprotinin, 10 mg/ml pepstatin, and 10 mg/ml
leupeptin). In some cases, a double-stranded oligodeoxynucleotide as competitor or antibodies against MAZ (Tsutsui et
al., 1996) or against c-jun/AP-1 (Santa Cruz Biotechnology
Inc., Santa Cruz, CA, USA) or against Sp1 (Santa Cruz
Biotechnology Inc.) were added to the reaction mixture. The
puri®ed Sp1 protein from Hela cells was obtained from Santa
Cruz Biotechnology Inc. The mixture was incubated on ice
for 5 min, and incubation was continued at 48C for 6 h
without or with antibodies, then it was incubated for 25 min
at 258C in the presence of a 32P-labeled double-stranded
oligodeoxynucleotide and ®nally resolved on a 4.5%
polyacrylamide gel that had been prerun at 110 V for 2 h
in 0.256TEA bu€er (40 mM Tris (pH 7.8), 1.1 mM EDTA,
37 mM sodium acetate (pH 8.0)). Electrophoresis of the
loaded gel was performed at 200 V for 90 min, and then the
gel was dried and subjected to autoradiography on X-Omat
®lm (Eastman Kodak Company, Rochester,USA) at 7808C.
We determined the radioactivity of complex C relative to the
total radioactivity of the DNA probe. The normalized
radioactivity of the DNA-protein complex that corresponded to band C from the extract of undi€erentiated P19
cells was taken arbitrarily as 100% (12.5% of the total
radioactivity was shifted on the gel). Each cited result
represents the mean+s.d.) of results from four experiments.
Immunohistochemical staining
Immunohistochemical staining was performed to determine
the phenotypic features of the di€erentiated P19 cells.
Cells, plated on a glass coverslip, were cultured in the
presence or absence of RA at 378C and ®xed in precooled
methanol at 7208C. Cells on coverslips were washed with
phosphate-bu€ered saline plus 0.05% Tween 20-(PBS-T),
incubated in blocking solution that contained 0.05%
Tween 20 and 1% BSA for one hour at room
temperature, and then incubated with monoclonal antibodies against neuro®lament 160 kDa protein (NF-160K)
(Boehringer-Mannheim, Mannheim, Germany) that had
been diluted 1: 200 in 1% BSA in PBS-T for 2 h at room
temperature. After several washes with PBS-T, the sample
was incubated with 500-fold diluted biotin-conjugated
rabbit antibodies against mouse IgG (Zymed Laboratories) in blocking solution that contained 3,3'-diaminobenzidine (Sigma). Photographs were taken with a PM-30
automatic system (Olympus, Tokyo; photomicrographs6200).
Acknowledgements
The authors are grateful to T Murata, K Ogura and C
Koga for critical reading of the manuscript. This work was
supported by the Special Coordination Funds of the
Science and Technology Agency, by Life Science Project
Funds from RIKEN and by grants from the Ministry of
Education, Science and Culture of Japan.
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