From the Howard Hughes Medical Institute, Department of Biochemistry and Kaplan Cancer Center, New York University Medical Center, New York, New York 10016
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The basic region/helix-loop-helix/leucine zipper
(B-HLH-LZ) oncoprotein c-Myc is abundant in proliferating cells and
forms heterodimers with Max protein that bind to E-box sites in DNA and
stimulate genes required for proliferation. A second B-HLH-LZ protein,
Mxi1, is induced during terminal differentiation, and forms
heterodimers with Max that also bind E-boxes but tether the mSin3
transcriptional repressor protein along with histone deacetylase
thereby antagonizing Myc-dependent activation. We show that
Mxi1 also antagonizes Myc by a second pathway, repression of
transcription from the major c-myc promoter, P2. Repression was independent of Mxi1 binding to mSin3 but dependent on the Mxi1 LZ
and COOH-terminal sequences, including putative casein kinase II
phosphorylation sites. Repression targeted elements of the
myc P2 promoter core ( The c-myc gene is an immediate-early response gene,
which is activated in quiescent cells by mitogenic stimuli.
c-myc is required for the G0 to G1
transition (reviewed in Refs. 1-3), but in contrast to other
immediate-early response genes, its expression continues beyond the
G0-G1 transition at a lower constitutive level
throughout the cell cycle, as cells proliferate (4-6). Myc also exerts
G1-S control by enhancing cyclin E/cdk2 activity (7, 8).
Because inhibition of c-myc in proliferating cells arrests
growth (9, 10), c-myc expression is required for continuous
cell proliferation. Deregulated overexpression of c-Myc blocks terminal
differentiation of a number of cell types. Likewise, cellular
differentiation is normally accompanied by a decrease of c-Myc in a
variety of cell lines (reviewed in Refs. 1-3). This suggests that the
cellular choice between growth and differentiation depends upon a fine balance between c-myc stimulation and c-myc repression.
c-Myc is a basic region/helix-loop-helix/leucine zipper
(B-HLH-LZ)1 transcription
factor, which forms heterodimers with Max that bind to E-box Myc sites
(11-15). Myc-Max heterodimers transactivate promoters with E-boxes
(13, 14, 16), which in most cases are found in growth-promoting genes,
including the ornithine decarboxylase gene (17-19), cdc25A gene (20),
and the Myc-Max heterodimers form one arm of the Max interacting regulatory
network. The other arm of this network is provided by Mad-Max
heterodimers (reviewed in Ref. 2). Mad family proteins include Mad1,
-3, and -4 and Mxi1 (Mad 2) (23-25). Mad-Max heterodimers also bind
E-boxes, but repress transcription through their interaction with
mSin3, a homolog of a yeast general transcriptional repressor (25-28).
Histone deacetylases associated with mSin3 mediate Mad or Mxi1
transcription repression (29-31). Stimulation of transcription by
Myc-Max at E-boxes is therefore balanced by transcription repression by
Mad-Max.
Given the opposing actions of Myc-Max and Mad-Max, the cell may alter
the activities of E-box-dependent genes by altering the
relative levels of Myc and Mad, and this in turn may control proliferation. Indeed, in many instances of terminal differentiation, Mxi1 levels increase and c-Myc levels decrease as cells stop
proliferating and differentiate (23-25, 32-37). Furthermore, Mxi1
antagonizes the Myc transformation function (27, 37-40). Mxi1-knockout
mice demonstrate a cancer-prone phenotype, and an enhanced ability for
several Mxi1-deficient cell types to proliferate (41). Thus, Mxi1/Mad
proteins are essential in the regulation of normal and neoplastic growth.
The mechanism of inverse expression of the c-myc and
mxi1 genes is not known. However, in vivo,
c-myc expression ceases in many tissues during terminal
differentiation, at the time that the mxi1 gene is
activated. The c-myc gene is regulated at multiple levels
including repression of transcription (42, 43). c-myc transcripts are initiated from two major start sites, P1 and P2, which
are separated by 162 base pairs in the human c-myc gene. Because the great majority of c-myc transcripts arise from
P2 (43), P2 is a likely target for transcriptional repression.
Myc is known to repress its own promoter, providing an autoregulatory
loop that limits c-myc transcription (44-47). Myc also represses other promoters including the adenovirus-2 major late promoter and the promoters of the C/EBP In this report, we show that the Mxi1 protein is a repressor of the
c-myc promoter. Mxi1 represses the chromosomal
c-myc gene 30-fold, even in the absence of other aspects of
terminal differentiation. Repression of c-myc depends on a
novel regulatory domain within the Mxi1 COOH terminus and targets the
major c-myc promoter, P2. This repression is independent of
the Mxi1 NH2 terminus and mSin3, and thus differs from the
E-box repression function of Mxi1. Thus, Mxi1 represses genes required
for proliferation through two distinct pathways: repression of
E-box-dependent genes by a mechanism dependent on the Mxi1
amino terminus interaction with mSin3, and repression of the
c-myc P2 promoter by a mechanism dependent on the Mxi1 carboxyl-terminal domain.
Plasmids--
To express Mxi1 in vivo, CMV-Mxi1 was
constructed by subcloning a fragment from the full-length Mxi1
cDNA (a 2.4-kb EcoRI fragment (24) generated by PCR
using a 5' primer 5'-CGGGATCCACCATGGAGCGGGTGAAGAT (mx1f) and
a 3' primer (5')-GGAATTCCCACTGTTATGTCATGCTGGG (mx2r) into
the BamHI and EcoRI sites of the expression
vector pcDNA3 (Invitrogen). The following mutations were generated
by PCR and expressed in pcDNA3. CMV-Mxi1NT36 was generated using a
5' primer 5'-CGGGATCCATGCCGAGCCCCCGACTGCA and the same 3' primer as in
the wild-type Mxi1 construction. CMV-Mxi1CT149 and CMV-Mxi1CT187 were constructed using the same 5' primer as the wild-type construction and
a 3' primer, (5')-GGAATTCCTAACCCTGCAGCTGTTCCAG or
(5')-GGAATTCCTAGGAGAACTCTGTGCTTTC, respectively. CMV-Mxi1
M4tk-luc was derived from M4MinCAT (gift of L. Kretzner) by replacing
the chloramphenicol acetyltransferase reporter gene with the luciferase
gene. Tk-luc was constructed by deleting the four tandem E-box repeats
from the upstream promoter region of m4tk-luc. P2(
Plasmid MT-mxi1 was constructed by inserting the human Mxi1 coding
sequence (aa 1-228) tagged with a NH2-terminal
Flag-epitope (DYKDDDDK) obtained by PCR amplification of a fragment
from the human Mxi1 cDNA (24) into MT-neo (also known as pMTH
Cell Culture--
NIH3T3 and 3T3L1 cells (ATCC) were grown in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Life
Technologies, Inc.) supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin. Differentiation of 3T3L1 cells was performed as described in published procedures (55).
Transfection Assays--
Transient transfections of NIH3T3 cells
were performed in 10-cm dishes using the calcium phosphate
co-precipitation method (56). A total of 25 µg of plasmid DNA was
used in each transfection, consisting of 3-8 µg of reporter plasmid,
0.1 µg of CMV-
Northern and Western Analyses--
Northern analyses were
performed as described previously (52) using human cDNA probes for
human c-myc, human mxi1, and human gapdh labeled with [
Western blotting was performed as described previously (52). Amounts of
total cellular protein from transiently transfected cells were
normalized to Flow Cytometric Analysis--
Cell cycle distribution
experiments were performed as described previously (52) using a FACScan
flow cytometer (Becton & Dickinson). Briefly, 5 × 105
cells were pelleted by centrifugation and resuspended in 0.5 ml of
phosphate-buffered saline supplement with 2% (v/v) fetal calf serum,
and fixed in 100% ethanol. The fixed cells were pelleted and
resuspended in 0.75 ml of phosphate-buffered saline (with 2% fetal
calf serum), stained with propidium iodide, and treated with RNase A. These cells were then analyzed by flow cytometry for DNA content.
mSin3-independent Repression of the c-myc Promoter by
Mxi1--
The 3T3L1 cell line differentiates in culture from a
proliferating preadipocyte cell to a non-proliferating postmitotic
adipocyte (Fig. 1A) (57).
Between days 1 and 7 of 3T3L1 differentiation, c-myc
mRNA levels decrease and mxi1 messenger RNA expression
is strongly induced (Fig. 1B). These changes are reminiscent
of the inverse relationship of mxi1 and c-myc
gene expression in other differentiation systems (24, 34, 36) and
suggest a role for Mxi1 in the repression of c-myc. We
therefore asked whether Mxi1 can repress the c-myc promoter.
For these and subsequent experiments that employ transient
transfection, we made use of NIH3T3 cells, which are the parent cells
of 3T3L1. This was necessary because transfection of 3T3L1
preadipocytes was extremely inefficient, while NIH3T3 cells were
readily transfected. We transiently transfected NIH3T3 cells with
increasing amounts of the Mxi1 expression plasmid, CMV-Mxi1,
and the repression target plasmid, P2( Repression Requires the Mxi1 LZ and COOH Terminus--
The ability
of Mxi1 to repress c-myc in the absence of the
NH2-terminal mSin3 binding domain suggested that a
different Mxi1 domain repressed c-myc. To locate this
domain, we assayed repression of the c-myc promoter by
mutants of Mxi1, which had an altered DNA binding domain or an altered
carboxyl terminus. Western blotting confirmed that the mutants, which
were epitope-tagged to distinguish them from endogenous Mxi1, were
expressed at comparable levels (Fig. 4E).
Mxi1
The COOH-terminal domain of Mxi1 contains 80 amino acids distal to the
LZ and is rich in serine and acidic residues. Within this region lie
five potential casein kinase II (CKII) phosphorylation sites, grouped
in three locations (Ser170/Ser172,
Ser187, and Ser198/Ser200).
Mxi1CT149, which lacks the entire COOH-terminal tail (
Mutant Mxi1 Mxi1 Repression of c-myc Requires Elements of the P2 Core
Promoter--
Although c-myc transcription is initiated
from start sites P1 and P2, P2 accounts for over 90% of the activity
(43). Plasmid P1myc-luc (residues
We next defined components of P2 required for repression. P2TATA-luc
(residues
The c-Myc protein also targets c-myc P2 core promoter
elements to bring about its own negative autoregulation (47, 59). Myc
repression of the adenovirus-2 major late promoter and the C/EBP Mxi1, but Not Its COOH-terminal Deletion Mutant CT149, Antagonizes
USF-mediated Transactivation of P2--
c-Myc core promoter repression
(48-51, 59, 60) can be reversed by a second B-HLH-LZ protein, USF,
which stimulates core promoter activity (49, 61, 62). USF stimulated
the c-myc P1 and P2 promoters 2.7-fold in plasmid P2
( Regulated Expression of Mxi1 Can Repress the Endogenous c-myc
Gene--
To measure repression of the chromosomal c-myc
promoter by Mxi1 under more physiologically relevant conditions, we
constructed cell lines derived from NIH3T3 and 3T3L1 cells with a
stably transformed, Zn2+-inducible Mxi1 gene (MT-mxi1). In
the clonal cell line MT-mxi1.7 (Fig.
7A), exogenous mxi1
mRNA (0.8 kb) is induced by Zn2+ 18-fold, but the
endogenous Mxi1 transcript (2.4 kb) was not seen. No expression of
either endogenous or exogenous Mxi1 mRNA was seen in control cells
(Fig. 7B). Western analysis (Fig. 7C) indicated
that Mxi1 protein was expressed by 3 h and peaked at 10 h
following Zn2+ treatment.
To test whether Mxi1 represses the wild-type c-myc gene, the
MT-mxi1.7 cell line, and a control cell line, MT-neo, which contains a
Zn2+-regulated empty vector, were both deprived of serum
for 48 h to induce the quiescent state. The growth medium was
replenished with 20% fetal calf serum at time 0 h. To assure high
levels of metallothionein promoter induction at the time of serum
stimulation, ZnCl2 (40 µM) was added to both
cell lines 2 h prior to serum addition (
To determine whether Mxi1 can limit cell cycling, we employed four
additional cell lines, L1-MT.neo, L1-MT.mxi1, L1-MT-nt36, and
L1-MT-ct149, derived from 3T3L1 cells, the preadipocyte cell line in
which Mxi1 expression is activated during terminal differentiation (Fig. 1). These lines express the wild-type or mutant forms of Mxi1
protein that were studied above by transfection. Fig.
9A shows that the S + G2/M phase population of cells had increased 2-4-fold
18 h following serum stimulation of zinc-treated MT-neo, MT-nt36,
and MT-ct149 cells. In contrast, however, zinc-treated MT-mxi1 cells
showed no increase in the percentage of cells in S + G2/M
phases following serum stimulation. Western and Northern analysis shows
that the mxi1 gene is zinc-inducible in each cell line,
except the control line L1-MT.neo (Fig. 9, B and
C). These observations demonstrate that Mxi1, in addition to
repressing the c-myc promoter, blocks serum-induced cell
entry into S phase following serum stimulation. Furthermore, expression
of NT36 did not block cell cycle progression, although in transient
assays it was capable of repressing the c-myc promoter.
Likewise, mutant CT149 also did not block cell cycle progression,
although it repressed E box-dependent transcription in
transient assays. These results indicate that inhibition of cell
cycling by Mxi1 requires both the c-myc repression function
provided by the Mxi1 COOH terminus and the E-box repression function
provided by the Mxi1 NH2 terminus.
Effect of Mxi1 Expression on c-myc Gene Expression during Terminal
Differentiation--
Expression of the c-myc gene maintains
the ability of cells to proliferate and blocks or retards cellular
differentiation. Because reduction of c-myc induces cell
differentiation (2, 3) and constitutive c-myc expression
blocks differentiation in a variety of cell lineages, including
pre-adipocyte 3T3L1 cells (65), c-myc down-regulation may be
a prerequisite for differentiation.
A second aspect of terminal differentiation is the induction of Mxi1,
which was observed here during terminal differentiation of 3T3L1 cells,
and which was coincident with a decrease of c-myc mRNA.
Indeed, the mxi1 (or mad) and c-myc
genes are inversely regulated in a large number of terminally
differentiating systems, including leukemia cells (24, 32, 34),
epidermal cells (25, 33, 36, 37), and neurons (25), as well as during
mouse and zebrafish embryogenesis (35). A consequence of the decline in
Myc and increase in Mxi1 and other Mad family proteins is a change in
the relative levels of Myc-Max relative to Mad-Max heterodimers (32).
This change leads to repression of E-box-dependent genes.
The current work shows that Mxi1 represses the c-myc
promoter. This provides a second mechanism for repression of
Myc-dependent functions. In differentiating HL60 and U937
cells, repression of the c-myc promoter has been attributed
to a shut-off of c-myc transcription initiation (66). This
mechanism is consistent with P2 repression shown here.
Regulation at the P2 Core Promoter--
During the differentiation
of monocytic cells (67, 68), two domains of the myc
promoter, one between residues Regulatory Functions of Mxi1, Myc, and USF--
USF is a
ubiquitous cellular transcription factor (69, 70), which we show
stimulates the c-myc P2 core promoter. USF also stimulates
the Ad-2 major late promoter and the C/EBP
USF may provide a relatively constant positive contribution to the
basal activity of the c-myc promoter. Both Myc and Mxi1 appear to oppose this USF action. However, Myc and Mxi1 are likely to
provide this action under different circumstances. During cell proliferation, when Myc is elevated and Mxi1 and other Mad family members are low, Myc would negatively regulate P2. During terminal differentiation, when Mxi1 and other Mad proteins are high and Myc is
low, Mxi1 is likely to be the major repressor of P2. During the shift
from proliferation to differentiation, repression would switch from the
Myc-mediated mechanism to the Mxi1-mediated repression. We find
in vivo a 10-20-fold difference between c-myc
basal promoter activity in the unrepressed, USF-induced state and the
Mxi1-repressed state. Myc autoregulation could control myc
gene activity during continuous cell proliferation (9, 10). USF may
provide a constitutive positive stimulus for c-myc
expression under normal conditions, which can be regulated by Myc or
Mxi1 depending on cell state. During terminal differentiation,
reduction of c-Myc to the low levels (72) that are required for growth
arrest would, by this model, be the consequence of Mxi1 repression of
P2. Mxi1 would also repress E- box-dependent,
growth-promoting genes via Mxi1-Max heterodimers.
Transcriptional Regulation by Mxi1: NH2- and
COOH-terminal Functions--
The NH2-terminal domain of
Mxi1 binds mSin3, the mammalian homolog of the yeast repressor, Sin3
(26, 27) and is required for E-box repression (25, 26, 40). Because a
Mxi1 NH2-terminal truncation mutant repressed the
c-myc promoter, c-myc promoter repression by Mxi1
must be independent of mSin3.
The carboxyl-terminal sequences of Mxi1 distal to the leucine zipper
are required for Mxi1 repression of the c-myc promoter. The
COOH-terminal 41 amino acids of Mxi1 appear to be particularly important for myc repression, but are dispensable for E-box
repression. The leucine zipper, however, is required for repression of
both the c-myc promoter and E-boxes. c-Fos repression of the
c-fos promoter requires phosphorylated, COOH-terminal 27 aa
residues of c-Fos (73, 74). With Mxi1, CKII phosphorylation sites in the COOH terminus are also implicated in repression. These are grouped
in three locations: I, Ser170/Ser172; II,
Ser187; and III, Ser198/Ser200.
Double mutations (Ser/Ser to Ala/Ala) of group I and III CKII sites in
the Mxi1 COOH terminus resulted in 60% or greater loss of wild-type
Mxi1 repression activity. Removal of peptide sequences containing the
group II and III sites in mutant Mxi1CT187 eliminated repression
activity altogether. The COOH terminus may bind a corepressor analogous
to mSin3 that recruits a histone deacetylase. Alternatively, the Mxi1
COOH terminus might interact directly with a histone deacetylase as
does Rb to repress transcription (75-77). It is also possible that the
COOH terminus has the intrinsic ability to repress when in the
phosphorylated state.
Biological Roles for Mxi1 in Cell Cycle Exit--
Autoregulation
of the c-myc gene may provide a fine control of Myc
expression in proliferating cells. However, autoregulation would not be
capable of establishing a definitive and complete shut down of Myc
expression. During terminal differentiation, a more complete repression
may be achieved by switching the mechanism of c-myc promoter
repression from Myc autoregulation to repression by Mxi1. Because Mxi1
is induced during terminal differentiation and its expression is often
accompanied by growth arrest, Mxi1 may provide a more complete and long
lasting block of c-myc transcription than c-myc
autoregulation, which restricts Myc protein to very low levels and
potentiates the Mxi1 NH2-terminal transcriptional repressor
function. The G1 phase cell cycle block by Mxi1 may result
from effects of both repression of c-myc and effects of the
Mxi1 NH2-terminal transcriptional repressor function.
Finally, our results are consistent with the observations that ectopic expression of Mad1 inhibits cell proliferation at the G1
phase in human tumors (78), and in response to growth factor
stimulation (79).
35/+10), where it reversed
transactivation by the constitutive transcription factor, USF. We show
that Zn2+ induction of a stably transfected,
metallothionein promoter-regulated mxi1 gene blocked the
ability of serum to induce transcription of the endogenous
c-myc gene and cell entry into S phase. Thus, induction of
Mxi1 in terminally differentiating cells may block Myc function by
repressing the c-myc gene P2 promoter, as well as by
antagonizing Myc-dependent transactivation through
E-boxes.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-prothymosin gene (21, 22).
, gas1, and
gadd45 genes (48-53). In fact, Myc may induce cell
proliferation, at least in part, by repressing genes with growth arrest
activity (54). This repression is opposed by USF, an ubiquitous
B-HLH-LZ transcription factor (49, 50). The autoregulation of the
c-myc promoter may be controlled by a similar
Myc-dependent repression mechanism in continuously
proliferating cells. However, an autoregulatory mechanism is inherently
incapable of the complete shut down of c-myc promoter
activity that takes place during terminal differentiation. Thus other
mechanisms for c-myc regulation are likely to operate during
terminal differentiation.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
BR was
constructed by subcloning from CMV-Mxi1 a PCR-generated fragment made
in two steps: first, by amplifying the NH2-terminal
fragment with the 5' primer mx1f and 3' primer
(5')-AAGGCGCAGATGAGCTCTGTTGGCAGTGCT, and the COOH-terminal fragment
with 5' primer 5'-AGCACTGCCAACAGAGCTCATCTGCGCCTT and 3' primer
mx2r; then, by generating the full deletion mutant by mixing
the two reactions and amplifying with mx1f and
mx2r primers. Mutant Mxi1 expressing plasmids CMV-Mxi1
LZ,
CMV-Mxi1
162-185, CMV-Mxi1
170/172[SS-AA], and
CMV-Mxi1
198/200[SS-AA] were similarly constructed. The primers
used for generating the NH2- and COOH-terminal fragments
were: for
LZ, mxif and
(5')-TTCCATCTCCTGAGGGATGTGTGCTTTGGC, and
5'-GCCAAAGCACACATCCCTCAGGAGATGGAA and mx2r, respectively; for
162-185, mx1f and
(5')-TTCTCCATGGGAGAAAATGCTGTCCATTCG, and 5'-CGAATGGACAGCATTTTCTCCCATGGAGAA and mx2r, respectively;
for
170/172[SS-AA], mx1f and
(5')-CTCTCGCTCTGCATCAGCACGATC, and 5'-GATCGTGCTGATGCAGAGCGAGAG and
mx2r, respectively; and for
198/200[SS-AA],
mxif and (5')-ATCAATGTCAGCGATGGCGGTGGT, and
5'-ACCACCGCCATCGCTGACATTGAT and mx2r, respectively.
Mxi1 wild-type and mutants (except for
170/172[SS-AA] and
198/200[SS-AA]) were tagged at their amino termini with the FLAG
epitope in order to distinguish their in vivo expression
from that of endogenous Mxi1. Flag-Mxi1 had the same phenotype as Mxi1
in repression assays (data not shown).
2489)-luc was
constructed by cloning
2489 to +352 (relative to P2) with
HindIII linkers into the HindIII site of pGL2Basic (Promega). P2(
514) was derived from a PvuII
digestion of the
2489/+352 fragment and cloning into the
PvuII/HindIII site of pGL2Basic. P2(
70)-luc
contains
70 to +10 relative to P2 and was constructed by PCR
amplification using 5' primer 5'-CCGCTCGAGCGCTTGGCGGGAAAAAGAACG and 3'
primer (5')-CCCAAGCTTGTACAGCGAGTTAGATAAAGC, and cloned into
XhoI/HindIII digested pGL2Basic. P1P2myc-luc,
P2myc-luc, and P1myc-luc contain c-myc promoter fragments
266 to +352,
96 to +352, and
266 to
96 (relative to P2),
respectively, and were constructed by cloning the insert from the
appropriate restriction enzyme digestion of P2(
2489)-luc into
pGL2Basic. P2TATA-luc, P2SVL-luc, and SVL-luc were kindly provided by
J. Lang.
globin.neo, kindly provided by S. Segal) in the sense orientation.
gal as an internal control plasmid to monitor
transfection efficiency, a variable amount of expression plasmid
adjusted with empty vector plasmid to equalize the amount of effector
plasmid, and pBS SK plasmid as carrier DNA. For stable transfections
into NIH3T3 and 3T3L1 cells, 5 µg of MT-mxi1 or MT-neo were used.
Stable transfectants were selected in the presence of 800 or 1500 µg/ml G418 (Geneticin, Life Technologies, Inc.), respectively.
Individual clones were expanded and used for experiments or cryopreserved.
-Galactosidase activity was measured using the
Galacto-LightTM chemiluminescent reporter assay system
(Tropix, Inc.) and performed according to the manufacturer's protocol
with the modification that 100 µl of lysis buffer were used to lyse
cell pellets prepared from 10-cm dishes. Ten microliters of the lysate
were mixed with 100 µl of reaction buffer and 100 µl of light
emission accelerator. The same lysate (50 µl) was used for
measurement of luciferase activity in a Berthold Lumat LB9501
luminometer as described previously (49). Luciferase activity was
normalized to
-galactosidase activity to account for variations in
transfection efficiency. Normalization between experiments was done by
setting the activity obtained with the empty expression vector equal to
1. -Fold change is calculated as the inverse of relative luciferase
activity. A positive -fold change represents stimulation; a negative
-fold change, repression. At least four determinations were made for each reporter/activator combination.
-32P]dCTP by random
priming (Boehringer Mannheim).
-galactosidase activity to adjust for variations in
transfection efficiency, and protein lysates representing equivalent
-galactosidase activity were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose by electroblotting. Otherwise, 50 or 100 µg of total protein per lane were loaded on
SDS-polyacrylamide gels. Detection was performed with the Amersham ECL
system according to the manufacturer's instructions using anti-FLAG M2
monoclonal antibody (International Biotechnologies, Inc) or goat
anti-Mad2 (Mxi1) polyclonal antibody (Santa Cruz Biotechnology) to
detect Mxi1. Rabbit polyclonal antibody to c-Myc (Santa Cruz
Biotechnology) was used to detect c-Myc.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
2489)-luc (Fig.
2), which contains residues
2489 to
+352 of the human c-myc promoter region (numbering relative
to P2) linked to the firefly luciferase reporter gene. A cotransfected
CMV-
gal expression plasmid provided an internal control for
variations in transfection efficiency. In an initial experiment (Fig.
2), transient expression of Mxi1 reduced c-myc promoter
activity 3-fold, but the tk promoter, a control, was
relatively unaffected. Repression of c-myc by Mxi1 differed
from repression of an E-box. While the Max protein is required for Mxi1
repression of the E-box (23, 24, 26, 27), Max expressed from the vector
CMV-Max progressively reversed Mxi1 repression of c-myc
(Fig. 3A), rather than aiding
the repression. Indeed, Max expression in the absence of Mxi1 greatly
stimulated the c-myc promoter (Fig. 3A). As a
control, Max synergized with Mxi1 to repress
E-box-dependent transcription from plasmid m4tk-luc (Fig.
3B). Furthermore, the Mxi1 mutant, Mxi1NT36, which lacks the
Mxi1 amino-terminal 35 amino acids required for mSin3 interaction (Fig.
4A) and for
mSin3-dependent repression of E-boxes (26, 40), repressed
c-myc promoter activity to the same extent as wild-type Mxi1
(Fig. 4B), although as expected, it could not repress the
E-box-dependent activity of m4tk-luc (Fig. 4B).
These observations indicate that Mxi1 represses the c-myc
promoter and E-boxes by different mechanisms.
View larger version (58K):
[in a new window]
Fig. 1.
Terminal differentiation of 3T3L1
pre-adipocytes to adipocytes. A, photomicrographs of
cells on day 0 and day 7 of the terminal differentiation program. Note
the accumulation of cytoplasmic fat droplets in day 7 terminally
differentiated cells (55). Original magnification, ×100 or ×320 as
indicated. B, Northern analysis of RNA obtained on different
days of 3T3L1 differentiation. Twenty micrograms of total RNA per
condition were analyzed with cDNA probes to human mxi1
and then to c-myc after stripping. Ethidium bromide staining
of the gel showed equivalent loading of lanes as indicated by the 28 S
rRNA band. Lane 1, day 0; lane 2, day 1;
lane 3, day 4; lane 4, day 7; lane 5,
day 10; lane 6, day 14.
View larger version (16K):
[in a new window]
Fig. 2.
Repression of the c-myc promoter
by Mxi1. Reporter plasmid P2( 2489)-luc (5 µg) or tk-luc (10 µg) was co-transfected with the indicated amounts of CMV-Mxi1. Total
amount of CMV expression plasmid was held constant by adjusting with
empty CMV expression vector. Luciferase activity was normalized to
-galactosidase activity. The basal activity is indicated as a
relative -fold repression of 1. Data points are the mean of two to five
experiments with standard error bars. Vertical
bars indicate S.E. Reporter constructs are indicated on the
right.
View larger version (21K):
[in a new window]
Fig. 3.
Effect of Max on Mxi1 transcriptional
repression. A, Mxi1 repression of P2( 2489)-luc is
antagonized by Max. Three micrograms of P2(
2489)luc was
co-transfected with CMV-Mxi1 and/or CMV-Max in the amounts as
indicated. Total amount of CMV expression plasmid was held constant
with empty CMV expression vector. B, Max and Mxi1 potentiate
each other's transcriptional repression of m4tk-luc. Two micrograms of
m4tk-luc were co-transfected with the indicated amount of CMV-Max with
10 µg of either CMV-Mxi1 (+) or empty vector (
). M4tk-luc contains
four E-boxes linked in tandem upstream of the herpes simplex virus
thymidine kinase (tk) minimal promoter driving a luciferase
reporter gene. Total amount of CMV expression plasmid was held constant
with empty CMV expression vector.
View larger version (30K):
[in a new window]
View larger version (25K):
[in a new window]
Fig. 4.
Effect of wild-type and mutant Mxi1 on
P2( 2489)-luc or m4tk-luc. A, structure of Mxi1
wild-type and mutants. Mxi1 mutants were generated as described under
"Experimental Procedures" and cloned into pcDNA3.
,
COOH-terminal putative casein kinase II phosphorylation sites.
SID, mSin3 interaction domain. B-HLH-LZ, basic
region/helix-loop-helix/leucine zipper DNA binding domain for
interaction at the E-box. B, the amino-terminal 35-aa domain
(containing the mSin3 interaction domain) is required for
transcriptional repression of m4tk-luc, but not of P2(
2489)-luc. Ten
micrograms of CMV-Mxi1, CMV-Mxi1NT36, or pcDNA3 were co-transfected
with 5 µg of reporter plasmid P2(
2489)-luc or m4tk-luc. The
experimental conditions that involved m4tk-luc described here and in
C additionally contained CMV-Hm (2 µg) and CMV-Max (2 µg) which elevated the basal activity of m4tk-luc. The data indicate
the average of at least three experiments with standard errors of the
mean. C, the Mxi1 COOH-terminal domain (aa 149-228) is
necessary for repression of P2(
2489)-luc, but not of m4tk-luc,
whereas the LZ is required for repression of both promoters. The
indicated amounts of CMV-Mxi1, CMV-Mxi1
BR, CMV-Mxi1
LZ,
CMV-Mxi1CT149, or CMV-Mxi1CT187 were co-transfected with 5 µg of
P2(
2489)-luc or m4tk-luc. These results are the averages of at least
three experiments with standard errors of the mean. The basal activity
is indicated as a -fold change of 1. D, COOH-terminal
phosphorylation of Mxi1 is necessary for efficient repression of the
c-myc promoter. Ten micrograms of the indicated Mxi1
expression plasmid were co-transfected with 5 µg of P2(
2489)-luc or
m4tk-luc. As in C, these results are the averages of at
least three experiments with standard errors of the mean. The basal
activity is indicated as a -fold change of 1. Data for Mxi1 wild-type,
CT149, and CT187 are reproduced here for ease of comparison.
E, in vivo expression of Mxi1 mutants. Cellular
protein, normalized to equivalent
-galactosidase activity from
transient tranfections of NH2-terminal Flag-tagged Mxi1
expression vectors, was immunoblotted with the monoclonal anti-Flag
antibody to determine expression of the relevant transfected genes.
Epitope tagging was performed so that protein expression could be
determined independent of endogenous Mxi1 levels.
LZ, which lacks the leucine zipper (
aa 117-149), failed to
repress either c-myc or the E-box-dependent
plasmid, m4tk-luc (Fig. 4C). Mxi1
BR, which lacks the
basic region (
aa 70-80), did not repress m4tk-luc (Fig.
4C), but retained approximately 50% of the wild-type
activity for repression of P2(
2489)-luc (Fig. 4C). Thus,
the Mxi1 leucine zipper, but not the basic region, was necessary for
c-myc promoter repression.
aa 150-228),
was nearly inactive as a repressor of the c-myc promoter (Fig. 4, C and D), although it retained 50% of
the wild-type E-box repression activity. The mutant, Mxi1CT187, in
which the COOH-terminal 41 aa (including two CKII sites) are lacking
and a third CKII site is disrupted by severing Ser187 from
its CKII recognition sequence, also failed to repress c-myc (Fig. 4, C and D). However, the internal deletion
mutant Mxi1
162-185 (
aa 162-185) repressed P2 (
2489)-luc to
50% of the extent of the wild-type (Fig. 4D). Thus, the
COOH-terminal 41 amino acids of Mxi1 (aa 187-228) contain a
c-myc promoter repression domain that depends upon the
adjacent serine-rich and acidic regions for full activity.
170/172[SS-AA], in which serines within CKII
phosphorylation sequences at positions 170 and 172 were replaced by
alanines, retained 42% of the repression activity while mutant Mxi1
198/200[SS-AA], in which the CKII consensus phosphorylation sites at Ser198 and Ser200 were changed to
alanine, retained 36% (Fig. 4D). Thus, repression of the
c-myc promoter was dependent upon CKII consensus
phosphorylation sites within the COOH-terminal domain.
266 to
96; numbering relative to
P2) contains the c-myc P1 promoter while P2myc-luc (residues
96 to +352) contains P2. P1P2myc-luc, which contains both P1 and P2
(
266 to +352), and P2myc-luc both had much higher basal levels of
activity than P1myc-luc and were repressed by Mxi1 to the same extent
(Fig. 5A). In contrast,
P1myc-luc was relatively resistant to Mxi1 repression. This indicates
that the major target of Mxi1 is the P2 promoter.
View larger version (24K):
[in a new window]
Fig. 5.
Mxi1 represses the c-myc promoter
through P2 core promoter elements. A, Mxi1 repression
of c-myc promoter activity is mediated almost entirely by
P2. The structure of c-myc promoter luciferase constructs
containing both P1 and P2, and P1 or P2 alone are shown. The
c-myc P2 promoter is responsible for initiating
transcription of greater than 90% of c-myc mRNAs (43).
Ten micrograms of either empty expression vector or CMV-Mxi1 were
co-transfected with 3 µg of the indicated reporter plasmids.
Luciferase activity was normalized to -galactosidase activity. Shown
are the averages of two separate transfections. B, Mxi1
repression of P2 is mediated by P2 core promoter elements. The P2 core
promoter reporter and control constructs are shown. Ten micrograms of
empty expression vector or CMV-Mxi1 were co-transfected with 3 µg of
the indicated reporter plasmid. Shown are the averaged results of at
least three experiments with vertical bars
indicating standard error of the mean. The basal activity is indicated
as a -fold change of 1. C, repression of the P2 promoter is
mediated at least in part by the P2 initiator element. The Inr
sequences of pP2Inr.wt and mutants m1 and m2 are shown. The indicated
quantities of CMV-Mxi1 were co-transfected with 8 mg of pP2Inr.wt,
pP2Inr.m1, or pP2Inr.m2. The basal activity is indicated as a -fold
change of 1.
150 to +10), which contains P2 but not P1, was also
strongly repressed by Mxi1 (Fig. 5B), limiting the target of
Mxi1 to a 160-residue region. When the core promoter, which encompasses
the P2 TATA box, and the initiator (Inr) site (residues
35 to +10 of
P2TATA-luc) (58) were replaced with the SV40 late Inr region (SV40
residues +199 to +399 relative to the SV40 origin) (59), repression by
Mxi1 was reduced (Fig. 5B). The Inr region of the SV40 late
promoter was used in this replacement because the SV40 late promoter is
not subject to core promoter repression by Myc
(59).2 The residual
repression of P2SVL-luc was also seen with plasmid SVL-luc (Fig.
5B), which contains the SV40 late promoter initiator alone,
and was therefore unrelated to control through c-myc
promoter elements. Thus, Mxi1 represses the c-myc promoter
through P2 core promoter elements located between residues
35 to
+10.
promoter is mediated by their respective Inr elements (48, 49). Since
the c-myc promoter has a candidate Inr for Myc-mediated transcriptional repression, we asked whether Mxi1 repression of the P2
promoter might involve the initiator element. To test this, two mutants
of the P2 Inr region were assayed for repression by Mxi1. Mutant
P2Inr.m1 contains a triple point mutation at residues
1,
2, and
3, and P2Inr.m2 has a triple point mutation at residues +2, +3, and
+4. Both sets of mutations reduced Mxi1 repression (Fig.
4C). The fact that repression was not entirely abolished suggests that other residues may be involved. Our observations suggest
that Mxi1 repression of the P2 promoter involves, at least in part, an
intact P2 Inr.
514)-luc (residues
514 to +352) and P2 alone 8-fold in plasmid
P2(
70)-luc (residues
70 to +10) (Fig.
6, A and B).
Significantly, Mxi1 reversed the activation of the c-myc
promoter by USF. Mxi1 repressed the USF activated P1 plus P2 promoters
6-fold, and the USF activated P2 promoter 14-fold (Fig. 6, A
and B). Indeed, Mxi1 reduced the activities of both
promoters to below basal levels. In contrast, mutant Mxi1CT149, which
lacks the Mxi1 COOH-terminal tail (Fig. 4A) and did not repress basal c-myc promoter activity (Fig. 4, C
and D), was also ineffective in repressing USF-stimulated
c-myc promoter activity (Fig. 6, A and
B). These results indicate that USF strongly activates the
c-myc P2 promoter, and that Mxi1 antagonizes this
stimulation by a mechanism dependent on the Mxi1 COOH-terminal domain.
This regulation can provide a 14-fold change in c-myc
promoter activity.
View larger version (18K):
[in a new window]
Fig. 6.
USF activates the c-myc P2
promoter and antagonizes Mxi1 transrepression. CMV-USF (10 µg)
was cotransfected with 10 µg of empty vector, CMV-Mxi1, or
CMV-Mxi1CT149, with 8 µg of either P2( 514)-luc (contains P1 and P2,
panel a) or P2(
70)-luc (contains only P2, panel
b). Luciferase activity was normalized to
-galactosidase
activity. Shown are the results of at least three separate
transfections with vertical bars indicating
standard error of the mean. A, USF activated a P1 and P2
containing promoter fragment 2.7-fold. Mxi1 repressed this
USF-activated state 6-fold. The COOH-terminal truncation mutant
Mxi1CT149 had no effect on repression. B, P2 is activated
8-fold by USF. This activation was repressed 14-fold by Mxi1. Mxi1CT149
demonstrated no repression capability. USF expression was confirmed by
Western analysis (not shown).
View larger version (40K):
[in a new window]
Fig. 7.
Zinc induction of mxi1 expression
in stable NIH3T3 cell lines containing a transfected metallothionein
promoter-regulated mxi1 cDNA coding sequence or
controls. A, zinc-dependent mxi1
expression in MT-mxi1 cell line. The cell MT-mxi1.7 was stimulated with
0, 25, or 40 µM ZnCl2 for 20 h at which
time total RNA was extracted. Ten micrograms of total RNA per lane were
analyzed with a human mxi1 cDNA probe. Note that the
signal is approximately 0.8 kb, in contrast to 2.4 kb, which is the
size of the endogenous mxi1 mRNA transcript. -Fold
induction was calculated after relative mRNA expression
(mxi1/gapdh) was determined by densitometric analysis.
B, control cell lines did not express the exogenous
mxi1 gene. The MT-mxi1.7 cell line and control cell lines,
containing either the metallothionein promoter empty vector (MT-neo) or
a transfected metallothionein promoter-regulated max gene
(MT-max), were stimulated with 40 µM ZnCl2
for 20 h. Northern analysis was performed as above with 10 µg of
total RNA. The 28 and 18 S rRNA sizes are indicated on the
right of each panel. C, time course of
zinc-induced Flag-Mxi1 protein expression by the MT-mxi1.7 cell line.
One hundred micrograms of whole cell lysate was loaded per lane on a
SDS-polyacrylamide (16.5%) gel. Mxi1 protein expression was detectable
at 1 h on longer exposure (not shown), but clearly present by
3 h, and peaked at 10 h following zinc addition.
2 h; Fig.
8, A and B). In the
MT-neo control cell line, c-myc mRNA reached high levels
by 1.5 h after serum addition (Fig. 8, A and
B), consistent with serum induction of c-myc in
quiescent cells (63, 64). In contrast, induction of c-myc
mRNA was blocked in the Mxi1-expressing cells. A low level
c-myc activity at the time of serum addition resulted from
the addition of Zn2+ (compare
2 and 0 h lanes of
MT-neo and MT-mxi1.7, Fig. 8A). Western analysis confirmed
that c-Myc protein expression was also blocked (Fig. 8D).
Serum induction of c-myc was also blocked by Mxi1 in
MT-mxi1.7 cells, relative to an uninduced MT-mxi1.7 cell control (Fig.
8, E-G). In this experiment, the induction of
c-myc mRNA was greatly attenuated by Mxi1, with
densitometric quantitation revealing a 30-fold repression.
View larger version (24K):
[in a new window]
View larger version (33K):
[in a new window]
Fig. 8.
Induction of exogenous mxi1
expression blocked serum induction of the chromosomal c-myc
in quiescent cells. A, induction of
mxi1 expression in cell line MT-mxi1.7, but not MT-neo, by
zinc prevented serum induction of the c-myc gene. Cell lines
MT-neo and MT-mxi1.7 were serum-deprived in Dulbecco's modified
Eagle's medium supplemented with only 0.5% fetal calf serum for
48 h, then treated with ZnCl2 (40 µM)
2 h prior to adding fetal bovine serum (final concentration of
20%) to each. Time 0' indicates the time of serum addition.
Northern analysis was performed with cDNA probes to
mxi1, c-myc, and gapdh. The filter was
stripped after each hybridization. B and C,
relative mRNA expression for c-myc and mxi1,
respectively. Expression of mxi1 and c-myc
relative to that of gapdh was determined by densitometric
analysis. D, Western blots of cells treated as in
A, demonstrating that zinc-induced Mxi1 expression blocks
serum induction of c-Myc protein expression. E-G, treatment
with zinc in the MT-mxi1.7 cell line abrogated the serum induction of
c-myc gene expression. The cell line MT-mxi1.7 was deprived
of serum as above, then treated with or without ZnCl2 (40 µM), and serum (20%) was added 2 h later. Time
0' indicates the time of serum addition. Northern analysis
and densitometric analyses were performed as in panels
A, B, and C.
View larger version (24K):
[in a new window]
Fig. 9.
Induction of Mxi1 prevents S phase entry
following serum stimulation of 3T3L1 preadipocytes. A,
individual 3T3L1 clonal cell lines stably transfected with vectors
pMT-neo, pMT-mxi1, pMT-nt36, or pMT-ct149 were grown to confluence.
Monolayers were washed twice with phosphate-buffered saline and then
incubated in Dulbecco's modified Eagle's medium containing 0.5%
fetal calf serum for 72 h. ZnCl2 (40 µM,
final concentration) was added at 2 h, and 2 h later (0 h) the
cells were stimulated with fetal calf serum (20% final concentration).
Cells were harvested at times 0 and 18 h, fixed, and stained with
propidium iodide. Cell cycle profiles were determined by
fluorescence-activated cell sorting analysis. The absolute percentages
of cells in S + G2/M phases are shown. One-sided
error bars are presented for clarity and
represent 1 standard deviation. B, the expression of
wild-type or mutant Mxi1 by the MT.mxi1, MT.nt36, and MT.neo cell lines
was assayed by Western blotting with an antibody directed to the Mxi1
COOH-terminal domain. C, the expression of mutant Mxi1
mRNA by the MT.ct149 and control MT.neo cell lines was assayed by
Northern analysis using a Mxi1 coding sequence-specific probe.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
606 and
101, upstream of P1, and a
second between residues
2392 and
1396 (68) function in
myc promoter repression. Our work defines a third domain,
which overlaps the P2 transcription start site, a position where RNA
polymerase II is retained in differentiating HL60 cells while
c-myc mRNA decreases in vivo (42, 67). Myc also autoregulates its own expression through P2 (47, 59).
promoter and reverses
Myc-mediated repression (49, 61). The transcription factor TFII-I
interacts with USF and with Myc to form complexes that bind promoter
initiator elements which mediate activation or repression of
transcription, respectively (48, 71). The opposing effects of USF and
Mxi1 noted in this report suggest the possibility of the formation of
USF and Mxi1 core promoter complexes.
![]() |
ACKNOWLEDGEMENTS |
---|
The full-length mxi1 cDNA was a gift from A. Zervos. We thank L. Kretzner for providing plasmid M4Min-CAT. The luciferase reporter plasmids P2TATA, P2SVL, and SVL were a gift from J. Lang. We thank Dr. John Hirst for assistance in flow cytometry. We thank Linheng Li, Gareth Inman, Chris Daly, Tom Hornyak, Joey Perez, and members of the Ziff laboratory for helpful discussions. We also thank Dr. Lennart Philipson for critical reading of the manuscript. We thank Latika Khatri for excellent technical assistance and Terry Serra for preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant RO1 AG13620 from the NIA, National Institutes of Health (to E. Z.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by National Institutes of Health Grant CA-01713 and a
Skirball Institute award. Physician-Scientist Scholar of the Skirball
Institute of Biomolecular Medicine.
§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Dept. of Biochemistry and Kaplan Cancer Center, New York University Medical Center, 550 First Ave., New York, NY 10016. Tel.: 212-263-5774; Fax: 212-683-8453.
The abbreviations used are: B-HLH-LZ, basic region/helix-loop-helix/leucine zipper; PCR, polymerase chain reaction; CMV, cytomegalovirus; kb, kilobase pair(s); aa, amino acid(s); Inr, initiator; CKII, casein kinase II.
2 L. Li and E. Ziff, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|