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INTRODUCTION |
The c-myc gene encodes a transcription factor, c-Myc,
that dimerizes with Max to serve as a master regulator of cell growth, cell division, and cell differentiation in all metazoans (reviewed in
Ref. 1). Mutations that disrupt the regulated expression of
c-myc (or the related N-myc and L-myc
genes) are common, causative lesions in many human and animal cancers
(2). In addition to its prominent link to cancer, myc is
also required for timely progression through the cell cycle and is an
essential downstream effector of mitogenic signals (3-5). The Myc
family proteins function as transactivators when assayed on reporter
constructs containing consensus binding sites or when the Myc N
terminus is tethered to DNA through a heterologous DNA binding domain
(6, 7). Recognition of specific chromosomal targets primarily involves the basic region, which forms an extended
-helix with helix 1 in the
Max homodimer crystal structure (8, 9). Myc/Max heterodimers recognize
a preferred consensus site ACCACGTGGT, although other related sites are
also bound with varying affinity (10-12). The helix-loop-helix and
leucine zipper domains dictate the highly specific dimerization of Myc
with Max, which prevents dimerization with numerous other transcription
factors that have related
HLH1 and/or leucine zipper
motifs (13). An increasing number of cellular targets for Myc/Max
heterodimers have been defined (14) with compelling evidence that Myc
protein can be cross-linked to target sites in vivo using
chromatin immunoprecipitation (15-18).
More recently, the Max superfamily was extended to include the Mad and
Mxi proteins that form alternate heterodimers with Max (reviewed in
Ref. 19). Unlike Myc, Mad/Mxi proteins function as repressors of
transcription and inhibitors of both cell proliferation and oncogenic
transformation (20-22). Mad/Max heterodimers recognize consensus
binding sites in vitro that are indistinguishable from those
recognized by Myc/Max, suggesting that these alternate Max complexes
may activate and repress common target genes. Although this is an
appealing model, there remains only limited evidence to support it.
Whereas Myc protein is expressed in all proliferating cells, Mad
protein expression is more restricted and primarily found in terminally
differentiated cells (23-26). Thus, in mitogen-deprived cells where
Myc levels are low, there is no evidence that Mad supplants Myc to
repress target genes. Some specific gene promoters, tert and
cyclinD2, have been shown to bind to Mad1 protein in vivo, using HL60 cells, which differentiate and express high
enough levels of Mad protein to cross-link (16, 18). In
undifferentiated cells that express Myc rather than Mad, Myc was bound
to the same promoters, suggesting that Myc and Mad can bind
sequentially to common promoters. However, these are isolated examples
so far, and no other promoters were examined. Further support for
antagonistic functions of Myc and Mad/Mxi comes from mouse gene
knockout experiments in which Mad and Mxi knockouts exhibit minor
hyperproliferative disorders or occasional tumors, suggesting that
these genes can function as tumor suppressors (27-29). However, no
homozygous mutations in mad or mxi genes have
been found in human or animal cancers to establish any direct role for
these proteins as tumor suppressors.
Previous studies compared Myc and Mxi/Mad binding with cellular
promoters through either the introduction of Mad/Mxi1-specific amino
acid substitutions into the Myc basic region or the complete substitution of the Mad1 basic/HLH/LZ region into Myc (30, 31). Since
the basic region is responsible (when dimerized with Max) for the
recognition of DNA sequence elements at chromosomal sites, it was
suggested that these mutants would offer insight into Myc versus Mad/Mxi target gene recognition and biological
function. One study noted a substantial decrease in the transforming
activity of the Myc(Mxi1-BR) exchange mutants and a shift in the
spectrum of genes regulated in human fibroblasts (30). From this, it was suggested that the biological activities of Myc and Mxi1 involve the regulation of both common and distinct sets of target genes governing diverse biological processes. A second study with a complete
B/HLH/LZ exchange came to the different conclusion that DNA binding
activity and target gene regulation in terms of cell proliferation were
similar for the Myc and Mad1 proteins but that the induction of
apoptosis was substantially different (31).
We were interested in a comparison of target gene regulation and
biological activities of the Myc and Mad/Mxi basic regions. In
particular, we noted that the Mad/Mxi1 proteins lacked conserved basic
residues in the N-terminal half of the basic region that are common to
the three Myc family proteins but that Mad/Mxi proteins have several
highly conserved basic amino acids farther toward the N terminus that
are not found in Myc proteins. To assess the basic region of Mad/Mxi1
proteins, we constructed a hybrid Myc protein in which the entire basic
region of Myc was exchanged for the corresponding region of Mad1 while
retaining the Myc HLH/LZ domain. We find that this Myc(Mad-BR) hybrid
protein has the ability to oncogenically transform cells, induce
apoptosis, and regulate cellular promoters that is indistinguishable
from that of Myc family proteins. We also showed that Mad1 protein can
bind to several additional Myc target genes in vivo.
Therefore, we conclude that there is no inherent structural difference
between the Myc and Mad/Mxi1 basic regions that would promote the
regulation of distinct sets of cellular target genes.
 |
MATERIALS AND METHODS |
Vectors--
Expression plasmids were created by standard
methods in retroviral expression vector pLXSH and verified by sequence
analysis. Details of individual constructs are available upon request.
Cell Culture, and Retroviral Infection--
The human
promyelocytic leukemia cell line HL60 was grown in RPMI 1640 medium
containing 10% fetal calf serum and 2 mM
L-glutamine. To induce cell differentiation, cells at a
density of 0.4 × 106 cells/ml were treated with
1.25% v/v Me2SO for 48 h. Normal human fibroblasts
(IMR90), rat c-myc-null fibroblasts (HO15.19 cells), and
retroviral producer PhoeNX cells were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum. Retroviral
infection of HO15.19 and IMR90 cells was performed according to
published protocols (32), using PhoeNX cells. PhoeNX cells were
transfected via the calcium phosphate method. To obtain a transduced
cell population, cells transduced with LXSH-based retroviruses were
selected for resistance to hygromycin (150 µg/ml) for 7 days.
Fluorescence-activated Cell Sorter--
To induce apoptosis,
~106 cells were incubated in Dulbecco's modified
Eagle's medium supplemented with 0.1% calf serum. Floating and
adherent cells were harvested after 48 h of starvation. The cells
were pelleted, and the pellet was resuspended in 500 µl of
phosphate-buffered saline + 0.5% fetal calf serum and fixed in 70%
ethanol at
20 °C overnight. The fixed cells were pelleted and
resuspended in 500 µl of phosphate-buffered saline +0.5% fetal calf
serum and an equal volume of the DNA extraction buffer (192 ml, 0.2 M Na2HPO4; 8 ml, 0.1 M
citric acid, final pH of 7.8) was added to each sample followed by
incubation for 5 min at room temperature. The cells were pelleted and
resuspended in 800 µl of phosphate-buffered saline +0.5% fetal calf
serum supplemented with 8 µl of propidium iodide (1 mg/ml in 20 mM sodium citrate) and 4 µl of RNase A (DNase
free, 10 mg/ml) in a 1-ml total volume. The cells were incubated for 30 min at 37 °C and analyzed on a BD Biosciences FACScan
fluorescence activated cells sorter.
Chromatin Immunoprecipitation--
Chromatin immunoprecipitation
assay on logarithmically growing and differentiating HL60 cells
was performed according to Xu et al. (18). Conditions for
the chromatin immunoprecipitation assay on HO15.19 cells were described
previously (33). The following sets of primers were used: rat
nm23-specific primers NM23a (5'-GGTCGTTCTCGTCTCTGCTC-3') and
NM23b (5'-TTCTGCTCGAATCGCTTGAT-3'); rat mSHMT-specific
primers RmSHMT1 (5'-CTGGATGACCAGTGGAAAGG-3') and RmSHMT2
(5'-GACCCTTGCGTGATGAAAGT-3'); rat nucleolin-specific primers NUCLa
(5'-CTGGGAGGGCGATGTAGAGT-3') and NUCLb (5'-GGAAGGGGGTTATCTCGAAG-3');
rat glucokinase-specific primers GLUa (5'-TGCCCGATTTTCATCTTCTT-3') and
GLUb (5'-CCAAGGACTTCCGCACTAAC-3'); hsp60-specific primers
HSPa (5'-GGCTGGGGATAAGTCTGTCA-3') and HSPb (5'-GACGGTGCAAAACCCTCTAA-3'); human mSHMT-specific primers SC-25 (5'-CGAGTTGCGATGCTGTACTTCTCT-3') and SC-26
(5'-GCTCGGTTGCATCATCTGCA-3'); human cad-specific primers
hCAD-CPs (5'-CCAGTTCCCATTGGTGTTGTTGCC-3') and hCAD-CPa
(5'-GAGAGGCGCATCACAGAGTGGGATAA-3'); human hsp60-specific primers hHSP60-CPs (5'-CTCCGCCTCAGACTCGTACT-3') and hHSP60-CPa (5'-AGAGGAGGAAGGCCCACTC-3'); human cdk4-specific primers
hCDK4-CPa (5'-CTCTGGGTGGCCTAGGTTG-3') and hCDK4-CPb
(5'-TAGAGAGGCCCCCTCACC-3').
To normalize samples by the amount of nonspecific DNA, we amplified a
region in the promoter of the rat pcna gene, PCNa
(5'-CGAAGCACCCAGGTAAGTGT-3') and PCNb (5'-ATCGTATCCGTGGTTTGAGC-3'), or
in the third intron of the human
-globin gene, SC-46
(5'-ATCTTCC-TCCCACAGCTCCT-3') and SC-47 (5'-TTTGCAGCCTCACCTTCTTT-3').
One of the primers in each pair was end-labeled with
[
-32P]ATP. Amplification was performed in a T3
Thermocycler (Biometra®) for 31 cycles at 94 °C for
45 s, 60 °C for 45 s, and 72 °C for 60 s followed
by a final extension step at 72 °C for 5 min. The optimal number of
cycles for exponential amplification was determined by kinetic
analysis. PCR products were resolved on a 4 or 6% polyacrylamide gel.
PCR products were quantitated and normalized using the Amersham Biosciences phosphorimaging system.
Reverse Transcription and PCR--
Total RNA was isolated from
infected cells using TRIzol reagent (Invitrogen). Reverse transcription
of 1-3 µg of RNA was performed using SuperScriptTM
first-strand synthesis system (Invitrogen) according to the
manufacturer's instructions. PCR was performed on cDNA
using previously described oligonucleotide sets (33, 34). For the
quantitative analysis of PCR products, one of the oligonucleotides in
each pair was end-labeled with [
-32P]ATP.
Amplification was performed in a T3 Thermocycler
(Biometra®) for various number of cycles at 94 °C for
45 s, 60 °C for 45 s, and 72 °C for 60 s followed
by a final extension step at 72 °C for 5 min. The optimal number of
cycles for exponential amplification was determined by kinetic
analysis. All quantitative PCR tests were repeated at least two times.
PCR products were resolved on 6% polyacrylamide gel and quantitated
using the Amersham Biosciences phosphorimaging system.
 |
RESULTS |
Mad Binds to Myc Target Genes in Vivo--
To explore the binding
of Mad1 protein in vivo, we took advantage of HL60 cells,
which produce Mad1 protein after being induced to differentiate with
Me2SO. We analyzed the binding of Mad1 protein to a number
of chromosomal Myc targets in vivo by chromatin
immunoprecipitation (Fig. 1). We found
that Mad1 protein could be efficiently cross-linked to the
cad, cdk4, hsp60, mSHMT,
and Id2 promoters in differentiated HL60 cells where Mad1
expression is induced, but not in the same cells before inducing
differentiation where Mad1 is not expressed. All of these genes have
been shown previously to bind to Myc in several different cell types.
Nonspecific PCR amplification of DNA from the
-globin gene, which
does not bind to Myc, served as a negative control for normalization.
These data add to the previous demonstration that Myc and Mad1 bind to
the promoters of tert and cyclinD2 in
logarithmically growing and differentiated cells, respectively (16,
18).

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Fig. 1.
Mad1 binds to the promoter of Myc-responsive
genes. Logarithmically growing (not expressing Mad1) or
Me2SO (DMSO)-treated, differentiated HL60 cells
(expressing Mad1) were cross-linked and lysed, and chromatin was
immunoprecipitated with Mad1-specific antibodies followed by the
reversal of the cross-linking and DNA isolation. Isolated DNA was used
in PCR with radiolabeled oligonucleotides flanking Myc binding sites in
the promoter of the genes indicated on the right side. For a
negative control, PCR was carried out with oligonucleotides
complementary to the third intron of the human -globin gene.
Quantitation of the PCR products was performed using the Amersham
Biosciences phosphorimaging system. Numbers below a
panel indicate the fold difference in the signal intensity
determined by dividing the PCR signal in the DMSO column by
a corresponding signal in the Log column and by the ratio of
these signals for -globin.
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Exchange of Myc and Mad Basic Regions--
Different antibodies
are used for Myc and Mad1 chromatin immunoprecipitation; therefore, we
cannot determine whether binding to cellular targets is quantitatively
equal between the two proteins. To dissect the contribution of the
basic DNA recognition domain alone, we decided to test the DNA binding
activity of the Myc and Mad basic regions through functional assays. A
comparison of the basic regions of the Myc and Mad/Mxi families is
shown in Fig. 2. Both protein families
have a bipartite basic region that extends ~20-25 amino acids
N-terminal to helix 1. The only crystal structure from this gene family
is of Max/Max homodimers in which the proximal basic residues are in a
helical structure and have defined base contacts (8, 9). However,
phosphorylation of serine 11 in Max inhibits DNA binding activity (35),
indicating that amino acids distal to the basic region can dramatically
influence its function. Both Myc and Mad/Mxi have 6 basic
residues proximal to helix 1; however, the Mad/Mxi basic region extends
over 19 amino acids, whereas the Myc basic region extends over only 13 amino acids. The Myc and Mad/Mxi basic regions are both bounded by
proline or glycine, which are likely positions for a domain boundary.
Given these structural considerations, we decided to test the Mad basic
region function by exchanging 24 amino acids of mouse c-Myc with 21 amino acids of mouse Mad1, using an N-terminal proline as one boundary
and an invariant leucine in helix 1 as the C-terminal boundary. Within
the domain exchanged, 9 out of the 25 amino acids are identical or
conservative substitutions.

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Fig. 2.
Schematic representation of wild-type Myc,
Mad1, and Myc with the Mad1 basic region. The shaded
region on the Myc(Mad-BR) line represents the extended
basic region of Mad that was exchanged into the c-Myc protein. Basic
residues are indicated in bold.
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Ectopic transient expression of WT c-Myc and Myc(Mad-BR) gave
equivalent levels of protein and equivalent binding to E-box elements
in electrophoretic mobility shift assay (data not shown). To assess the
ability of the two proteins to promote cell proliferation, we
reconstituted Myc-null rat fibroblasts with each Myc protein and
established that they were expressed at similar levels (Fig. 3A). We then examined the cell
morphology (Fig. 3B) and growth rate (Fig. 3C). Both WT
c-Myc and Myc(Mad-BR) fully rescued the growth defect in Myc-null
fibroblasts, giving doubling times of 18 and 19 h, respectively,
which is equivalent to the doubling time (18 h) of the parental cells
with WT c-myc genes. In contrast, the Myc-null fibroblasts have a
doubling time of ~45 h. We also tested the relative fraction of cells
in each phase of the cell cycle by propidium iodide staining (Fig.
4). When compared with the pronounced
accumulation of cells in G1 in the Myc-null cells (69%),
both c-Myc and Myc(Mad-BR) had an equivalent reduction in
G1 fraction (52 and 50%), respectively. Cells
reconstituted with c-Myc and Myc(Mad-BR) had similar increases in S
phase cells (20 and 21% respectively) when compared with myc-null
cells (11%). Thus, the Myc and Myc(Mad-BR) proteins rescue the cell
cycle defect in Myc-null cells to an equivalent extent.

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Fig. 3.
Phenotypes of wild-type Myc and Myc(Mad-BR)
in cell growth and primary cell transformation. A,
expression of Myc and Myc(Mad-BR) proteins in stably reconstituted
HO15.19 cells. Equal amounts of nuclear extract were immunoprecipitated
(IP) with anti-FLAG antibody and probed with anti-Myc (N262;
Santa Cruz Biotechnology). B, colony size and morphology of
HO15.19 cells infected with empty vector or Myc- or
Myc(Mad-BR)-expressing vectors. C, doubling times of
Myc-null cells reconstituted with the indicated Myc-derived proteins.
D, oncogenic activity of Myc and Myc(Mad-BR) in primary rat
embryo cells. Constructs expressing Myc and Myc(Mad-BR) were
cotransfected with H-rasG12V into early passage rat embryo
fibroblasts, and foci were scored after 18 days. The data are the
average of three experiments.
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Fig. 4.
Wild-type Myc and Myc(Mad-BR) induce
apoptosis in Myc-null cells. A, representative flow
cytometry profiles for HO15.19 cells infected with the indicated
expression constructs following normal growth (10% serum) or serum
starvation (0.1%) for 48 h. Both floating and adherent cells were
harvested and analyzed by flow cytometry to determine the percentage of
apoptotic cells. M1, M2, M3, and
M4 represent gating for sub-G1, G1,
S, and G2/M populations, respectively. B,
quantitation of the fraction within each gate in panel
A.
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To compare the oncogenic activity of Myc(Mad-BR) with WT c-Myc, we
tested each construct for focus formation in primary rat embryo cells
in cooperation with an H-ras(G12V) oncogene. Both WT c-Myc
and Myc(Mad-BR) induced equivalent numbers of foci, ~15/dish for each
(Fig. 3D). Similar results were obtained for three
independent assays. The morphology of the foci was indistinguishable
between WT and the exchange mutant (data not shown). We conclude that the Mad basic region has an equivalent biological function to that of
c-Myc. Since the proliferative and oncogenic activities of c-Myc are
presumably a manifestation of the complex sets of target genes that it
regulates, we infer that all of the critical functional targets
recognized by the c-Myc basic domain are also recognized by the Mad
basic domain.
Myc and Myc(Mad-BR) Induce Equivalent Apoptosis--
As a final
test of biological activity, we examined the induction of apoptosis by
the Myc and Myc(Mad-BR) proteins. The Myc-null cells were
reconstituted with each protein using a long terminal repeat-driven
reconstitution vector, which is sustained in expression when serum
survival factors are withdrawn. This constitutive Myc expression
induces apoptosis, unlike in the parental Rat1 (thioguanine-resistant (TGR) subclone) cells where Myc is down-regulated upon serum
withdrawal. As reported previously, the Myc-null cells have no
detectable apoptosis when starved in 0.1% serum for 24-48 h,
whereas cells reconstituted with a constitutively expressed WT c-Myc
protein exhibit a substantial amount of apoptosis (23%) as measured by the appearance of floating cells with a sub-G1 DNA content
(Fig. 4A). Notably, there was no difference in the fraction
of cells that activated the apoptotic death program with WT c-Myc
when compared with Myc(Mad-BR) (20%) (Fig. 4B). Therefore,
the basic region of Mad is capable of activating the same apoptotic
pathway as that of the WT c-Myc protein.
Myc and Mad Basic Regions Recognize Equivalent Chromosomal
Targets--
The data above support a model in which the Myc and Mad
basic region recognize an equivalent set of chromosomal target genes. To address this question more directly, we analyzed the expression of
several target genes in reconstituted Myc-null fibroblasts (Fig.
5A). The expression of five
different c-Myc target genes (hsp60, nm23,
nucleolin, mSHMT, and cad) was analyzed by
reverse transcription-PCR, and all genes exhibited equivalent
induction in response to both WT c-Myc and Myc(Mad-BR). In each case,
target gene expression is stimulated by ectopic Myc expression above the level in the Myc-null cells (Fig. 5A). gapdh
levels were unchanged by ectopic Myc expression and were used as a
negative control. A much more sensitive assay for the activation of
chromosomal targets by c-Myc is the activation of the tert
gene in primary human fibroblasts (36). The tert gene is
tightly suppressed in primary cells and activated by ectopic Myc
expression through the recruitment of TRRAP-containing chromatin
modifying complexes (33). We found that both WT c-Myc and Myc(Mad-BR)
activated the endogenous tert gene to the same extent (Fig.
5A, bottom panel).

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Fig. 5.
Myc and Myc(Mad-BR) bind to and activate the
same cellular targets. A, wild-type Myc and Myc(Mad-BR) regulate
the same set of genes. Quantitative PCR was performed with cDNA
synthesized on total RNA isolated from cells expressing the empty
vector, Myc, or Myc(Mad-BR) proteins using radiolabeled
oligonucleotides specific for the rat genes shown on the
right. Quantitation of the PCR products was performed using
an Amersham Biosciences phosphorimaging system. Numbers
below a panel indicate the fold induction determined by
dividing an hsp60, nm23, nucleolin,
mSHMT, cad, or gapdh-specific signal
by a corresponding gapdh-specific signal and by the ratio of
these signals in the vector lane. B, wild-type
Myc and Myc(Mad-BR) bind equivalently to the same chromosomal sites in
rat fibroblasts. HO15.19 cells expressing empty vector
(vector), WT c-Myc (indicated by Myc), and
Myc(Mad-BR) were cross-linked and lysed, and chromatin was
immunoprecipitated with c-Myc-specific (M) or Gal4-specific
(G) antibodies followed by the reversal of the cross-linking
and DNA isolation. Isolated DNA was used in PCR with radiolabeled
oligonucleotides flanking Myc binding sites in the promoter of the
genes indicated on the right side. For negative controls,
two types of PCR were performed. The first used oligonucleotides
flanking CACGTG sites that do not interact with c-Myc in the promoter
of rat glucokinase gene (GLU). Another PCR was performed
with oligonucleotides flanking a DNA region containing no CACGTG sites
in the rat pcna gene. Quantitation of the PCR products was
performed by using the Amersham Biosciences phosphorimaging system.
Numbers below a panel indicate the fold
difference in the signal intensity determined by dividing a given PCR
signal by a corresponding pcna-specific signal and by the
ratio of these signals in the Gal4 lane.
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As a definitive test of the in vivo function of the basic
region, we assayed the direct binding of the different proteins to
chromosomal sites by chromatin immunoprecipitation. We analyzed the
binding of Myc proteins to chromosomal promoters in reconstituted Myc-null cells, with cells that lack Myc protein as controls. Formaldehyde cross-linked chromatin was immunoprecipitated with anti-Myc antiserum or a control anti-GAL4 antiserum, and the
immunoprecipitated DNA was analyzed for the presence of four target
genes (hsp60, nm23, nucleolin, and
mshmt). For comparison, we analyzed a CACGTG site in the
glucokinase gene, which does not bind to Myc and also the
pcna gene, which lacks Myc binding sites (Fig.
5B). There was a reproducible enrichment (4-11-fold) of Myc
target gene sequences in the anti-Myc immunoprecipitations in
Myc-reconstituted cells when compared with the signal with the control
anti-GAL4 antiserum. No similar enrichment was observed for the two
genes that do not bind to Myc in vivo. Most significantly,
the binding of the Myc(Mad-BR) protein to chromosomal promoters was
indistinguishable from that of WT c-Myc. The small differences in
signal between c-Myc and Myc(Mad-BR) for the specific experiment shown
were not found reproducibly, and the signals for different genes were
not consistently higher for one protein versus the other. No
significant enrichment of any gene sequences was observed with the
anti-Myc antiserum in the original Myc-null cells when compared
with the anti-GAL4 control.
 |
DISCUSSION |
In this study, we have conducted a thorough analysis of the DNA
recognition properties of the Myc and Mad1 basic regions. We conclude
that there is no measurable difference in DNA site recognition in
vivo between the Myc and Mad1 basic regions, and therefore, that
these two domains can recognize equivalent sets of target genes. The
precise fusion proteins that we analyzed and their resulting
biological activities differ significantly from two previous
reports (30, 31) that addressed similar questions. It is informative to
compare and contrast each study to understand the precise contribution
of the basic region to target gene selection by the Myc/Max/Mad family
of proteins.
Our conclusions differ most significantly from a previous study by
O'Hagan et al. (30) that attempted to create a Mxi-like basic region within Myc through the mutation of 2-4 amino acids in the
Myc basic region to those found at the corresponding positions in Mxi
(and Mad) proteins. The most disruptive mutation in this study was
R357S (numbered from the human c-Myc initiation methionine), which
changes one of the basic amino acids in Myc into a non-basic residue.
It seems quite possible that target gene recognition by Myc is
dependent on an extended set of basic amino acids within this region,
and thus, this mutation debilitates Myc function. The basic region
exchange used in our study included two additional basic amino acids
farther toward the N terminus. We speculate that these amino acids
provide important nonspecific DNA interactions with the phosphate
backbone to increase target gene recognition in vivo. It is
also possible that other conformational aspects of the Mad1 basic
region are preserved in the more extended Mad/Myc exchange. This
interpretation is supported by the partial restoration of transforming
activity in the Myc(R357S/V361E) double mutant, which exchanges two
positions of the Myc basic region with the corresponding residues from
Mxi1 and Mad1 (30). The indistinguishable transforming and
proliferative activity of the Myc and Myc(Mad-BR) exchange mutant in
our study argues that the basic regions of Myc and Mad1 can recognize
the same critical target genes in vivo.
Our findings also contrast a more recent report that the Myc and Mad1
B/HLH/LZ domains differ in apoptotic potential but not in proliferative
activity (31). We find that the c-Myc and Myc(Mad-BR) proteins induce
indistinguishable levels of apoptosis, implying that these two proteins
activate or repress the same target genes that promote apoptosis in the
absence of survival factors. The simplest explanation for the
differences between the two studies is the potential contribution of
the HLH/LZ domain to biological activity since our study exchanged only
the extended basic region and not the entire DNA binding/Max
dimerization domain. Structural studies imply that the helix 1 and loop
domains can affect DNA binding activity (37, 38), although no
differences in DNA binding between WT c-Myc and the Mad bHZ
exchange were found using in vitro assays (31). Of more
interest are reported differences in protein interactions with the
HLH/LZ domains, where Myc can bind to Miz1 and Mad1 cannot (39). We
also note a significant difference in the ability of the previously
described bHZ exchange protein to rescue the growth defect in Myc-null
cells. We find indistinguishable cell doubling times for Myc and
Myc(Mad-BR), whereas the growth rate for the Myc(MadbHZ) exchange was
less than for WT c-Myc (31). Even greater differences were found in the
rescue of the cell cycle defect. In the Myc(MadbHZ) study, 48% of
cells were in G1 for the Myc(MadbHZ) exchange when compared with 58% for Myc-null cells and 28-29% for both Myc reconstituted and parental Rat1 cells (31). Similarly, the S phase fraction was 25%
for Myc-null, 43% for WT c-Myc, and 37% for Myc(MadbHZ). In contrast,
we find indistinguishable profiles for
G1-S-G2/M between Myc and Myc(Mad-BR), similar
to the results for cell doubling time. Thus, the Myc(MadbHZ) exchange
protein only rescues part of the growth defect in Myc-null cells. These
differences between our study and the bHZ exchange study may be
attributable to specific protein-protein interactions with the HLH
and/or LZ domains rather than to any differences in target gene
recognition. It is also possible that the defect in apoptosis noted in
the Myc(MadbHZ) mutant is coupled to the defect in cell cycle rescue.
The most important question that remains unresolved is whether or not
Myc and Mad/Mxi regulate the same target genes under normal
developmental conditions. Our study provides the most extensive evidence to date for the direct binding of Myc and Mad1 to the same
target genes in vivo. Since the endogenous Myc and Mad/Mxi proteins differ in antibody reactivity, it is impossible to provide an
accurate quantitative assessment of the relative binding efficiency of
each protein at individual targets, and it is also difficult to assess
the activity of each protein once binding has occurred. However, in the
differentiating HL60 cells that we studied, Mad1 binds to all of the
Myc target genes we tested under the differentiated/growth arrest
conditions where Mad1 is induced.
Our data support a model in which Myc/Max and Mad/Max heterodimers
recognize equivalent target genes in vivo. The primary biological difference between these complexes is their precise regulation and their recruitment of nuclear cofactors, i.e.
activators versus repressors (reviewed in Ref. 19). Mad/Mxi
family proteins are mainly expressed at distinct stages of development
and differentiation, almost all of which are associated with growth
arrest. An exception to this is the nearly ubiquitous expression of
Mxi1 and the S phase-specific expression of Mad3. In addition,
Mxi1-deficient primary cells have a higher colony-forming ability when
plated at low density, suggesting an enhanced proliferative capacity (27). In contrast, Myc proteins are expressed in all dividing cells and
repressed under virtually all growth arrest conditions. Thus, in many
cells, target genes will be down-regulated in non-dividing cells by the
absence of Myc activation but not through active repression by Mad/Mxi.
Furthermore, we note that most Myc target genes are still expressed
basally even in the complete absence of Myc, such as in Myc-null
fibroblasts (33, 40). This Myc-independent target gene expression is
likely due to promoter elements that bind other nuclear factors, and
the Myc-dependent induction of its targets is usually quite
modest (3-fold) in log phase cells (33, 40). One factor with a
potential role in this system is Mnt, which is simultaneously expressed
in dividing cells with Myc. Mnt can bind the Sin3A corepressor and
block Myc function (41), and Mnt/Max complexes are significantly more
abundant than Myc/Max complexes in nuclear extracts (26, 42). It is not
known whether Mnt/Max heterodimers constitutively repress Myc target
genes and act to down-regulate their expression when Myc is repressed.
Moreover, induction of Mad/Mxi under conditions where Myc is already
down-regulated may recruit additional repressive cofactors to shut down
target gene promoters below their level of basal transcription. Yet
another complication in this system is the upstream stimulatory
factor (USF) transcription factor, which has been shown to bind to many
Myc/Max binding sites under conditions when Myc is not expressed (43).
Our data are consistent with a model in which all Myc target genes can
be suppressed by Mad/Mxi when the latter proteins are induced in
development and/or differentiation.