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Oncogene (2001) 20, 4650 ± 4664
2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00
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Dynamic in vivo interactions among Myc network members
Xiao-ying Yin1, Melanie F Landay1, Weiping Han2, Edwin S Levitan2, Simon C Watkins3,5,
Richard M Levenson6, Daniel L Farkas6,7 and Edward V Prochownik*,1,4,5
1
Section of Hematology/Oncology, Department of Pediatrics, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, PA
15213, USA; 2Department of Pharmacology, The University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, PA 15213,
USA; 3Department of Cell Biology and Physiology, The University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, PA
15213, USA; 4Department of Molecular Genetics and Biochemistry, The University of Pittsburgh Medical Center, Pittsburgh,
Pennsylvania, PA 15213, USA; 5The University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, PA 15213, USA; 6Center
for Light Microscope Imaging and Biotechnology, Carnegie-Mellon University, Pittsburgh, Pennsylvania, PA 15213, USA;
7
Department of Bioengineering, The University of Pittsburgh, Pittsburgh, Pennsylvania, PA 15213, USA
Members of the Myc oncoprotein network (c-Myc, Max,
and Mad) play important roles in proliferation, di€erentiation, and apoptosis. We expressed chimeric green
¯uorescent protein (GFP) fusions of c-Myc, Max, and
three Mad proteins in ®broblasts. Individually, c-Myc
and Mad proteins localized in subnuclear speckles,
whereas Max assumed a homogeneous nuclear pattern.
These distributions were co-dominant and dynamic,
however, as each protein assumed the pattern of its
heterodimeric partner when the latter was co-expressed
at a higher level. Deletion mapping of two Mad
members, Mad1 and Mxi1, demonstrated that the
domains responsible for nuclear localization and speckling are separable. A non-speckling Mxi1 mutant was
also less e€ective as a transcriptional repressor than
wild-type Mxi1. c-Myc nuclear speckles were distinct
from SC-35 domains involved in mRNA processing.
However, in the presence of co-expressed Max, c-Myc,
but not Mad, co-localized to a subset of SC-35 loci.
These results show that Myc network proteins comprise
dynamic subnuclear structures and behave co-dominantly
when co-expressed with their normal heterodimerization
partners. In addition, c-Myc-Max heterodimers, but not
Max-Mad heterodimers, localize to foci actively engaged
in pre-mRNA transcription/processing. These ®ndings
suggest novel means by which Myc network members
promote transcriptional activation or repression. Oncogene (2001) 20, 4650 ± 4664.
Keywords: Max; Mad Mxi1; green ¯uorescent protein
(GFP); SC-35; transcription
*Correspondence: EV Prochownik, Section of Hematology/
Oncology, Department of Pediatrics, Children's Hospital of
Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA;
E-mail: [email protected]
Received 16 January 2001; revised 3 May 2001; accepted 9 May
2001
Introduction
c-Myc, a basic ± helix ± loop ± helix ± leucine ± zipper
(bHLH ± ZIP) transcription factor, plays critical roles
in proliferation, di€erentiation, transformation, and
apoptosis (Henriksson and LuÈscher, 1996; Facchini
and Penn, 1998; Nesbit et al., 1999). c-Myc is highly
expressed during proliferation, in contrast to the low
levels associated with cellular quiescence and terminal
di€erentiation (Henriksson and LuÈscher, 1996; Facchini and Penn, 1998). Following removal of growth
factors, c-Myc mRNA and protein rapidly decline
(Blanchard et al., 1985; Rabbits et al., 1985). Mitogenic
stimulation of quiescent cells is accompanied by a
rapid re-induction of c-Myc mRNA and protein with
immediate-early response kinetics (Kelly et al., 1983;
Campisi et al., 1984).
In common with certain other bHLH, bZIP, and
bHLH ± ZIP proteins, c-Myc neither homodimerizes
nor binds DNA well in vitro (Smith et al., 1990), and cMyc homodimers have not been detected in vivo.
Rather, the biologically active form of the protein
exists in heterodimeric association with another nuclear
bHLH ± ZIP protein, Max (Blackwood and Eisenman,
1991; Prendergast et al., 1991; Amati et al., 1992;
Blackwood et al., 1992). Because Max is an extremely
stable protein (Blackwood et al., 1992; Wagner et al.,
1992; Dunn et al., 1994), the relative abundance of cMyc ± Max heterodimers is determined to a large
degree by the absolute amount of c-Myc.
In addition to c-Myc's bHLH ± ZIP domain, which
promotes dimerization with Max and DNA binding
(Blackwell et al., 1990; Blackwood and Eisenman,
1991; Van Antwerp et al., 1992), the protein contains a
nuclear localization signal (NLS) and a transcriptional
activation domain (TAD) (Henriksson and LuÈscher,
1996; Facchini and Penn, 1998). These regions are
required for all known c-Myc functions (Henriksson
and LuÈscher, 1996).
Whereas c-Myc ± Max heterodimers activate c-myc
target genes, transcriptional repression is mediated by
heterodimers between Max and at least four other
related bHLH ± ZIP family members known as Mad
Myc network interactions
X-y Yin et al
proteins (Ayer et al., 1993; Zervos et al., 1993; Hurlin
et al., 1995a). Repression by Mad proteins is an active
process, requiring a highly conserved amino-terminal
motif (the SID domain) (Ayer et al., 1995; SchreiberAgus et al., 1995). Mad members also show highly
variable levels of tissue-speci®c expression and regulation (Larsson et al., 1994; Hurlin et al., 1995a,b;
VaÈstrik et al., 1995; Queva et al., 1998). Studies in
mad1 and mxi1 nullizygous mice have demonstrated
distinct roles for the encoded proteins in tissue-speci®c
negative control of cellular proliferation (Foley et al.,
1998; Schreiber-Agus et al., 1998).
Despite the well-described interactions of Myc
network proteins, little is known about their distribution within the nucleus, or how they seek out one
another, and dimerize in an appropriate, timely, and
controlled manner. Such studies are hindered by the
fact that no cells express all the proteins simultaneously, and by the often low abundance of those
proteins which are expressed. In the current study, we
have used green ¯uorescent protein (GFP) fusions of cMyc, Max, and three Mad proteins (Mad1, Mxi1, and
Mad3) to study their subnuclear locations and
interactions. Our ®ndings indicate that, depending
upon the relative abundance of their heterodimeric
partners, Myc network proteins may either occupy
distinct compartments (`speckles') or be di€usely
distributed throughout the nucleus. We show that for
Mad proteins, speckling requires a speci®c domain
within the polypeptide, distinct from the NLS, and
necessary for e€ective transcriptional repression.
Furthermore, c-Myc nuclear speckles overlap a subset
of sites engaged in pre-mRNA transcription and
processing. Our studies suggest that dynamic subnuclear tracking of Myc network proteins is
intimately associated with their function as transcriptional activators or repressors.
Results
Myc network members show distinct patterns of
subnuclear localization
To localize each Myc network protein within the
nucleus, the complete coding sequences of c-Myc,
Max(L), Max(S), Mad1, Mxi1, and Mad3 were
expressed as GFP fusions in three di€erent ®broblast
lines. So as to avoid over-expression artifacts (Hedley
et al., 1995), short transfection times (2 ± 4 h) and posttransfection incubation periods (3 ± 6 h) were used.
Cells were then ®xed and examined by epi¯uorescence
microscopy (Figure 1). Transfection with the pEGFPC1 parental vector alone produced a di€use, primarily
cytoplasmic ¯uorescence (not shown), whereas each of
the Myc network fusions localized exclusively to the
nucleus in all three cell lines. In the case of GFP ± cMyc, the fusion protein assumed a speckled subnuclear
appearance that was most readily appreciated in Cos-7
cells (Figure 1). In contrast, both GFP ± Max(L) and
GFP ± Max(S) produced an extremely homogeneous
nuclear staining. Pronounced speckling pattern were
also seen with Mad1, Mxi1, and Mad3. Mad1 and
Mad3 fusion proteins tended to form the most
pronounced speckles, whereas those formed by
GFP ± Mxi1 tended to be somewhat smaller and more
numerous. Examination of several hundred cells
transfected with each construct con®rmed the nuclear
patterns shown in Figure 1.
From the above experiments, we conclude that each
of the GFP ± Myc network proteins was expressed in
distinct subnuclear patterns in three di€erent cell types.
Experiments in several other ®broblast cell lines have
shown similar patterns of expression (not shown).
In order to rule out the possibility that the fusion of
GFP to the amino terminus of Myc network members
was responsible for their distinct subnuclear patterns,
each protein was expressed in the pEGFP ± N3 vector
as a carboxy terminal fusion with GFP. Expression of
these constructs in all three cell lines produced
patterns identical to those obtained with the
pEGFP ± C1-based amino terminal fusions depicted
in Figure 1. In still other experiments, each protein
was epitope tagged at the carboxy terminus and
detected by indirect immuno¯uorescence. Although
staining was less intense than with GFP fusions
owing, at least in part, to di€erences in promoter
strength and the method of detection, the subnuclear
patterns observed were identical to those seen with
GFP fusions (not shown).
Finally, we addressed the possibility that the patterns
shown in Figure 1 were the result of ®xation or
overexpression artifacts. Several di€erent methods of
®xation, performed at various times after transfection,
were employed to examine this. In all cases, identical
subnuclear localization patterns were seen. In other
studies, time-lapse video UV-microscopy was used to
follow the synthesis and nuclear targeting of GFPtagged molecules in live cells as soon as the proteins
became observable. In each case, we were able to
identify newly synthesized GFP fusion proteins in the
cytoplasm, and to follow their immediate transport to
the nucleus where they rapidly assumed the patterns
shown in Figure 1 (not shown).
From the above studies, we conclude that the
patterns of expressed proteins were not the artifactual
result of GFP tagging, of cellular ®xation, or of
excessive overexpression, but rather re¯ected intrinsic
properties of the Myc network proteins themselves.
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Some Myc network proteins co-localize and act
co-dominantly
The localization of c-Myc and mad family members to
the nucleus in discrete, speckled patterns raised the
question of whether these proteins occupied the same
subnuclear space. To address this, we created new
fusions between certain Myc network proteins and
mutant forms of GFP that possess di€erent excitation/
emission spectra (Tsien, 1998). These mutants have
been dubbed `Sapphire' (Sap) and `Topaz' (Top) (Yin
et al., 1999). In initial experiments shown in Figure 2,
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Figure 1 Nuclear patterns of Myc network proteins. The indicated cell lines were transfected with Myc network-GFP expression
plasmids and visualized 3 ± 6 h later after ®xing in PBS ± 2% paraformaldehyde. Max(L) and Max(S) indicate the 160 and 151 amino
acid isoforms of the Max protein, respectively (Blackwood and Eisenman, 1991; Prendergast et al., 1991). All photos:61000
transfection of Cos-7 cells with only the single Sap ±
Mad1 construct produced the expected speckled
pattern when examined under optimal Sap conditions
(Figure 2a). In contrast, minimal or no ¯uorescence
was detected using optimal Top conditions (Figure 2b).
Similarly, under Top conditions, we observed bright
speckled ¯uorescence following single transfections
with Top ± c-Myc or Top ± Mad 3 (Figure 2c,e) whereas
minimal or no ¯uorescence was detected using Sap
detection conditions (Figure 2d,f).
The above vectors were next used to determine
whether the subnuclear compartments of co-expressed
proteins were identical or spatially distinct. In the ®rst
case, Cos-7 cells were co-transfected with expression
vectors for Sap ± Mad1 and Top ± Mad3 at a 1 : 1 molar
ratio. Examination of single Sap- and Top-speci®c
images, as well as the digitally superimposed images,
showed that the two proteins co-localized (Figure 2g ±
i). Similarly, a transfection in which Sap ± Mad1 and
Top ± c-Myc were co-expressed indicated that these
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proteins also localized to identical sub-nuclear compartments (Figure 2j ± l). Co-localization of Top ± cMyc and Sap ± Mxi1 was also seen (not shown).
Because c-Myc and Mad proteins neither homodimerize nor heterodimerize with one another, we conclude
that their co-localization does not require their direct
interaction.
Max, di€usely expressed in the nucleus (Figure 1), is
the only member of the Myc network that heterodimerizes with each of the other members. It thus
performs a unique and central role in the control of
both transcriptional activation and repression (Henriksson and LuÈscher, 1996). To determine whether the
expression of Max could in¯uence the subnuclear
location of c-Myc and Mad proteins and vice versa,
we constructed a Sap ± Max(L) vector and co-expressed
it at di€erent ratios together with Top-c-Myc, Top ±
Mad1, or Top ± Mad3 in Cos-7 cells. As with single
transfection studies using other Sap constructs (Figure
2), Sap ± Max was readily detectable under optimal Sap
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Figure 2 Nuclear co-localization of Myc and Mad member proteins. Two spectral variants of GFP, Sapphire (Sap) and Topaz
(Top) (Tsien, 1998) were fused to each of the indicated Myc network members. NIH3T3 cells were transfected with the appropriate
1 : 1 molar ratio of the plasmids and then ®xed as described in the legend to Figure 1. Individual GFP-tagged proteins were then
visualized under Sapphire or Topaz conditions. (a) Cells expressing only Sap-Mad1 only and visualized under Sapphire conditions;
(b) the same ®eld visualized under Topaz conditions; (c) cells expressing Top ± c-Myc only and visualized under Topaz conditions;
(d) the same ®eld visualized under Sapphire conditions; (e) cells expressing Top ± Mad3 only and visualized under Topaz conditions;
(f) the same ®eld visualized under Sapphire conditions; (g) cells expressing with Sap ± Mad1 and Top ± Mad3 and visualized under
Sapphire conditions; (h) the same ®eld visualized under Topaz conditions; (i) digitally superimposed image of g and h; (j) cells
expressing Sap ± Mad1 and Top ± c-Myc and visualized under Sapphire conditions; (k) the same ®eld visualized under Topaz
conditions; (l) digitally superimposed image of j and k. (61000)
conditions whereas it was barely detectable under
Topaz conditions (compare Figure 3a and b).
We then co-expressed Sap ± Max with either Top ± cMyc, Top ± Mad1, or Top ± Mad3 at 5 : 1 molar ratios.
Under these conditions of relative Max excess, its
nuclear pattern remained di€use and homogeneous as
previously demonstrated in Figure 1 (Figure 3c,e,g).
Under Top detection conditions, however, the patterns
of c-Myc, Mad1 and Mad3 now appeared homogeneous rather than punctate (Figure 3d,f,h). When the
ratios of co-transfected plasmids were reversed, SapMax assumed a speckled pattern (Figure 3i,k,m). As
expected, the patterns of c-Myc, Mad1, and Mad3,
when viewed under Top conditions, remained speckled
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Figure 3 Co-dominant behavior of Myc network proteins. NIH3T3 cells were co-transfected with the indicated ratios of plasmids
encoding Sapphire- or Topaz-tagged members of the Myc network as described in the legend to Figure 2. Proteins were then visualized
under Sapphire or Topaz conditions. (a) Cells transfected with Sap ± Max(L) only and visualized under Sapphire conditions; (b) the
same ®eld visualized under Topaz conditions; (c) cells transfected with Sap ± Max(L)+Top-c-Myc (5 : 1 molar ratio) and visualized
under Sapphire conditions; (d) the same ®eld visualized under Topaz conditions; (e) cells transfected with Sap ± Max(L) and Top ±
Mad1 (5 : 1 ratio) and visualized under Sapphire conditions; (f) the same ®eld visualized under Topaz conditions; (g) cells transfected
with Sap ± Max(L) and Top ± Mad3 (5 : 1 ratio) and visualized under Sapphire conditions; (h) the same ®eld visualized under Topaz
conditions; (i), cells transfected with Sap ± Max(L)+Top ± c-Myc (1 : 5 ratio) and visualized under Sapphire conditions; (j) the same
cells visualized under Topaz conditions; (k) cells transfected with Sap ± Max(L)+Top ± Mad1 (1 : 5 ratio) and visualized under
Sapphire conditions; (l) the same ®eld visualized under Topaz conditions; (m) cells transfected with Sap ± Max(L)+Top ± Mad3 (1 : 5
ratio) and visualized under Sapphire conditions; (n) the same ®eld visualized under Topaz conditions. 61000
(Figure 3j,l,n). These experiments establish that Max
and its normal heterodimerization partners act in a codominant manner, with each assuming the pattern of
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whichever co-expressed protein is the more abundant.
These experiments also indicate that appropriate
heterodimeric interactions between the HLH ± ZIP
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X-y Yin et al
domains of Max and other Myc network members
occur despite the presence of GFP tags (Blackwood
and Eisenman, 1991; Ayer et al., 1993; Zervos et al.,
1993; Hurlin et al., 1995a).
Mad nuclear and subnuclear targeting sequences are
separable
The observation that, in three di€erent cell lines, Mad
member proteins localized in a speckled pattern
following their transport into the nucleus (Figure 1)
a€orded the opportunity to determine whether the
sequences responsible for imparting these attributes
were distinct and genetically separable. Accordingly,
we constructed a series of deletion mutants of Mad1
and Mxi1 and expressed each in the pEGFP-C1 vector
as amino terminal-linked GFP fusions. Most mutants,
along with their full-length counterparts, were tested in
more than one cell line and gave consistent results.
Only the results with NIH3T3 cells are shown here.
In the case of Mad1 (Figure 4), two deletions from
the carboxy terminus of the protein (Mad11 ± 178 and
Mad11 ± 141) retained both the ability to be targeted to
the nucleus and to be expressed, like the full-length
protein, in a speckled pattern. Further deletion from
the carboxy terminus (Mad11 ± 124), caused loss of the
speckled pattern, although the ability to localize to the
nucleus remained intact. Deletion beyond this point
(Mad11 ± 91) also showed loss of the speckled pattern
and a partial loss of nuclear retention.
Deletions from the amino terminus of Mad1 were
also revealing. Thus, elimination of the ®rst 42 amino
acids (Mad143 ± 221) did not a€ect nuclear localization
but instead caused a marked loss of speckling. Further
deletion from the amino terminus (Mad191 ± 221) caused
a substantial, although once again incomplete, loss of
nuclear targeting. From these experiments, we conclude
that nuclear targeting is most in¯uenced by residues
centered around amino acid 91 of Mad1. In contrast,
signals for speckling appeared to be bipartite, consisting of residues between amino acids 1 ± 42 and 124 ±
141. Although both of these regions were necessary for
the protein to assume a speckled appearance, neither
appeared to be sucient. Thus, in the case of Mad1,
the nuclear localization and speckling signals map to
distinct and separable domains.
Similar experiments performed with Mxi1 demonstrated that a construct containing signi®cant deletions
of both the amino and carboxy termini (Mxi138 ± 192)
was targeted exclusively to the nucleus and assumed
the typical speckled pattern of the wild-type protein
(Figure 5). In contrast, a construct containing the
complete amino terminus but with a longer C-terminal
deletion (Mxi11 ± 152), while also targeted to the nucleus,
failed to localize in a speckled pattern. The construct
Mxi138 ± 175 also failed to form a speckled pattern. This
suggested that at least one region of Mxi1 responsible
for speckling was located between residues 175 and
192. Consistent with these ®ndings, two additional
mutants, Mxi138 ± 147, and Mxi138 ± 131 were also
targeted to the nucleus but in a di€use pattern.
Additional deletions of the C-terminus provided
evidence for the location of the nuclear localization
and speckling signals. Although nuclear localization
was retained with the Mxi170 ± 228 deletion, a substantial, although incomplete, loss of speckling was
observed. Mxi181 ± 228 showed a signi®cant amount of
cytoplasmic staining. Although the nuclear pattern was
somewhat obscured by overlapping cytoplasmic staining, examination of multiple individual cells indicated
that a speckled appearance was not generally maintained, despite the presence of the previously identi®ed
speckling sequences between residues 175 and 192. An
even more profound e€ect on nuclear localization was
seen with Mxi1131 ± 228. These results suggested that a
strong nuclear localization signal in Mxi1 resides
between residues 81 and 131 with perhaps an
additional weaker component centered around or just
upstream of residue 81. Thus, as in the case of Mad1,
the speckling signal in Mxi appears bipartite with
important residues residing in the regions 38 ± 81 and
175 ± 192.
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Speckling signals are necessary for normal Mad protein
function
To determine whether the ability to localize in a
speckled pattern was important for Mad protein
function, we created a mammalian expression vector,
pSVLneo ± Mxi11 ± 152 that encoded a C-terminally
truncated deletion mutant of the Mxi1 protein. Because
this mutant does not a€ect the bHLH ± ZIP or SID
domains, it retains the ability to dimerize with Max
and recruit the mammalian co-repressor mSin3 and its
associated proteins (Ayer et al., 1995; Schreiber-Agus
et al., 1995). However, based upon the ®ndings
presented in Figure 5, we predicted that this protein
would assume a di€use rather than a speckled pattern
upon its translocation to the nucleus. We compared the
ability of Mxi11 ± 152 and Mxi1(WT) to suppress a cMyc-responsive reporter construct. As seen in Figure
6a, a c-Myc expression vector stimulated the reporter
about fourfold in comparison to the empty vector
alone. As we and others have previously shown (Hurlin
et al., 1995a; Gupta et al., 1998; Yin et al., 1999), coexpression of Mxi1(WT) resulted in a dose-dependent
suppression of reporter activity, consistent with the
prevailing model that Mad ± Max heterodimers compete with c-Myc ± Max heterodimers for the same
binding sites, and mediate transcriptional repression.
In contrast, the truncated Mxi11 ± 152 protein showed
signi®cantly less ability to repress the reporter. This
was not the result of less protein being produced in the
case of Mxi11 ± 152 as indicated by Western analysis of
the same cells using an anti-Mxi1 antibody (Figure
6B). Finally, as expected, both bacterially expressed
full-length Mxi1 and Mxi11 ± 152 dimerized with Max
and bound an E-box-containing oligonucleotide (Figure 6c). Taken together, these studies suggest that
proper subnuclear localization is necessary for members of the Mad family to ful®l their roles as
transcriptional repressors.
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Figure 4 Deletion mapping of Mad1. PCR products encoding the indicated amino acids were cloned into the pEGFP ± C1 vector
to allow expression of the appropriate fusion protein. That each vector was in-frame with the GFP coding sequence and encoded the
indicated residues was veri®ed by DNA sequencing. WT indicates the full-length Mad1 protein (Figure 1). The bHLH ± ZIP domain
is indicated by the open box. Relevant restriction sites, used in the construction of some fusions, are also noted. Each plasmid DNA
was then transiently expressed in Cos-7 cells. (a) Fusion proteins expressed in a nuclear or speckled pattern, respectively, are
indicated by (+); those proteins expressed in a partially nuclear or partially speckled pattern are expressed as (+), whereas those
expressed in neither a nuclear nor speckled pattern are denoted (7). (b) Representative images of cells expressing the indicated
fusion protein.61000
Western blot confirmation of GFP fusion protein
expression
To demonstrate that all of the foregoing GFP
constructs expressed the predicted fusion proteins,
Western analyses were performed using lysates from
transfected Cos-7 cells. Fusion protein expression was
then detected using an anti-GFP antibody. As seen in
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Figure 7, each construct encoded a chimeric GFP
protein of the predicted size.
Association of c-Myc ± Max complexes with sites of
mRNA processing
Some pre-mRNA transcription and splicing occurs in
distinct subnuclear domains that can be identi®ed with
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X-y Yin et al
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Figure 5 Deletion mapping of Mxi1. Fusion constructs were created and veri®ed as described in the legend to Figure 4 and transiently
expressed in Cos-7 cells. Relevant restriction sites, used in the construction of some fusions, are also noted. (a) (+), (+) and (7) indicate
protein patterns as described in the legend to Figure 4. (b) Representative images of cells expressing the indicated fusion proteins.61000
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Figure 6 Comparison of Mxi1(WT) and Mxi11 ± 152 function. (a) Transient transfections were performed in NIH3T3 cells as
previously described (Zhang et al., 1997; Gupta et al., 1998) using the indicated amounts of each plasmid. Di€erences in amounts of
input DNAs were adjusted for by the addition of the empty pSVLneo parental vector. Following transfection, cells were grown for
an additional 2 days at which time they were harvested and assayed for b-galactosidase and luciferase activities (Zhang et al., 1997;
Gupta et al., 1998). The results shown are the average of three separate experiments, each performed in duplicate plates,+1 s.e. (b)
In separate transfections, 5 mg each of the parental pSVLneo vector, pSVLMxi1(WT) or pSVLMxi1(1 ± 152) were transiently
expressed in NIH3T3 cells. Cells were lysed after 2 days, total lysates were resolved by SDS ± PAGE, and the Western blot was
probed with an anti-Mxi1 polyclonal antibody (Gupta et al., 1998) (upper panel) or an anti-actin polyclonal antibody to serve as a
control for protein loading (lower panel). (c) Each of the above proteins, along with full-length Max(S) was expressed in E. coli,
puri®ed by nickel-agarose anity chromatography and tested for its ability to bind to a double-stranded 32P-labeled oligonucleotide
containing a c-Myc binding site (Gupta et al., 1998)
the SC-35 spliceosome assembly factor monoclonal
antibody (Fu and Maniatis, 1990). These domains are
of approximately the same size and abundance as Mmyc and Mad protein speckles (Huang and Spector,
1991; Spector et al., 1991; Hemavathy et al., 2000;
Smith et al., 1999). To determine whether SC-35
domains co-localize with c-Myc and Mad speckles,
Cos-7 cells were transfected with individual GFP ± cMyc or GFP ± Mad3 expression vectors, ®xed, and
immunostained with the SC-35 monoclonal antibody.
Nuclei were then visualized by laser confocal microscopy. As seen in Figure 8 (rows 1 and 2) GFP ± cMyc and GFP ± Mad3 localized to the previously
described nuclear speckles. SC-35 domains were also
detected (row 2) but were spatially separate from
those of GFP ± c-Myc and GFP ± Mad3 (merged
images, row 3). The examination of several hundred
transfected cells in each experiment revealed no
instances of co-localization of GFP ± c-Myc or
GFP ± Mad3 with SC-35 domains. The absence of
even a random pattern of overlap between c-Myc or
Mad and SC-35 domains indicated an active process
serving to maintain strong segregation between these
structures.
Because c-Myc and Mad3 must heterodimerize with
Max in order to activate or repress transcription, we
investigated the possibility that the exclusion of GFP ±
c-Myc and GFP ± Mad3 from SC-35 domains re¯ected
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their excess concentration relative to that of endogenous Max. GFP ± c-Myc and GFP ± Mad3 vectors were
co-transfected with a non-GFP-tagged Max expression
plasmid (Zhang et al., 1997). The latter was added at a
molar ratio (generally 2 : 1 ± 3 : 1) that allowed for the
incipient transition of GFP ± c-Myc or GFP ± Mad3
speckles into the more homogenous, previously
described, `Max-like' pattern (Figures 1 and 3). The
cells were then ®xed, stained for SC-35 domains and
examined for GFP and SC-35 co-localization. Under
these conditions, we observed that a subset of the
speckles now co-localized with SC-35 domains (Figure
8, row 3). In most cases, the overlap occurred at the
periphery of the SC-35 domains, which are the regions
within which actual mRNA transcription has been
proposed to occur (Xing et al., 1993, 1995; Smith et al.,
1999). Examination of several hundred SC-35 domains
from several independent experiments indicated that
approximately 15% of them co-localized with GFP-cMyc signals.
In contrast to the ®ndings above, co-expression of
GFP ± Mad3 and Max failed to demonstrate any
overlap between with SC-35 domains in several
experiments (Figure 8, row 4). These studies suggested
that, unlike the co-localization of GFP ± c-Myc and
GFP ± Mad (Figure 2) c-Myc ± Max and Mad-3 ± Max
complexes occupy distinct nuclear compartments.
These experiments also further indicated that the
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X-y Yin et al
tion. In the cases of c-Myc and all three members of the
Mad family studied, these patterns consist of dots or
speckles, whereas the distribution of Max is homogeneous. A second conclusion is that c-Myc and Mad
members co-localize. Although these proteins are
generally expressed exclusively of one another, clear
exceptions exist (Larsson et al., 1994; Zervos et al., 1993;
Hurlin et al., 1995b). Since none of these proteins has
been shown to homodimerize or to heterodimerize with
one another (Henriksson and LuÈscher, 1996), it seems
unlikely that a direct interaction is necessary for their
proper subnuclear targeting. This co-localization may
therefore indicate a common nuclear compartment in
which these monomeric proteins, whether co-expressed
or not, are sequestered prior to their association with
Max. Depending upon the proliferative state of the cell,
its tissue of origin, and its degree of di€erentiation, this
compartment may contain only c-Myc, only one or more
Mad proteins, or a combination of c-Myc and Mad
proteins. Following their association with Max, these
proteins may then be dispersed throughout the nucleus if
Max is in excess, or remain within the nuclear speckles if
Max is less abundant. Thus, a third conclusion is that
Myc network proteins are co-dominant, with the less
abundant member of any given pair assuming the pattern
of its more abundant natural partner. We also conclude
that, at least in the case of Mxil, its ability to localize in a
speckled pattern is closely linked to its function as a
transcriptional repressor of c-Myc target genes.
4659
Distinct nuclear localization and `speckling' signals
Figure 7 Western blot analysis of GFP fusion proteins. Plasmids
encoding each of the indicated full-length (a), or various Mad1 or
Mxi1 deletions (b and c) was transiently expressed in Cos-7 cells.
Two days later, 25 mg of total cell lysate was resolved by standard
10% SDS ± PAGE and Western blotted. GFP proteins were
detected with a polyclonal anti-GFP antibody
c-Myc ± Max and SC-35 overlap patterns observed
above did not simply re¯ect random events.
Discussion
Subnuclear localization of Myc network members
One conclusion of this study is that Myc network
proteins possess distinct patterns of subnuclear localiza-
The c-Myc NLS has been previously shown to be
bipartite (Dang and Lee, 1988). However, these
regions, when separated from other regions of the
molecule, are unable to impart a speckled appearance
to the nuclearly localized protein. These observations
are consistent with the conclusions of this study,
namely, that the signals responsible for nuclear
targeting and subsequent subnuclear localization of
Mad family members comprise distinct elements.
Based on the deletions depicted in Figure 4, the NLS
of Mad1 appears to be centered around amino acid 91,
which is within the loop region of the bHLH domain.
Since both Mad11 ± 91 and Mad191 ± 221 retained signi®cant nuclear localization capability, it is possible
that, like c-Myc, the NLS of Mad1 is bipartite. Indeed,
within the region spanning amino acids 43 ± 124 are
several potential NLSs. These include the region
K45RRNK49, originally proposed as the NLS (Ayer
et al., 1993), K49SKK52, E65KNRR69, E77KLKG81,
and K100AKLHIKK107. Despite this ambiguity, it is
clear from our studies that none of the constructs
containing these regions was able to recapitulate the
speckled patterns of the wild-type protein unless
additional amino terminal and carboxy terminal
regions were included.
The NLS of Mxi1 also localizes to the loop region
and appears bipartite. One NLS is encoded by the 81 ±
131 region whereas a weaker one is encoded by the
70 ± 81 region. Homologies to the putative NLS's of
Oncogene
Myc network interactions
X-y Yin et al
4660
Figure 8 Co-localization of c-Myc ± Max complexes and SC-35 domains. Cos-7 cells were transfected with 1 mg of either the
GFP ± -c-Myc expression vector (row 1) or the GFP ± Mad3 expression vector (row 2). A soon as the GFP signal became detectable,
the cells were ®xed and stained with the anti-SC-35 monoclonal antibody and examined by confocal microscopy. The merged images
show a complete lack of c-Myc or Mad3 and SC-35 co-localization. In rows 3 and 4, the above experiments were repeated except
that a non-GFP-tagged Max expression vector (Zhang et al., 1997) was added at a 2 ± 3-fold molar excess. This amount was
sucient to initiate the early transition of c-Myc and Mad3 from a speckled to the more di€use pattern assumed by Max. Staining
for SC-35 revealed its co-localization with c-Myc ± Max in a subset of SC-35 domains (row 3, arrows). Co-localization was still not
observed with SC-35 and Mad3-Max complexes (row 4)
Mad1 are seen in both segments. For example, the
sequences E76KNRR80 and K111AKAHIKK118 in
Mxi1 are identical or nearly identical to the sequences
E65KNRR69 and K100AKLHIKK107, respectively, in
Mad1. Of note is that these sequences are also well
conserved in Mad3 and Mad4. In contrast, the
originally proposed Mad1 NLS (K45RRNK49, Ayer
et al., 1993) has no counterpart in other Mad proteins
(Hurlin et al., 1995a).
Additional regions in Mad1 and Mxi1 serve to direct
the proteins to speci®c subnuclear speckles. As with the
NLS's, these regions appear to be bipartite. In the case
Oncogene
of Mad1, two regions, between amino acids 1 ± 42 and
124 and 141 are essential for speckling. Similarly, the
regions of Mxi1 necessary for speckling also localize to
separable regions of the molecule between amino acids
38 ± 81 and 175 ± 192 (Figure 4).
A large number of nuclear proteins assume a
speckled or dot-like pattern, and often physically
interact with and co-localize with other proteins with
which they share common functions (Spector, 1993;
Leonhardt and Cardoso, 1995; Mortillaro et al., 1996;
Kim et al., 1997; Cardoso and Leonhardt, 1998;
Scully and Livingston, 2000). In the case of some
Myc network interactions
X-y Yin et al
splicing factors, a common Ser/Arg-rich motif is
necessary for subnuclear localization and is distinct
from the NLS (Li and Bingham, 1991; Hedley et al.,
1995). Similarly, unrelated proteins such as DNA
methyltransferase (Leonhardt et al., 1992; AML ±
CBFa (Zeng et al., 1997), and DNA ligase (Cardoso
et al., 1997) also contain separable NLS's and
subnuclear targeting sequences. Our ®ndings with
Mad1 and Mxi1 are thus in keeping with this overall
theme.
Despite the presence of homologous subnuclear
targeting signals in splicing factors, most other proteins
that co-localize to nuclear speckles share neither these
nor other recognizable motifs to explain their common
topographical distribution. This suggests that only a
small number of the proteins comprising a speckle
contain an actual subnuclear targeting sequence. These
proteins could serve as nucleation centers for other
factors that would then be passively recruited to the
speckles. Indeed, such interactions have been observed
among splicing factors (Hedley et al., 1995). Such a
hierarchy of di€erentially interacting Myc network
proteins might explain how unrelated factors such as
mSin3, histone deacetylase and other proteins that
constitute the Mad ± Max transcriptional repressor
complex, and that interact through di€erent domains,
might co-localize without actually possessing distinct
speckling signals (Alland et al., 1997; Heinzel et al.,
1997).
Depending upon the level of Max expression, both cMyc and Mad nuclear speckles appear to be quite
ephemeral. Similar behavior is seen with the PML
RING-®nger `POD' domains, which are disrupted in
acute promyelocytic leukemia but reassembled in the
presence of retinoic acid (Dyck et al., 1994; Weiss et
al., 1994; Shulman and Evans, 1997). In another
example, DNA methyl-transferase localizes to replication foci during S phase but is dispersed during G1 and
G2/M (Leonhardt et al., 1992). Finally, the BRCA1
tumor suppressor protein localizes to a small number
of nuclear dots during S phase. DNA damaging agents
cause loss of BRCA1 from these foci and its reaccumulation at sites of DNA replication (Chen et al.,
1996; Scully et al., 1997a,b).
Myc network proteins and sites of transcription
SC-35 domains are thought to represent sites at which
factors necessary for pre-mRNA transcription, splicing, and polyadenylation are allowed to accumulate
at locally high concentrations (Spector et al., 1991;
Blencowe et al., 1994; Bregman et al., 1995; Smith et
al., 1999). The edges of these domains also represent
sites of active transcription of some, but not all, genes
(Xing et al., 1993, 1995; Moen et al., 1995; Clemson
and Lawrence, 1996; Smith et al., 1999). In contrast,
the observation that class I and II histone deacetylases, along with the nuclear co-repressors N-CoR and
SMRT, localize to nuclear bodies distinct from SC-35
domains (`matrix-associated deacetylase bodies'
(Downes et al., 2000), suggests that transcription and
repression may be segregated. Our ®nding that cMyc ± Max, but not Mad ± Max heterodimers, colocalize to the periphery of a subset of SC-35
domains, is in keeping with the above observations.
This raises the possibility that the transcription of
multiple c-Myc-regulated genes may occur in close
physical proximity to one another within a subset of
SC-35 domains, but that these domains exclude Mad
proteins.
It is possible to suggest a model that accounts for
our observations with myc network proteins. In this
model, c-Myc ± Max heterodimers localize to sites of
ongoing transcription of c-Myc-target genes. Mad ±
Max dimers localize to di€erent areas, most likely in
association with factors devoted to transcriptional
repression. This need not occur concurrently; indeed,
c-Myc and Mad proteins tend to be inversely
regulated (Hurlin et al., 1995a,b; Henriksson and
LuÈscher, 1996). The notion that c-Myc ± Max is
concentrated at sites of active transcription is
consistent with previous ®ndings showing c-Myc's
co-localization with small ribonucleoprotein particles
(Spector et al., 1987). Max would be drawn into
nuclear speckles and co-localize with c-Myc only
when the level of the latter protein exceeded a certain
threshold (Ayer and Eisenman, 1993; Baudino and
Cleveland, 2001). Mad proteins, generally expressed
at much lower levels during such times, would
associate with Max in a more di€use and transcriptionally ine€ectual pattern. The net result would be
the formation of the desired, transcriptionally active
c-Myc ± Max heterodimers at sites of active c-Myc
target gene expression. During times when c-Myc
levels are low and Mad levels are high, the opposite
pattern would be observed with c-Myc becoming
distributed homogeneously throughout the nucleus
and Mad proteins co-localizing with Max to form the
heterodimeric associations necessary to e€ect gene
repression. Under these circumstances it would be
expected that other proteins necessary to e€ect
transcriptional repression would also localize to these
same regions (Alland et al., 1997; Heinzel et al.,
1997). Proteins unable to concentrate in speckles
would be predicted to be less e€ective as transcriptional regulators, as we have indeed shown (Figure
6). Such regulation might provide a means by which
Max, in addition to its previously described functions, could serve to disperse c-Myc and Mad
proteins from their sites of transcriptional regulation
at times when they were least needed.
In summary, our results show that c-Myc network
members constitute a group of proteins that participates in the formation of distinct subnuclear structures.
The relative concentrations of c-Myc and Mad
proteins, relative to that of free Max, is at least one
of the factors dictating which types of heterodimers
will be localized to these sites. In the most extreme
circumstance, the nuclear speckles may represent sites
of mutually exclusive gene transcription or repression.
These sites are thus both physically and functionally
dynamic.
4661
Oncogene
Myc network interactions
X-y Yin et al
4662
Materials and methods
Plasmids and recombinant DNA techniques
Two GFP expression vectors were employed to localize
individual members of the Myc network. The ®rst, pEGFPC1 (Clontech, Palo Alto, CA, USA), allowed for the
creation of a fusion between GFP and the amino terminus
of each protein, whereas the second, pEGFP ± N3 fused
GFP to the carboxy terminus. The Myc network proteins
used consisted of c-Myc (Zhang et al., 1997), Max (both
151 and 160 amino acid isoforms, referred to as Max(S)
and Max(L), respectively) (Blackwood and Eisenman,
1991), Mad1, Mxil, and Mad3 (Ayer et al., 1993; Zervos
et al., 1993; Hurlin et al., 1995a). The entire coding region
of each cDNA was ampli®ed using the polymerase chain
reaction (PCR) with speci®c primers designed to allow inframe fusion of the PCR product with the GFP coding
sequence. In the case where cDNAs were cloned into the
pEGFP ± C1 vector, forward primers included the initiator
methionine codons, and reverse primers included the
naturally occurring termination codons. For cloning into
pEGFP ± N3, the same forward primers as above were
used, whereas the reverse primers without termination
codons were used to allow the reading frame to extend
into the GFP coding region. Each vector was analysed by
DNA sequencing to con®rm the identity of the cloned
fragment, its orientation, and its reading frame in relation
to that of GFP. For deletion analysis of Mad1 and Mxi1,
identical procedures were used except that the PCR primers
were designed to allow for ampli®cation of those regions of
the proteins indicated. Although only results with the
pEGFP-C1 constructs are shown here, identical results were
obtained with fusions in the pEGFP-N3 vector (not
shown).
For co-localization studies, we used variant forms of GFP
(`Topaz' [ExcitationMax=514 nm; EmissionMax=527 nm] and
`Sapphire': [ExcitationMax=395 nm; EmissionMax=508 nm])
(Packard Instruments, Meriden, CT, USA) (Tsien, 1998) in
the `C1' vector (Yin et al., 1999).
In some experiments, we used epitope tagged Myc network
proteins to con®rm the results obtained with GFP vectors.
Each of the above Myc network cDNAs was again ampli®ed
by PCR using the same reverse primers lacking termination
codons described above. After digestion with XhoI and/or
SalI, the fragments were cloned into the XhoI site of the
PSVLMTneo vector. This vector was constructed from the
previously described pSVLneo vector (Zhang et al., 1997) by
inserting a coding sequence for the Myc epitope EQKLISQQDL into its unique XhoI cloning site (not shown).
Expression of an appropriately sized, epitope-tagged protein
was con®rmed by transient transfection in Cos-7 cells
followed by Western blotting using the anti-c-Myc epitope
9E10 monoclonal antibody (Santa Cruz Biotechnology, Santa
Cruz, CA, USA). Immunostaining was then performed as
described below.
Cell lines, transfections and Western blotting
NIH3T3 ®broblasts, Rat- 1a ®broblasts, and Cos-7 cells
were grown under standard conditions in Dulbecco'smodi®ed Eagle's minimal essential medium (D-MEM)
containing 10% supplemented calf serum (GIBCO ± BRL,
Grand Island, NY, USA), 2 mM glutamine; 100 mg/ml
streptomycin and 100 units/ml penicillin G. Transfections
was performed by either a standard calcium phosphate
procedure or with Lipofectamine (GIBCO-BRL). In brief,
Oncogene
cells were seeded onto glass coverslips in 12-well tissue
culture plates 1 ± 2 days prior to transfection and allowed to
achieve logarithmic growth. Plasmid DNAs were applied
and allowed to remain on the cells for 2 ± 4 h. Coverslips
were then ®xed for microscopy at a time previously
determined to be the earliest point at which GFP could be
reproducibly observed (generally 3 ± 6 h after removal of the
transfection mix), thus avoiding overexpression artifacts
(Hedley et al., 1995). Coverslips were routinely ®xed in
2% paraformaldehyde ± PBS, although experiments using
other protocols or in live cells indicated that none of the
procedures altered the distribution of the proteins under
study. For immunolocalization using the 9E10 antibody,
cells were ®xed in 2% paraformaldehyde ± PBS. Antibody
was applied at a 1 : 500 dilution for 1 h followed by
application of FITC-conjugated polyclonal rabbit antimouse IgG (1 : 1000, Santa Cruz). The procedure for SC35 immunostaining was essentially identical except that the
primary antibody (Sigma) was used at a dilution of 1 : 1000
and the secondary antibody was conjugated with biotin
(Sigma). Detection was with avidin-conjugated Texas Red
(GIBCO ± BRL).
To con®rm the expression of GFP plasmids in vivo, Cos-7
cells were transfected with the indicated vectors using
Lipofectamine. The cells were harvested 2 days later and
prepared for Western analysis as previously described (Nesbit
et al., 1999). Membranes were incubated with a rabbit
polyclonal anti-GFP antibody (1 : 1000 dilution; Clontech)
followed by a horseradish peroxidase-labeled mouse antirabbit seconday antibody. Blots were developed using an
enhanced chemiluminescence system (Renaissance Plus, New
England Nuclear, Boston, MA, USA).
The method of transfection of the p3xmycE1b-luciferase
vector, along with pSVLneo, and pSVLMxil1 ± 152 into
NIH3T3 cells has been previously described (Zhang et al.,
1997; Gupta et al., 1998). The pSVLMxil1 ± 152 vector was
created by amplifying the relevant codons from a full-length
Mxil cDNA using standard PCR techniques and cloning into
the pSVLneo vector. All plasmid DNAs were puri®ed on
Qiagen columns (Qiagen, Chatsworth, CA, USA). The
plasmid pCMV ± b-gal (Clontech) was included in all
experiments to allow normalization of transfection eciencies. Expression of Mxil (WT) and Mxi1 (1 ± 152) in NIH3T3
cells was detected by Western blotting using a polyclonal
anti-Mxi1 antibody (Gupta et al., 1998).
Recombinant protein expression and electrophoretic mobility
shift assays (EMSAs)
Max(S), full-length Mxi1 [mxi1(WT)] and Mxi11 ± 152 were
cloned into the QE10 expression vector and expressed in E.
coli as His6-tagged recombinant proteins (Gupta et al., 1998;
Yin et al., 1999). Proteins were puri®ed by nickel-agarose
anity chromatography to 490% purity (not shown). 20 ng
of each protein were used for EMSAs along with 10 000
d.p.m. (approx. 20 ng) of the 32P-labeled `EO(GAC)' E-boxcontaining double-stranded oligonucleotide (Prochownik and
Van Antwerp, 1993).
Microscopy and subcellular localization
Epi¯uorescence microscopy was performed with Nikon
Diaphot inverted and Eclipse 800 upright microscopes
equipped with xenon arc lamps for excitation, a cooled
CCD camera and a Sutter Lambda 10-2 ®lter wheel for
changing excitation ®lters (400+20 nm for Sapphire,
505+5 nm for Topaz). Light intensity was regulated by use
Myc network interactions
X-y Yin et al
of a neutral density of ®lters. A 535+25 nm emission ®lter
was used for imaging GFP-tagged proteins. Laser confocal
microscopy was performed with a Leica TCS NT confocal
LSM microscope (Rockleigh, NJ, USA) and images were
collected using the 406objective. 0.5 mm thick optical
sections were typically obtained (usually 8/cell) and a
minimum of 20 ± 30 cells were scanned. The images presented
are representative of those obtained.
Acknowledgments
We thank Ciprian Almonte and Sean Albert for confocal
microscopy, to Dorothea Becker for comments on the
manuscript, and to Ron Berezney and Suri Somanathan
for helpful advice and discussions. This work was
supported by NIH grants HL33741 and CA 78259 to EV
Prochownik and by an NCI Cancer Center Core Grant to
The University of Pittsburgh Cancer Institute.
4663
References
Alland L, Mahle R, Hou H, Potes J, Chin L, Schreiber-Agus
N and DePinho R. (1997). Nature, 387, 49 ± 55.
Amati B, Dalton S, Brooks MW, Littlewood TD, Evan GI
and Land HT. (1992). Nature, 359, 423 ± 426.
Ayer DE and Eisenman RN. (1993). Genes Dev., 7, 2110 ±
2119.
Ayer DE, Kretzner L and Eisenman RN. (1993). Cell, 72,
211 ± 222.
Ayer DE, Lawrence QA and Eisenman RN. (1995). Cell, 80,
767 ± 776.
Baudino TA and Cleveland JL. (2001). Mol. Cell Biol., 21,
691 ± 702.
Blackwell TK, Kretzner K, Blackwood EM, Eisenman RN
and Weintraub H. (1990). Science, 250, 1149 ± 1151.
Blackwood EM and Eisenman RN. (1991). Science, 251,
1211 ± 1217.
Blackwood E, LuÈscher B and Eisenman RN. (1992). Genes
Dev., 6, 71 ± 80.
Blanchard J-M, Piechaczyk M, Dani C, Chambard J-C,
Franchi A, Pouyssegur J and Janteur P. (1985). Nature,
317, 443 ± 445.
Blencowe BJ, Nickerson JA, Issner R, Penman S and Sharp
PA. (1994). J. Cell Biol., 127, 593 ± 607.
Bregman DB, Du L, van der Zee S and Warren SL. (1995). J.
Cell. Biol., 129, 287 ± 298.
Campisi J, Gray HE, Pardee AB, Dean M and Sonenshein
GE. (1984). Cell, 36, 241 ± 247.
Cardoso MC, Joseph C, Rahn HP, Reusch R, Nadal-Ginard
B and Leonhardt H. (1997). J. Cell Biol., 139, 579 ± 587.
Cardoso MC and Leonhardt H. (1998). J. Cell. Biochem., 70,
222 ± 230.
Chen YM, Farmer AA, Chen CF, Jones DC, Chen PL and
Lee WH. (1996). Cancer Res., 56, 3168 ± 3172.
Clemson CM and Lawrence JB. (1996). J. Cell. Biochem., 62,
181 ± 190.
Dang CV and Lee WHF. (1988). Mol. Cell. Biol., 8, 4048 ±
4054.
Downes M, Ordentlich P, Kao H-Y, Alvarez JGA and Evans
RM. (2000). Proc. Natl. Acad. Sci. USA, 97, 10330 ±
10335.
Dunn BK, Cogliati T, Cultrano CM, Bar-Ner M and Segal S.
(1994). Cell Growth Di€er., 5, 847 ± 854.
Dyck GA, Maul GG, Miller WH, Chen JD, Kakizuka A and
Evans RM. (1994). Cell, 76, 333 ± 343.
Facchini LM and Penn LZ. (1998). FASEB J., 12, 633 ± 651.
Foley KP, McArthur GA, Queva C, Hurlin PJ, Soriano P
and Eisenman RN. (1998). EMBO J., 17, 774 ± 785.
Fu XD and Maniatis T. (1990). Nature, 343, 437 ± 441.
Gupta K, Anand G, Yin XY and Prochownik EV. (1998).
Oncogene, 16, 1149 ± 1159.
Hedley ML, Amrein H and Maniatis T. (1995). Proc. Natl.
Acad. Sci. USA., 92, 11524 ± 11528.
Heinzel T, Lavinsky RM, Mullen T-M, SoÈderstroÈm M,
Laherty CD, Torchia J, Yang WM, Brard G, Ngo SD,
Davie JR, Eisenman RN, Rose DW, Glass CK and
Rosenfeld MG. (1997). Nature, 387, 43 ± 48.
Hemavathy K, Guru SC, Harris J, Chen JD and Ip IT.
(2000). Mol. Cell. Biol., 26, 5087 ± 5095.
Henriksson M and LuÈscher B. (1996). Adv. Cancer Res., 68,
109 ± 182.
Huang S and Spector DL. (1991). Genes Dev., 5, 2288 ± 1302.
Hurlin PJ, Queva C, Koskinen PJ, Steingrimsson E, Ayer
DE, Copeland NG, Jenkins NA and Eisenman RN.
(1995a). EMBO J., 14, 5646 ± 5659.
Hurlin PJ, Foley KP, Ayer DE, Eisenman RN, Hanahan D
and Arbeit JM. (1995b). Oncogene, 11, 2487 ± 2501.
Kelly K, Cochran BH, Stiles CD and Leder P. (1983). Cell,
35, 603 ± 610.
Kim E, Du L, Bregman DB and Warren SL. (1997). J. Cell
Biol., 136, 19 ± 28.
Larsson L-G, Petterson M, Oberg F, Nilsson K and Luscher
B. (1994). Oncogene, 9, 1247 ± 1252.
Leonhardt H, Page AW, Weier HU and Bestor TH. (1992).
Cell, 71, 865 ± 873.
Leonhardt H and Cardoso MC. (1995). Int. Rev. Cytol.,
162B, 303 ± 335.
Li H and Bingham PM. (1991). Cell, 67, 335 ± 342.
Moen PT, Smith KP and Lawrence JB. (1995). Hum. Mol.
Genet., 4, 1779 ± 1789.
Mortillaro MJ, Blencowe BJ, Wei X, Nakayasu H, Du L,
Warren SL, Sharp PA and Berezney R. (1996). Proc. Natl.
Acad. Sci. USA, 93, 8253 ± 8257.
Nesbit CE, Tersak JM and Prochownik EV. (1999).
Oncogene, 18, 3004 ± 3016.
Prendergast GC, Lawe D and Zi€ EB. (1991). Cell, 65, 395 ±
407.
Prochownik EV and Van Antwerp ME. (1993). Proc. Natl.
Acad. Sci. USA, 90, 960 ± 964.
Queva C, Hurlin PJ, Foley KP and Eisenman RN. (1998).
Oncogene, 16, 967 ± 977.
Rabbits PH, Watson JV, Lamond A, Forster A, Stinson M,
Evan G, Fischer W, Atherton E, Sheppard R and Rabbits
TH. (1985). Nature, 314, 363 ± 366.
Schreiber-Agus N, Chin L, Chen K, Torres R, Rao G, Guida
P, Skoultchi AI and DePinho RA. (1995). Cell, 80, 777 ±
786.
Schreiber-Agus N, Meng Y, Hoang T, Hou H, Chen K,
Greenberg R, Cordon-Cardo C, Lee H-W and DePinho
RA. (1998). Nature, 393, 483 ± 487.
Scully R, Chen J, Plug A, Xiao Y, Weaver D, Feunteun J,
Ashley and Livingston DM. (1997a). Cell, 88, 265 ± 275.
Scully R, Chen J, Ochs RL, Keegan K, Hoekstra M,
Feunteun J and Livingston DM. (1997b). Cell, 90, 425 ±
435.
Oncogene
Myc network interactions
X-y Yin et al
4664
Oncogene
Scully R and Livingston DM. (2000). Nature, 408, 429 ± 432.
Shulman IG and Evans RM. (1997). Leukemia (Suppl.) 3,
376 ± 377.
Smith MJ, Charron-Prochownik DC and Prochownik EV.
(1990). Mol. Cell Biol., 10, 5333 ± 5339.
Smith KP, Moen PT, Wydner KL, Coleman JR and
Lawrence JB. (1999). J. Cell Biol., 144, 617 ± 629.
Spector DL. (1993). Ann. Rev. Cell Biol., 9, 265 ± 315.
Spector DL, Fu XD and Maniatis T. (1991). EMBO J., 10,
3467 ± 3481.
Spector DL, Watt RA and Sullivan NF. (1987). Oncogene, 1,
5 ± 12.
Tsien RY. (1998). Ann. Rev. Biochem., 67, 509 ± 544.
Van Antwerp ME, Chen DG, Chang C and Prochownik EV.
(1992). Proc. Natl. Acad. Sci. USA, 89, 9010 ± 9014.
VaÈstrik I, Kaipainen A, Penttila T-L, Lymboussakis A,
Alitalo R, Parvinen M and Alitalo K. (1995). J. Cell Biol.,
128, 1197 ± 1208.
Wagner AJ, LeBeau MM, Diaz MO and Hay N. (1992).
Proc. Natl. Acad. Sci. USA, 89, 3111 ± 3115.
Weiss K, Rambaud S, Lavau C, Jansen J, Carvalho T,
Carmo-Fonseca M, Lamond A and Dejean A. (1994).
Cell, 76, 345 ± 356.
Xing Y, Johnson CV, Dobner PR and Lawrence JB. (1993).
Science, 259, 1326 ± 1330.
Xing Y, Johnson CV, Moen PT, McNeil JA and Lawrence
JB. (1995). J. Cell. Biol., 131, 1635 ± 1647.
Yin X-Y, Gupta K, Han WP, Levitan ES and Prochownik
EV. (1999). Oncogene, 18, 6621 ± 6634.
Zeng C, Van Wijnen AJ, Stein JL, Meyers S, Sun W,
Shopland L, Lawrence JB, Penman S, Lian JB, Stein GS
and Hiebert SW. (1997). Proc. Natl. Acad. Sci. USA, 94,
6746 ± 6751.
Zervos A, Gyuris J and Brent J. (1993). Cell, 72, 223 ± 232.
Zhang H, Fan F and Prochownik EV. (1997). J. Biol. Chem.,
272, 17416 ± 17424.