Function and regulation of the transcription factors of the Myc/Max/Mad network
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Gene 277 (2001) 1–14
www.elsevier.com/locate/gene
Review
Function and regulation of the transcription factors of the
Myc/Max/Mad network
Bernhard Lüscher*
Abt. Biochemie und Molekularbiologie, Institut für Biochemie, Universitätsklinikum der RWTH, Pauwelstrasse 30, 52057 Aachen, Germany
Received 18 June 2001; received in revised form 17 August 2001; accepted 27 August 2001
Received by A.J. van Wijnen
Abstract
The members of the Myc/Max/Mad network function as transcriptional regulators. Substantial evidence has been accumulated over the
last years that support the model that Myc/Max/Mad proteins affect different aspects of cell behavior, including proliferation, differentiation,
and apoptosis, by modulating distinct target genes. The unbalanced expression of these genes, e.g. in response to deregulated Myc expres-
sion, is most likely an important aspect of Myc’s ability to stimulate tumor formation. Myc and Mad proteins affect target gene expression by
recruiting chromatin remodeling activities. In particular Myc interacts with a SWI/SNF-like complex that may contain ATPase activity. In
addition Myc binds to TRRAP complexes that possess histone acetyl transferase activity. Mad proteins, that antagonize Myc function, recruit
an mSin3 repressor complex with histone deacetylase activity. Thus the antagonism of Myc and Mad proteins is explained at the molecular
level by the recruitment of opposing chromatin remodeling activities. q 2001 Published by Elsevier Science B.V.
Keywords: Cell cycle; Chromatin; Differentiation; Oncogene; Signaling; transformation
1. Introduction activity of Myc proteins (Morgenbesser and DePinho, 1994;
Pelengaris et al., 2000). This appears to be the result of
The broad and lasting interest in myc genes and Myc Myc’s ability to stimulate cell proliferation and at the
proteins is based on the realization that they regulate deci- same time to inhibit cells to enter a resting state or to term-
sively various aspects of cell behavior. Foremost is the large inally differentiate. Thus a consequence of the constitutive
body of information that has been accumulated over the last presence of Myc is an increase in the respective cellular
twenty years which demonstrates a strong involvement of compartment which is thought to provide the base for addi-
myc in tumorigenesis (Marcu et al., 1992; Henriksson and tional genetic alterations that cooperate with Myc in tumor
Lüscher, 1996; Nesbit et al., 1999). Soon after the initial formation (Berns, 1991; Adams and Cory, 1992). Recently a
identification of v-myc in several transforming chicken more direct role of Myc in transforming cells has also been
retroviruses, genetic alterations of myc genes were found suggested. This is based on findings that overexpression of
in many different human malignancies, e.g. translocations Myc causes genomic destabilization potentially due to its
of the c-myc gene in Burkitt’s lymphoma and amplification ability to induce endoreplication (Mai et al., 1999; Taylor
of the N-myc gene in neuroblastoma. This results in general and Mai, 1998; Felsher and Bishop, 1999; Felsher et al.,
in a deregulated, enhanced expression of myc genes, a 2000). Knock-out experiments provide further strong
condition found in many if not most human tumors. In addi- evidence for a critical role of Myc for normal cell behavior
tion studies in animal models and in tissue culture systems (Stanton et al., 1992; Charron et al., 1992; Davis et al., 1993;
have provided compelling evidence for a tumor promoting Moreno de Alboran et al., 2001). In addition Myc proteins
can stimulate apoptosis, a finding that on first view does not
Abbreviations: bHLHZip, basic region/helix4oop-helix/leucine zipper; seem to be compatible with the other functions mentioned
CDK, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation; above (Evan et al., 1992; Thompson, 1998; Prendergast,
CKI, cyclin-dependent kinase inhibitor; GSK, glycogen synthase kinase; 1999). However it is now thought that the induction of
HAT, histone acetyl transferase; HDAC, histone deacetylase; INR, initiator apoptosis by Myc is part of a safeguard mechanism that
element; MBII, Myc box II; RB, retinoblastoma tumor suppressor protein;
helps restrict the dominant action of Myc proteins on cell
SID, mSin3 interaction domain; TAD, transactivation domain
* Tel.: 149-241-808-8850; fax: 149-511-808-2427. proliferation. In support tumor cells with elevated Myc
E-mail address: luescher@rwth-aachen.de (B. Lüscher). levels are frequently defective in apoptotic pathways.
0378-1119/01/$ - see front matter q 2001 Published by Elsevier Science B.V.
PII: S 0378-111 9(01)00697-72 B. Lüscher / Gene 277 (2001) 1–14 Another activity of Myc that has been demonstrated recently (Grandori and Eisenman, 1997; Facchini and Penn, 1998; is the ability to induce cell growth independent of cell cycle Boyd and Farnham, 1999). In particular the use of DNA progression in some systems (Johnston et al., 1999; Iritani microarray technology has made it possible to probe a and Eisenman, 1999; Schuhmacher et al., 1999). Together large number of genes and to define a set of potential target these findings define Myc as a highly versatile factor influ- genes that reflect Myc’s broad role in the control of cell encing many aspects of normal cell behavior. behavior (Coller et al., 2000; Guo et al., 2000; O’Hagan et For many years it was unclear how Myc proteins achieve al., 2000b; Schuhmacher et al., 2001). The targets include the diverse biological effects summarized above. From genes involved in the control of the cell cycle, protein and various studies, including the nuclear localization of these DNA biosynthesis, cell growth, cell adhesion, apoptosis, proteins, it was inferred that Myc could play roles in nuclear and immortality (Grandori et al., 2000). It will now be chal- architecture, replication, splicing, and/or transcription lenging to sort out the hierarchy of the importance of these (Lüscher and Eisenman, 1990). A hypothesis supporting genes for specific Myc functions, particularly for transfor- the latter was formulated towards the end of the 1980s mation. mainly on the basis of sequence homologies to other tran- The regulation of Myc expression and function is still far scription factors. The motif found in common is referred to from being understood. In particular the expression of myc as the basic region/helix-loop-helix/leucine zipper genes appears highly complicated. While a large number of (bHLHZip) domain and functions as a DNA binding and transcription factors have been defined that can regulate the protein-protein interaction domain (Lüscher and Larsson, c-myc promoter, no unifying concept exists that explains 1999). A break-through observation in defining a role of myc expression during the cell cycle and during the transi- Myc in transcription was the identification of the bHLHZip tions from resting to cycling and from cycling to differen- protein Max as a Myc dimerization partner 10 years ago tiating cells (Spencer and Groudine, 1991; Marcu et al., (Blackwood and Eisenman, 1991; Prendergast et al., 1991; 1997). Recent findings however provide some insight into Blackwood et al., 1992). Max is the essential heterodimer- c-myc regulation during the transition from G0 into G1 ization partner of Myc proteins for various biological activ- (Nasi et al., 2001). The tyrosine kinase Src has been impli- ities, including transformation, apoptosis, and cated in mediating growth factor signal-induced c-myc transcriptional activation (Amati et al., 1992, 1993a,b). In expression (Barone and Courtneidge, 1995; Blake et al., agreement with these findings Max is essential for normal 2000). Src appears to signal through the Rho GTPases rather mouse development (Shen-Li et al., 2000). However than Ras or mitogen-activated protein kinases to activate the whether Max is required for all molecular functions of c-myc promoter (Chiariello et al., 2001). Although the tran- Myc is presently not known. Nevertheless the identification scription factor targets of Src/Rho signaling remain unde- of Max and the finding that Myc/Max heterodimers bind fined, these findings provide a starting point to unravel specifically to E box DNA sequences with the consensus growth factor-dependent c-myc expression. core 5 0 -CACGTG and through such elements activate the Regarding the regulation of Myc proteins, recent progress expression of reporter genes supported the hypothesis that has been made in understanding the signals that impinge on Myc functions as a transcriptional regulator (Dang, 1999; Myc. In particular phosphorylation has been shown to affect Grandori et al., 2000). In addition a transactivation domain the stability of Myc, a protein with a short half life in the (TAD) was identified near the N-terminus of Myc (Facchini order of 20–30 min. Furthermore numerous Myc interaction and Penn, 1998; Dang, 1999). Both the bHLHZip and the partners have been identified, the functions of some support TAD are important for Myc function in the control of cell a role of Myc in the regulation of chromatin structure (Dang, behavior. Together these findings provide support for a role 1999; Grandori et al., 2000; Amati et al., 2001). One task for of Myc in gene transcription. Other activities, however, the coming years will be to determine the role of these cannot be excluded. different proteins for specific aspects of Myc function and How can Myc affect so profoundly different aspects of to connect these interactions to signaling pathways. In addi- cell behavior? To answer this question it will be important tion the essential Myc dimerization partner Max binds also to define the genes that are targeted by Myc and to under- to several other bHLHZip factors, forming what is referred stand how the expression and function of Myc is regulated. to as the Myc/Max/Mad network of transcriptional regula- Of particular interest is the question how Myc causes cell tors (Fig. 1). To understand the biological functions of Myc transformation in view of identifying targets that might be proteins requires also that the other components of this accessible to therapeutic strategies. During the early days of network are studied and their regulation and function deter- the search for Myc target genes it was hoped, somewhat mined since they impinge in one way or another on Myc naively from toady’s point of view, that one could identify function. ‘the Myc target gene for transformation’. It seems more Numerous excellent reviews have been published in realistic now that Myc will modulate the expression of recent years that describe various aspects of the function many genes to control cell behavior. A continuously and the biology of the Myc/Max/Mad network. Several of increasing number of target genes are being proposed these reviews are cited to summarize work that can not be some of which are thought to be relevant for transformation discussed in detail here. This review concentrates on novel
B. Lüscher / Gene 277 (2001) 1–14 3
Fig. 1. The Myc/Max/Mad network. The components of the Myc/Max/Mad network are indicated. Arrows define the known interactions between different
network members. The interactions are specified by the HLHZip dimerization domains. As far as characterized the members of the network function as
regulators of different aspects of cell behavior as indicated. More detailed information about individual components is given in the text.
aspects regarding the molecular functions and regulations of further expand the network laterally. Together with the
Myc and its network partners in gene transcription and the many proteins found associated with the Myc/Max/Mad
control of cell proliferation. network (see also Sections 3 and 4), an assemblage of
factors is being defined that is rapidly growing not only in
number of components but also in functional complexity.
2. The Myc/Max/Mad network: components and their Comparing the structure of the different network compo-
basic functions nents reveals similarities but also differences (Fig. 2). As
expected from the identification of these factors as dimer-
The identification of Max as binding partner of different ization partners, all components possess a bHLHZip motif
Myc proteins, including c-Myc, N-Myc, and L-Myc, as DNA binding/dimerization interphase. Max and Mlx are
initiated a search for additional bHLHZip dimerization part- capable of forming homodimers but none of the other
ners. This led to the description of the Mad proteins Mad1, components. All these dimers function as DNA binding
Mxi1, Mad3, and Mad4, as interaction partners of Max and competent complexes that recognize E box DNA sequences
together these factors define the Myc/Max/Mad network and have been suggested to function at least in part as tran-
(Fig. 1) (Henriksson and Lüscher, 1996; Grandori et al., scriptional regulators (Dang, 1999; Lüscher and Larsson,
2000). Later two additional Max partners, Mnt and Mga, 1999; Grandori et al., 2000). The importance of the
were found (Hurlin et al., 1997, 1999). Recently the identi- bHLHZip domains of both Myc and Mad proteins is
fication of Mlx, a Max-like protein that can heterodimerize supported by mutational analysis, demonstrating an essen-
with Mad1, Mad4, and Mnt, but neither with the other Mad tial role of this domain for all functions tested. Interestingly
proteins nor with Myc or Max, has been reported (Billin et as far as analyzed, only Myc proteins, MondoA, and
al., 1999; Meroni et al., 2000). The most recent extensions WBSCR14 carry a TAD (Fig. 2) and Myc proteins and
of the network are the cloning of MondoA as an Mlx bind- MondoA can transactivate reporter genes through E box
ing partner (Billin et al., 2000) and the identification of elements (Dang, 1999; Billin et al., 2000; Cairo et al., 2001).
WBSCR14 that also interacts with Mlx (Cairo et al., In addition to gene activation, Myc can also function as a
2001). The gene encoding WBSCR14 lies in the commonly repressor (Claassen and Hann, 1999; Grandori et al., 2000;
deleted region associated with Williams-Beuren syndrome Amati et al., 2001). This activity of Myc has remained,
(Cairo et al., 2001). Thus both Max and Mlx are the central however, largely unexplained at the molecular level until
components of network subdomains that are interconnected recently. One mechanism of Myc-dependent repression
by common interaction partners (Fig. 1). It is possible that functions through blocking of the positive acting, initiator
not all members of the network have been defined yet and it (INR) binding factor Miz-1 by Myc (Section 4) (Staller et
will be of interest to detail additional components that may al., 2001; Seoane et al., 2001). From many different studies4 B. Lüscher / Gene 277 (2001) 1–14
Fig. 2. Structure-function schematic of components of the Myc/Max/Mad network. The known functional domains of different network components are
summarized. Information regarding the expression and the loss of function phenotypes, as far as defined, is indicated. TAD: transactivation domain; I: Myc box
I; II: Myc box II; b: basic region; R: region involved in repression; HLH: helix-loop-helix domain; Zip: leucine zipper domain; SID: mSin3-interaction domain;
T-Domain: T-box DNA binding domain; TRD: transrepression domain; P: position of known phosphorylation sites. The numbers refer to amino acids of the
human proteins.
it appears that both repression and activation of transcription for numerous additional functional domains (Fig. 2). Inter-
are essential activities of Myc proteins (for a recent discus- estingly Mga carries two distinct DNA binding domains
sion see Amati et al., 2001). suggesting that it can regulate genes not only through E
In contrast to Myc, Mad proteins and Mnt possess an box elements but also through sequences recognized by its
mSin3 interaction domain (SID) which mediates transcrip- T-box DNA binding motif (Tada and Smith, 2001). It is
tional repression (Kiermaier and Eilers, 1997; Schreiber- likely that further analysis of the different components of
Agus and DePinho, 1998; Knoepfler and Eisenman, 1999). the network will reveal additional new and unexpected find-
Besides bHLHZip, the SID is critical for the function of ings.
Mad proteins. This is also true for Mnt, however this protein
has most likely additional domains relevant for function.
The former is suggested by the findings that Mad proteins 3. The Myc–Mad antagonism: opposing regulation of
with an impaired SID show little biological activity. In chromatin structure and gene transcription
support of the latter is that MntDSID gains transforming
activity in cooperation with an activated Ras oncoprotein, All the studies that have been reported today provide
whereas Mnt and Mad proteins with functional SIDs inter- evidence for a functional antagonism of Myc and Mad
fere with transformation (Grandori et al., 2000). Together proteins (Fig. 3). This has been proposed first from findings
these studies define the TAD of Myc proteins, the SID of showing that Mad proteins are expressed preferentially in
Mad proteins, and the bHLHZip of all components as essen- non-proliferating cells as opposed to Myc proteins which
tial domains for the function of the respective network are present almost exclusively in proliferating cells. Further-
members. However detailed structure-function analyses of more Mad proteins inhibit reporter genes that are activated
the proteins that have been identified more recently have not by Myc, interfere with transformation of rat embryo fibro-
been performed yet. This is certainly true for Mga which, as blasts, block cell growth, and prevent apoptosis (Henriksson
its name indicates, is a very large protein of just over 3000 and Lüscher, 1996; Grandori et al., 2000). The findings
amino acids (Hurlin et al., 1999). This already distinguishes summarized above, which are based mainly on overexpres-
Mga from the other members of the Myc/Max/Mad network sion studies, are supported by the analysis of mad knock-out
which in general are rather small molecules and offers room and mad transgenic mice (Schreiber-Agus et al., 1998;B. Lüscher / Gene 277 (2001) 1–14 5 Fig. 3. Model for the control of promoter activity by Myc/Max/Mad network members. Distinct dimeric complexes, i.e. Myc/Max, Max/Max, and Mad/Max, are thought to recruit different cofactors to promoters through specific interaction with E box DNA elements. Myc binds to a component of the SWI/SNF complex that contains an ATPase activity involved in nucleosome remodeling. In addition Myc interact with TRRAP complexes that possess HAT activity important for modulating the acetylation status of core histones and possibly other components associated with promoters, including transcription factors and subunits of the PolII complex. Max has not been reported to bind transcriptional cofactors. Thus Max/Max homodimers will most likely not influence directly promoter activity. Nevertheless Max/Max dimers are potentially able to compete for DNA binding with other E box binding factors. Mad proteins recruit an mSin3-dependent repressor complex that is associated with HDAC activity. This complex will deacetylate core histones and possibly other acetylated proteins. The results of binding of Myc/Max, Max/Max, or Mad/Max dimers to a promoter are summarized and a possible order of events is indicated. Myc/Max binding and recruitment of SWI/SNF may lead to alterations of a promoter’s chromatin structure by modulating nucleosomes. This may make binding sites available for other transcriptional regulators that may or may not cooperate with Myc in promoter modulation. The recruitment of a cofactor complex with HAT activity will further enhance promoter accessibility and in addition also provide acetylation-dependent binding sites on histone tails. It is not clear whether SWI/SNF and TRRAP complexes work on the same promoters and in which order. Myc interacts with components of the PolII complex and may therefore also be involved in PolII recruitment. The binding of Mad/Max and recruitment of the mSin3 repressor complex will lead to histone deacetylation and increased chromatin compaction. This is thought to result in decreased accessibility of transcription factors to their binding sites and of the PolII complex to the core promoter region. Besides HDAC other components of the mSin3 complex contribute to repression. This suggests that other functions, e.g. an activity that antagonizes SWI/SNF function, may be associated with this repressor. E box: refers to a DNA element that is recognized by Myc/Max/Mad network members; RE: response element for transcriptional regulators; Ac: acetylation of histones; TF: transcription factor; HAT: histone acetyl transferase; HDAC; histone deacetylase; Foley et al., 1998; Queva et al., 1999, 2001). However, in activators are important for the efficient recruitment and particular the knock-out studies have not provided the activation of the polymerase complex. In addition activators strong phenotypes originally expected, most likely due to may regulate the processivity of the polymerase complex. overlapping and redundant functions of Mad proteins in More recently it has become evident that these activities are different cell types. Indeed in all cells analyzed more than most likely downstream of effects on the chromatin struc- one Mad protein is expressed (Grandori et al., 2000). ture of gene loci and more specifically of promoter regions. Transcriptional regulators are thought to affect the tran- The structure of the chromatin appears to be a major deter- scription of specific genes at several different levels (Good- minant whether promoters are accessible to the polymerase rich et al., 1996; Struhl, 1996). Early findings suggested that complex and thus whether a gene can be transcribed or not
6 B. Lüscher / Gene 277 (2001) 1–14
(Kadonaga, 1998; Kornberg and Lorch, 1999; Wolffe and activity, one containing GCN5/PCAF the other Tip60/NuA4
Hansen, 2001). Repressors of transcription may also affect (Vassilev et al., 1998; Grant et al., 1998; Ikura et al., 2000).
the polymerase complex but it is thought that their primary Indeed Myc recruits GCN5 and HAT activity, most likely
effects are on compacting chromatin and thus making it through TRRAP (McMahon et al., 2000; Park et al., 2001).
inaccessible for positive acting factors as well as for poly- A potential involvement of Tip60 is also indicated by the
merases. How is chromatin affected? Two fundamentally identification of Tip48 and Tip49, both are part of the Tip60
distinct mechanisms have emerged. One involves the activ- complex, as Myc interaction partners (Wood et al., 2000).
ity of molecules that possess ATPase activity and possibly TRRAP interacts with the TAD of Myc, more precisely it
helicase activity and affect the structure and location of requires a conserved element in the TAD, the so called Myc
nucleosomes (Kingston and Narlikar, 1999; Peterson and box II (MBII) (McMahon et al., 1998). MBII is a small
Workman, 2000; Aalfs and Kingston, 2000; Fry and Peter- element that is essential for all biological activities of
son, 2001). The second mechanism targets histones. These Myc proteins. Although a role of MBII in gene transcription
proteins can be modified by, e.g. phosphorylation, acetyla- has been questioned (Xiao et al., 1998), the binding of this
tion and methylation, leading to a multitude of differentially Myc domain to HAT complexes suggests that MBII is
modified histones. Distinct modification patterns, referred to involved in the regulation of gene transcription. Indeed
as histone code, are thought to affect compaction of chro- recent studies using chromatin immunoprecipitation
matin and to provide docking sites for proteins. At least in (ChIP) assays, in which transcription factors and their cofac-
some cases certain histone modifications have been linked tors are crosslinked in vivo to their DNA binding sites and
to specific functional states of promoters (Strahl and Allis, subsequently the associated DNA is analyzed by gene-
2000; Cheung et al., 2000). specific PCR, have demonstrated that Myc recruits
Evidence is accumulating that the antagonism of Myc and TRRAP activity in a MBII-dependent manner to target
Mad proteins at the molecular level works at least in part by genes. This results in histone acetylation of responsive
affecting chromatin structure (Fig. 3). The first evidence that promoters and subsequently in gene expression (Bouchard
Myc/Max/Mad network proteins may indeed function in et al., 2001; Frank et al., 2001). The apparent conflict in
chromatin remodeling came from the findings that Mad regard of a role of MBII in transcription reflects most likely
proteins recruit through the SID a repressor complex that the difference between the analysis of transiently transfected
contains histone deacetylase (HDAC) activity (Hassig et al., reporter genes and endogenous, chromosome-embedded
1997; Alland et al., 1997; Sommer et al., 1997; Laherty et genes (Amati et al., 2001). The latter are dependent for
al., 1997). Several lines of evidence support the notion that expression on chromatin remodeling and histone modifica-
the recruitment of the repressor complex is essential for the tion activities, while reporter genes have more relaxed
biological effects of Mad1 described above. Deletion of the requirements. Together these findings define an important
SID impairs the activity of Mad proteins in transformation, role for MBII in the control of gene transcription by Myc
transactivation, cell growth inhibition, and apoptosis and thus support the concept that regulation of gene tran-
(Grandori et al., 2000; Baudino and Cleveland, 2001). scription is an essential aspect of Myc biology.
Since the bHLHZip is also important for these functions, Early on after the identification of Mad1 it was suggested
it is likely that these are, at least in part, the result of specific that Myc/Max and Mad/Max complexes form a molecular
target gene regulation. Although the widely expressed Mnt switch involved in the regulation of transitions between
also possesses a SID domain that binds mSin3 in vitro and different cell states, in which Myc/Max complexes are
its deletion affects Mnt function, it remains to be determined exchanged by antagonizing Mad/Max complexes, and vice
whether in vivo the SID of Mnt serves to recruit an HDAC- versa, on responsive E box DNA elements (Ayer et al.,
containing repressor complex. It is possible that the inter- 1993; Ayer and Eisenman, 1993). In support of this model
action of Mnt with the mSin3 complex is regulated in an as Myc/Max and Mad/Max complexes are expressed mainly in
yet unknown manner. Since Mnt is, in contrast to Mad growing and in resting and differentiated cells, respectively.
proteins, a stable protein expressed ubiquitously such a More recently the exchange of Myc/Max to Mad/Max
regulation might be desirable to modulate its function. complexes could be observed on two responsive promoters.
The findings that Mad proteins recruit a repressor Using ChIP assays it has been demonstrated that Myc/Max
complex with HDAC activity led to the suggestion that complexes bound to the promoters of hTERT and human
Myc proteins might recruit histone acetyl transferase cyclin D2 genes in exponentially growing HL-60 myelo-
(HAT) activity, thus providing a molecular explanation blast cells are replaced by Mad/Max complexes during
for the antagonism between Myc and Mad proteins. Indeed differentiation (Xu et al., 2001; Bouchard et al., 2001).
recent findings demonstrated that Myc interacts with a large Furthermore this switch from Myc/Max to Mad/Max
protein, TRRAP, that appears to function as coactivator complexes on cyclin D2 is associated with recruitment of
(McMahon et al., 1998; Saleh et al., 1998). Like mSin3 HDAC1, a decrease in histone acetylation, and a loss of
for Mad proteins, TRRAP can function as a platform for polymerase II binding to the cyclin D2 promoter (Bouchard
additional proteins involved in gene transcription. TRRAP et al., 2001). Thus the findings discussed above provide
is part of at least two large complexes that possess HAT evidence for and define one consequence of the proposedB. Lüscher / Gene 277 (2001) 1–14 7
switch, i.e. the alternate recruitment of HAT and HDAC Miz-1 depends on the retinoblastoma tumor suppressor, the
activity to target genes. This suggest that the distinct regula- expression of the Ink4 cyclin-dependent kinase inhibitors
tion of histone acetylation in response to Myc/Max/Mad was analyzed. Miz-1 binds to the Ink4b INR and activates
network members represents one level of the functional the expression of this gene. Importantly Myc is also asso-
antagonism of Myc and Mad proteins. ciated with the INR thus providing the first direct evidence
While at present there is little evidence for additional for the physical interaction of Myc with a negatively regu-
Mad-interacting proteins that are involved in the regulation lated promoter. Several genes are repressed by Myc through
of gene transcription, several other factors implicated in the core promoter and INR elements, including c-myc, p21 CIP1,
regulation of transcription have been identified that bind to C/EBPa, p27, and H-ferritin (Antonson et al., 1995;
Myc. The C-terminal domain of Myc interacts with INI1/ Facchini et al., 1997; Wu et al., 1999; Claassen and Hann,
hSNF5, a component of the multiprotein SWI/SNF 2000; Yang et al., 2001) suggesting an involvement of Miz-
complex, that is involved in chromatin remodeling in an 1. Other genes, including gadd45 and pdgf-b receptor, are
ATP-dependent manner (Cheng et al., 1999; Kingston and repressed in an INR-independent manner (Marhin et al.,
Narlikar, 1999; Peterson and Workman, 2000). Interestingly 1997; Oster et al., 2000; Izumi et al., 2001). Repression of
INI1 has been suggested to function as a tumor suppressor the pdgf-b receptor is mediated by NF-Y, a factor that is
and the two SWI2/SNF2 homologues, BRG1 and hBRM, inhibited by Myc (Izumi et al., 2001).
can induce cell cycle arrest (Versteege et al., 1998; How does Myc inhibit the activation of the Ink4b promo-
Muchardt and Yaniv, 1999). Thus it appears, somewhat ter by Miz-1? Miz-1 recruits the coactivator p300, an inter-
puzzling, that the proto-oncoprotein c-Myc interacts with action that is competed for by Myc (Staller et al., 2001).
a complex that has growth inhibitory activity. Although it Since the C-terminal HLHZip region of Myc is sufficient for
has been reported that recruitment of the SWI/SNF complex this inhibition, one would expect that Myc brings a func-
is necessary for Myc transactivation function in reporter tional TAD into Miz-1/Myc complex. However the Myc
gene assays, it is not yet clear what the role of this complex TAD appears not to function under these conditions, either
is for the regulation of chromosome embedded target genes. coactivators cannot be recruited efficiently or the Ink4a
It will now be of interest to determine the relative contribu- promoter is not responsive. Another possibility is that addi-
tion of the different complexes, including TRRAP and SWI/ tional domains in Myc are relevant for repression, including
SNF complexes, for Myc-specific gene regulation. MBII and the region spanning amino acids 96–106 (region
R in Fig. 2) as suggested previously (Philipp et al., 1994; Li
et al., 1994; Lee et al., 1997). As indicated above it will be
4. Myc and gene repression through initiator elements important to define whether other Myc-repressed promoters
are regulated by a Miz-1 dependent mechanism or whether
While evidence has steadily accumulated over the last Myc can regulate INR elements through additional means.
years that Myc can function as transcriptional activator,
the suggestion that Myc can also function as a transcrip-
tional repressor has been ill-defined at the molecular level 5. Myc and cell cycle control
until recently (Facchini and Penn, 1998; Claassen and Hann,
1999; Grandori et al., 2000; Amati et al., 2001). A main The products of a substantial number of the proposed
problem has been the difficulty in identifying response Myc target genes are involved in the control of cell prolif-
elements that mediate Myc repression and in defining the eration, i.e. in the regulation of cell-matrix interaction, of
mode of Myc function on such elements. Recent findings protein and DNA synthesis and of the transition from G1
however have shed light on Myc-mediated repression. Early into S phase of the cell cycle (Coller et al., 2000; Guo et al.,
on it has been suggested that the initiator element (INR), 2000; O’Hagan et al., 2000b; Schuhmacher et al., 2001).
which is part of some core promoters, responds to Myc (Li These genes and their products provide a mechanistic base
et al., 1994; Philipp et al., 1994; Mai and Martensson, 1995; to evaluate Myc’s role in stimulating cell proliferation,
Lee et al., 1996). At least three DNA binding proteins that particularly the G1-S transition and the inactivation of the
can bind to INR sequences, TFII-I, YY1, and Miz-1, are also G1-S checkpoint (or restriction point) (Fig. 4). Overcoming
interacting with Myc, leading to the proposal that these the latter is essential for cell proliferation. This requires the
factors mediate Myc repression (Peukert et al., 1997; Roy inactivation of pocket proteins, in particular the retinoblas-
et al., 1993; Hariharan et al., 1991; Seto et al., 1991; Shri- toma tumor suppressor protein (RB), and the activation of
vastava et al., 1993). While a role for TFII-I and YY1 in the cyclin E/cyclin-dependent kinase (CDK) 2 complex
Myc-dependent repression has not been firmly established, (Adams, 2001; Sherr and Roberts, 1999; Ekholm and
recent evidence suggests that Miz-1 mediates Myc repres- Reed, 2000). Early on it was shown that activation of Myc
sion. Miz-1 binds to and activates transcription from INR results in the activation of cyclin/CDK complexes (Jansen-
elements and inhibits cell cycle progression, both functions Dürr et al., 1993; Steiner et al., 1995; Perez-Roger et al.,
are antagonized by Myc (Peukert et al., 1997; Staller et al., 1997; Müller et al., 1997), leading to the suggestion that one
2001; Seoane et al., 2001). Since the cell cycle arrest by important activity of Myc in regulating cell proliferation is8 B. Lüscher / Gene 277 (2001) 1–14
With the identification of Myc as a regulator of the
expression and activity of cyclin/CDKs, one focus of subse-
quent research was the analysis of the mechanism of this
regulation. While some components of cyclin/CDK
complexes are direct targets of the Myc/Max/Mad network,
including cyclin D2 and possibly cyclin D1 and CDK4
(Bouchard et al., 1999; Bouchard et al., 2001; Perez-
Roger et al., 1999; Hermeking et al., 2000; Coller et al.,
2000), indirect effects of Myc also contribute to the activa-
tion of cyclin/CDK. Together the identification of these
target genes will now allow to define in more detail how
Myc proteins affect the activity of different cyclin D/CDK
and cyclin E/CDK2 complexes (Fig. 4). Both these cyclin/
CDK complexes are required to enter S phase (Sherr and
Roberts, 1999; Ekholm and Reed, 2000). Cyclin D/CDK
complexes are built from several different subunits includ-
ing cyclin D1, D2, and D3 and the two catalytic subunits
CDK4 and CDK6. The activation of cyclin D1 and D2 by
Myc is of particular importance since their expression not
only can positively influence cyclin D/CDK complexes but
these two cyclins have also been shown to sequester p27 KIP1,
an inhibitor of cyclin E/CDK2 (Bouchard et al., 1999;
Bouchard et al., 2001; Perez-Roger et al., 1999; Coller et
al., 2000; Dey et al., 2000). Furthermore the Ink4b gene,
encoding the cyclin-dependent kinase inhibitor (CKI)
p15 INK4b, is repressed by Myc as summarized above (Staller
Fig. 4. Regulation of G1–S phase progression by Myc. Accumulating et al., 2001; Seoane et al., 2001). The activation of different
evidence suggests that Myc regulates several target genes whose products Ink4 genes has been recognized as an important mechanism
are involved in controlling the transition from G1 into S. These interactions to negatively regulate cell proliferation by many different
are summarized (see Sections 4 and 5 for details). Myc can activate the agents (Vidal and Koff, 2000). In particular the Ink4b gene
genes encoding Id2, CDK4, Cyclin D2, Cul1, and Cdc25A. Id2 is a negative
is activated in response to TGFb, a function that is antag-
regulator of pocket proteins, including RB, and thus can stimulate progres-
sion through the restriction point. Induction of CDK4 and cyclin D2 stimu- onized by Myc (Massague et al., 2000; Amati, 2001).
lates both cyclin D/CDK and cyclin E/CDK complexes. Cul1 is a p15 INK4b is an inhibitor of cyclin D/CDK4/6 function.
component of the SCF complex that is responsible for signal-dependent Together these findings demonstrate that Myc can stimulate,
ubiquitination of p27 KIP1 which leads to its degradation. The phosphatase through the regulation of Ink4b, cyclin D2, and CDK4,
Cdc25A activates CDK2 by removing inhibitory phosphates. Furthermore
cyclin D/CDK function.
Myc represses genes that encode cyclin/CDK inhibitors, including p15 INK4b,
p21 CIP1, and p27 KIP1. These Myc-dependent regulations result in the stimu- The targets of cyclin D/CDK4/6 are pocket proteins. In
lation of cyclin D- and cyclin E-dependent kinase activities and in the particular the inactivation of the RB protein, a frequently
inhibition of pocket proteins. Together these effects enhance S phase mutated and by this inactivated tumor suppressor, is critical
progression and explain at least in part the S phase-inducing activity of for the regulation of the G1-S checkpoint (Bartek et al.,
Myc upon deregulated expression.
1997; Reed, 1997). Besides directly impinging on the activ-
ity of cyclin D/CDK complexes, Myc has also been shown
stimulating the G1-S transition. This transition is also a key to stimulate the expression of Id2, which encodes an HLH
target during oncogenesis (Bartek et al., 1997; Reed, 1997). protein that inhibits RB function (Lasorella et al., 2000).
Most if not all tumor cells have mutations that result in This provides an additional mechanism to release E2F tran-
altered control of the G1-S checkpoint and thus facilitate scription factor activity, which is negatively regulated by
the entry into the DNA replication mode. Once cells have RB, and thus to promote S phase progression (Harbour and
progressed into S phase, the remainder of the cell cycle Dean, 2000). The findings that Myc can interfere at multiple
proceeds independent of the presence of growth factors. levels with the function of RB proteins and the frequent
Thus under limiting amounts of such factors, advancing mutations in RB and in the upstream regulatory components
beyond the G1-S checkpoint is critical for proliferation. in human tumors suggest that this is an important function of
Myc is sufficient to overcome the restriction point and to Myc in tumorigenesis. Both gene activation and repression
induce S phase in the absence of growth factors (Eilers et al., are important for this activity of Myc.
1991). Therefore it is attractive to suggest that the activation The second cyclin/CDK activity that is required for enter-
of cyclin/CDK complexes is critical for Myc’s ability to ing S phase is cyclin E/CDK2. Although neither of the two
induce S phase. genes appear to be direct targets of Myc, multiple ways haveB. Lüscher / Gene 277 (2001) 1–14 9
evolved how Myc can influence cyclin E/CDK2 activity. the central acidic domain and one near the basic region, are
The cdc25A gene has been proposed to be a Myc target phosphorylated by protein kinase CK2 (Lüscher et al.,
(Galaktionov et al., 1996). Activation of Cdc25A, a dual 1989). Little is known about the regulation and function
specificity phosphatase, stimulates cyclin E/CDK2 activity of these phosphorylation sites. The third cluster is located
(Blomberg and Hoffmann, 1999; Sexl et al., 1999). In addi- in the transactivation domain, more specifically at and near
tion several pathways target the cyclin E/CDK2 inhibitor MBI (Seth et al., 1992, 1993; Pulverer et al., 1994; Henriks-
p27 KIP1. This CKI is an important regulator of cell prolifera- son et al., 1993; Lutterbach and Hann, 1994). Of particular
tion that is induced when growth factor levels are insuffi- importance are two sites, Thr-58 and Ser-62, because this
cient to sustain proliferation. Consistently p27 KIP1 is highly region is a mutational hot spot (Bhatia et al., 1993, 1994;
expressed in resting cells and downregulated upon serum Yano et al., 1993; Albert et al., 1994; Axelson et al., 1995).
induction (Sherr and Roberts, 1999). Importantly p27 KIP1 These mutations that are prevalent in Burkitt’s lymphomas,
is also repressed in response to Myc activation. First a tumor characterized by a translocated c-myc gene, affect
p27 KIP1 is sequestered by Myc-induced cyclin D2 and subse- predominantly phosphorylation of Thr-58. This site is phos-
quently degraded in response to cyclin E/CDK2-dependent phorylated in vitro by glycogen synthase kinase (GSK) 3
phosphorylation by a ubiquitin/proteasome dependent path- dependent on phosphorylation of the adjacent Ser-62
way (Bouchard et al., 1999; Bouchard et al., 2001; Perez- (Henriksson et al., 1993; Sears et al., 2000). This latter
Roger et al., 1999; Pagano et al., 1995). Also the latter site is targeted by MAP kinases and by cyclin/CDKs (Seth
process is activated by Myc (Steiner et al., 1995). A recently et al., 1992; Hoang et al., 1995; Pulverer et al., 1994; Lutter-
identified target gene of Myc is cul1 that is involved in bach and Hann, 1994; Henriksson et al., 1993; J. Vervoorts
p27 KIP1 degradation (O’Hagan et al., 2000a). Last but not and B. Lüscher, unpublished findings). For some time the
least Myc can also directly repress the expression of the functional relevance of these two phosphorylation sites and
p27 KIP1 gene (Yang et al., 2001). These findings are consis- the observed mutations in this region remained unresolved.
tent with the observation that immortal myc 2/2 cells have Early on it was suggested that phosphorylation at Ser-62
increased p27 levels (Moreno de Alboran et al., 2001). A stimulated the transcriptional potential of Myc (Seth et al.,
third CKI gene that is targeted by Myc is p21 CIP1 (Claassen 1992, 1993). However this finding is controversial (Henriks-
and Hann, 2000). This molecule inhibits cyclin E/CDK2 and son et al., 1993; Lutterbach and Hann, 1994). In in vitro
is upregulated in response to the activation of the tumor transformation assays small differences between wt c-Myc
suppressor p53 and in differentiating as well as aging cells and Thr-58 and Ser-62 mutants were detected (Pulverer et
as part of an antiproliferative program (Sherr and Roberts, al., 1994; Henriksson et al., 1993). Thus these findings did
1999). not allow to make strong conclusions regarding the function
Together these studies have defined the cyclin D and of the N-terminal phosphorylation sites.
cyclin E kinase complexes, that are essential regulators of However more recently, evidence has been provided indi-
the decision process whether or not a cell proceeds through cating that these phosphorylation sites are regulating Myc
the G1-S checkpoint and into a new round of cell division, stability. Myc is degraded by ubiquitin-mediated proteolysis
as targets of Myc. The identification of multiple Myc regu- (Salghetti et al., 1999; Bahram et al., 2000; Gregory and
lated genes whose products impinge on these kinases Hann, 2000), a process that involves covalent attachment
provide strong support for the notion that these two of ubiquitin to target proteins and subsequent proteolysis by
cyclin/CDK complexes are critical targets for Myc’s func- the proteasome (Varshavsky, 1997). In Myc the TAD deter-
tion in the regulation of cell proliferation. mines the short half life, in particular MBI and II have been
implicated in degradation (Flinn et al., 1998; Salghetti et al.,
1999; Gregory and Hann, 2000; Chen et al., 2000). The
6. Myc and signal transduction mutations at and around Thr-58 and Ser-62 found in
Burkitt’s lymphoma have been shown to result in increased
The conclusions that in normal cells the expression of stability of Myc (Salghetti et al., 1999; Bahram et al., 2000;
Myc is highly regulated and that deregulated expression Gregory and Hann, 2000). Consistent with this finding the
of Myc is important for tumor progression are well analysis of signaling that emanates from Ras and targets
supported by experimental evidence. The regulation of Myc has been shown to result in increased Myc stability
Myc at the posttranslational level has become more trans- (Sears et al., 1999, 2000). This appears to be the conse-
parent over the last several years. In particular regulation by quence of Ser-62 phosphorylation and at the same time
phosphorylation and protein stability have obtained consid- inhibition of Thr-58 phosphorylation by inhibiting a PI3K/
erable interest, also in view of their relevance for tumori- GSK3-dependent mechanism (Sears et al., 2000). This
genesis. Nevertheless some of the findings that have been increase in Myc stability may be relevant during the early
reported are still controversial and will require more phases of the cell cycle in which Ras-dependent signaling
detailed analysis. contributes to entry of cells into G1 from a resting state.
c-Myc is a highly phosphorylated protein with three clus- Since these findings have been obtained from Myc over-
ters of phosphorylation sites. Two of these clusters, one in expression studies, it will now be important to demonstrate10 B. Lüscher / Gene 277 (2001) 1–14
an increase in stability of endogenous Myc in response to sive role Myc plays in the control of cell proliferation one
growth factors. Ser-62 phosphorylation is not only triggered would suspect the former possibility.
by Ras signaling but also by cyclin/CDK complexes and The expression of the c-myc gene and the regulation of
possibly other kinases suggesting that changes in Myc turn- the protein are highly regulated. Recent progress implicates
over may also occur in other phases of the cell cycle. the Ras pathway in the control of the half life of Myc. Also
However such effects may be difficult to document since mutations in the TAD of c-Myc are associated with
at any given point in time only a subfraction of Myc may increased protein stability. These findings suggest that the
respond. Indeed the half life of the total Myc pool changes short half life is one way to interfere with Myc function by
little through the cell cycle with the exception of mitosis limiting its presence. The regulation of protein stability
(Lüscher and Eisenman, 1987; Gregory and Hann, 2000). It appears to be connected to signaling pathways that are
has also been suggested recently that transactivation and controlled by Ras. Since the majority of studies have been
degradation may be coupled (Thomas and Tyers, 2000; performed by using overexpression systems, it will be criti-
Tansey, 2001). Thus this may provide yet another signal cal in the future to establish whether similar events occur in
for Myc turnover, potentially through the same phosphory- cells that are not manipulated. This will be important since
lation sites. The emerging picture indicates that Myc protein overexpression of both, Myc and Ras, can have advert
turnover is a highly dynamic process we are just beginning effects on cell behavior. Thus indirect effects will have to
to understand. be excluded.
While more evidence regarding the regulation of Myc is Our knowledge regarding other Myc/Max/Mad network
becoming available (see above), there is still almost nothing members is still patchy. The prediction that Mad proteins
known about the regulation of Mad proteins. Nevertheless would function as tumor suppressors did not come true,
the short half life of Mad and the fact that these proteins are although the overexpression studies have indicated that
phosphorylated in vivo offers possibilities for regulation these proteins are potent inhibitors of cell proliferation.
that will have to be explored in the future. In addition However the analysis of knock out mice did not support
Max has been considered as the inert interaction partner these original findings. This may be due to overlapping
for Myc and Mad proteins. Max is ubiquitously expressed expression patterns and functions of different Mad proteins.
and constitutively phosphorylated by CK2 and little infor- Thus combinations of knock outs will hopeful provide more
mation regarding its regulation has been obtained (Henriks- insight into the function of these proteins.
son and Lüscher, 1996; Grandori et al., 2000). However We are witnessing an ever increasing list of Myc target
recent evidence indicates that during apoptosis several path- genes, mainly due to the use of DNA microarray screens. It
ways target Max resulting in its dephosphorylation and clea- will be a challenge to understand which of these target genes
vage by caspases (Krippner-Heidenreich et al., 2001). These are critical for tumor formation and thus may provide targets
findings suggest that the central component of the Myc/ for therapy. In this respect it will also be interesting to see
Max/Mad network is regulated under specific circum- whether Myc that is overexpressed, as in many human
stances. Further studies are required to define whether that tumors, regulates additional genes as compared to normal
affects the function of other components of the network, in physiological levels of Myc, or whether the difference is
particular Myc and Mad regarding their involvement in merely quantitative. Defining these differences will require
apoptosis. the input and work, preferentially concerted, of many
laboratories.
7. Outlook
Acknowledgements
Recent years have brought to light novel aspects of Myc
function and regulation. In particular a number of new inter- I apologize for the omission of many important and rele-
action partners have been identified that suggest an impor- vant papers due to space limitations. I thank many collea-
tant role of Myc in the control of chromatin remodeling. gues, in particular M. Eilers, L.-G. Larsson, and J. Lüscher-
Both SWI/SNF and TRRAP complexes can be recruited Firzlaff, and members of my laboratory for many helpful
by Myc, however, the order of events and many of the discussions. The work in my laboratory is supported by the
molecular details remain to be determined. As one example Deutsche Forschungsgemeinschaft and by the Fonds der
it is unclear which HAT activity is recruited to mediate the Chemischen Industrie.
acetylation of core histones of Myc responsive promoters. A
more fundamental question that has not been addressed yet
is whether Myc has the ability to open silent promoters, i.e. References
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