Function and regulation of the transcription factors of the Myc/Max/Mad network

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 expression, 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.

Introduction

The broad and lasting interest in myc genes and Myc proteins is based on the realization that they regulate decisively various aspects of cell behavior. Foremost is the large body of information that has been accumulated over the last twenty years which demonstrates a strong involvement of myc in tumorigenesis (Marcu et al., 1992; Henriksson and Lüscher, 1996; Nesbit et al., 1999). Soon after the initial identification of v-myc in several transforming chicken retroviruses, genetic alterations of myc genes were found in many different human malignancies, e.g. translocations of the c-myc gene in Burkitt's lymphoma and amplification of the N-myc gene in neuroblastoma. This results in general in a deregulated, enhanced expression of myc genes, a condition found in many if not most human tumors. In addition studies in animal models and in tissue culture systems have provided compelling evidence for a tumor promoting activity of Myc proteins (Morgenbesser and DePinho, 1994; Pelengaris et al., 2000). This appears to be the result of Myc's ability to stimulate cell proliferation and at the same time to inhibit cells to enter a resting state or to terminally differentiate. Thus a consequence of the constitutive presence of Myc is an increase in the respective cellular compartment which is thought to provide the base for additional genetic alterations that cooperate with Myc in tumor formation (Berns, 1991; Adams and Cory, 1992). Recently a more direct role of Myc in transforming cells has also been suggested. This is based on findings that overexpression of Myc causes genomic destabilization potentially due to its ability to induce endoreplication (Mai et al., 1999; Taylor and Mai, 1998; Felsher and Bishop, 1999; Felsher et al., 2000). Knock-out experiments provide further strong evidence for a critical role of Myc for normal cell behavior (Stanton et al., 1992; Charron et al., 1992; Davis et al., 1993; Moreno de Alboran et al., 2001). In addition Myc proteins can stimulate apoptosis, a finding that on first view does not seem to be compatible with the other functions mentioned above (Evan et al., 1992; Thompson, 1998; Prendergast, 1999). However it is now thought that the induction of apoptosis by Myc is part of a safeguard mechanism that helps restrict the dominant action of Myc proteins on cell proliferation. In support tumor cells with elevated Myc levels are frequently defective in apoptotic pathways. Another activity of Myc that has been demonstrated recently is the ability to induce cell growth independent of cell cycle progression in some systems (Johnston et al., 1999; Iritani and Eisenman, 1999; Schuhmacher et al., 1999). Together these findings define Myc as a highly versatile factor influencing many aspects of normal cell behavior.

For many years it was unclear how Myc proteins achieve the diverse biological effects summarized above. From various studies, including the nuclear localization of these proteins, it was inferred that Myc could play roles in nuclear architecture, replication, splicing, and/or transcription (Lüscher and Eisenman, 1990). A hypothesis supporting the latter was formulated towards the end of the 1980s mainly on the basis of sequence homologies to other transcription factors. The motif found in common is referred to as the basic region/helix-loop-helix/leucine zipper (bHLHZip) domain and functions as a DNA binding and protein-protein interaction domain (Lüscher and Larsson, 1999). A break-through observation in defining a role of Myc in transcription was the identification of the bHLHZip protein Max as a Myc dimerization partner 10 years ago (Blackwood and Eisenman, 1991; Prendergast et al., 1991; Blackwood et al., 1992). Max is the essential heterodimerization partner of Myc proteins for various biological activities, including transformation, apoptosis, and transcriptional activation (Amati et al., 1992, Amati et al., 1993a, Amati et al., 1993b). In agreement with these findings Max is essential for normal mouse development (Shen-Li et al., 2000). However whether Max is required for all molecular functions of Myc is presently not known. Nevertheless the identification of Max and the finding that Myc/Max heterodimers bind specifically to E box DNA sequences with the consensus core 5′-CACGTG and through such elements activate the expression of reporter genes supported the hypothesis that Myc functions as a transcriptional regulator (Dang, 1999; Grandori et al., 2000). In addition a transactivation domain (TAD) was identified near the N-terminus of Myc (Facchini and Penn, 1998; Dang, 1999). Both the bHLHZip and the TAD are important for Myc function in the control of cell behavior. Together these findings provide support for a role of Myc in gene transcription. Other activities, however, cannot be excluded.

How can Myc affect so profoundly different aspects of cell behavior? To answer this question it will be important to define the genes that are targeted by Myc and to understand how the expression and function of Myc is regulated. Of particular interest is the question how Myc causes cell transformation in view of identifying targets that might be accessible to therapeutic strategies. During the early days of the search for Myc target genes it was hoped, somewhat naively from toady's point of view, that one could identify ‘the Myc target gene for transformation’. It seems more realistic now that Myc will modulate the expression of many genes to control cell behavior. A continuously increasing number of target genes are being proposed some of which are thought to be relevant for transformation (Grandori and Eisenman, 1997; Facchini and Penn, 1998; Boyd and Farnham, 1999). In particular the use of DNA microarray technology has made it possible to probe a large number of genes and to define a set of potential target genes that reflect Myc's broad role in the control of cell behavior (Coller et al., 2000; Guo et al., 2000; O'Hagan et al., 2000b; Schuhmacher et al., 2001). The targets include genes involved in the control of the cell cycle, protein and DNA biosynthesis, cell growth, cell adhesion, apoptosis, and immortality (Grandori et al., 2000). It will now be challenging to sort out the hierarchy of the importance of these genes for specific Myc functions, particularly for transformation.

The regulation of Myc expression and function is still far from being understood. In particular the expression of myc genes appears highly complicated. While a large number of transcription factors have been defined that can regulate the c-myc promoter, no unifying concept exists that explains myc expression during the cell cycle and during the transitions from resting to cycling and from cycling to differentiating cells (Spencer and Groudine, 1991; Marcu et al., 1997). Recent findings however provide some insight into c-myc regulation during the transition from G0 into G1 (Nasi et al., 2001). The tyrosine kinase Src has been implicated in mediating growth factor signal-induced c-myc expression (Barone and Courtneidge, 1995; Blake et al., 2000). Src appears to signal through the Rho GTPases rather than Ras or mitogen-activated protein kinases to activate the c-myc promoter (Chiariello et al., 2001). Although the transcription factor targets of Src/Rho signaling remain undefined, these findings provide a starting point to unravel growth factor-dependent c-myc expression.

Regarding the regulation of Myc proteins, recent progress has been made in understanding the signals that impinge on Myc. In particular phosphorylation has been shown to affect the stability of Myc, a protein with a short half life in the order of 20–30 min. Furthermore numerous Myc interaction partners have been identified, the functions of some support a role of Myc in the regulation of chromatin structure (Dang, 1999; Grandori et al., 2000; Amati et al., 2001). One task for the coming years will be to determine the role of these different proteins for specific aspects of Myc function and to connect these interactions to signaling pathways. In addition the essential Myc dimerization partner Max binds also to several other bHLHZip factors, forming what is referred to as the Myc/Max/Mad network of transcriptional regulators (Fig. 1). To understand the biological functions of Myc proteins requires also that the other components of this network are studied and their regulation and function determined since they impinge in one way or another on Myc function.

Numerous excellent reviews have been published in recent years that describe various aspects of the function and the biology of the Myc/Max/Mad network. Several of these reviews are cited to summarize work that can not be discussed in detail here. This review concentrates on novel aspects regarding the molecular functions and regulations of Myc and its network partners in gene transcription and the control of cell proliferation.