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Mad1 Function Is Regulated through Elements within the Carboxy Terminus
Abstract
Myc and Mad are basic helix-loop-helix leucine zipper (bHLH-LZ) proteins that heterodimerize with Max to bind DNA and thereby
influence the transcription of Myc-responsive genes. Myc-Max dimers transactivate whereas Mad-Max-mSin3 complexes repress
Myc-mediated transcriptional activation. We have previously shown that the N-terminal mSin3 binding domain and the centrally
located bHLH-LZ are required for Mad1 to function during a molecular switch from proliferation to differentiation. Here we
demonstrate that the carboxy terminus (CT) of Mad1 contains previously unidentified motifs necessary for the regulation of
Mad1 function. We show that removal of the last 18 amino acids of Mad1 (region V) abolishes the growth-inhibitory function
of the protein and the ability to reverse a Myc-imposed differentiation block. Moreover, deletion of region V results in a
protein that binds DNA weakly and no longer represses Myc-dependent transcriptional activation. In contrast, deletion of the
preceding 24 amino acids (region IV) together with region V restores DNA binding and transcriptional repression, suggesting
a functional interplay between these two regions. Furthermore, phosphorylation within region IV appears to mediate this interplay.
These findings indicate that novel regulatory elements are present in the Mad1 CT.
... We also observed similar effects in the Rat-1 cell line (Figure 1B), suggesting that MAD1 function is inhibited by AKT. Although there has been debated regarding whether MDA-MD-435 is a breast cancer or melanoma cell line [26], one report indicated that MDA-MD-435 indeed originates from a breast cancer cell line that expresses melanocyte proteins [25]. ...
... It has been reported that MAD1 C-terminal Ser182 and Ser184 are phosphorylated by Casein Kinase II (CKII), and that phosphorylation also inhibits MAD1 DNA binding [26]. Additionally, a recent study reported that Ser145 of MAD1 is phosphorylated by S6K and RSK and that this phosphorylation mediates protein degradation [16]. ...
MAX dimerization protein 1 (MAD1) is a transcription suppressor that antagonizes MYC-mediated transcription activation, and the inhibition mechanism occurs mainly through the competition of target genes' promoter MYC binding sites by MAD1. The promoter binding proteins switch between MYC and MAD1 affects cell proliferation and differentiation. However, little is known about MAD1's regulation process in cancer cells. Here, we present evidence that AKT inhibits MAD1-mediated transcription repression by physical interaction with and phosphorylation of MAD1. Phosphorylation reduces the binding affinity between MAD1 and its target genes' promoter and thereby abolishes its transcription suppression function. Mutation of the phosphorylation site from serine to alanine rescues the DNA-binding ability in the presence of activated AKT. In addition, AKT inhibits MAD1-mediated target genes (hTERT and ODC) transcription repression and promotes cell cycle and cell growth. However, mutated S145A MAD1 abrogates the inhibition by AKT. Thus, our results suggest that phosphorylation of MAD1 by AKT inhibits MAD1-mediated transcription suppression and subsequently activates the transcription of MAD1 target genes.
... Instead, inputs received at promoter DNA in the form of multiple transcription factor binding events can mediate the recruitment of chromatin modifiers such as HATs, HDACs, histone kinases, and methyltransferases. These resultant epigenetic changes and posttranslational modifications occur on histone NH 2 -terminals in the form of specific patterns of acetylation, phosphorylation, methylation, or ubiquitination and thus extend the information content of the DNA code (35)(36)(37). Furthermore, a more sophisticated additional regulatory role in gene transcription is the idea that distinct acetylation sites in histone tails perform different roles. ...
... The Sin3 complex is brought to promoters through its interaction, either directly or indirectly, with sequence-specific transcription factors. For ex-ample, the Sin3 protein complex contributes to Mad-Max repression by interacting directly with the Mad family of proteins (35). Alternatively, the Sin3 complex represses nuclear receptor-mediated activation by interacting indirectly with liganded nuclear receptors through either N-CoR or SMRT corepressor proteins (36). ...
The transcription factor NF-kappaB (NF-kappaB) plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators including histone acetyltransferases (HATs) such as CBP/p300 and p/CAF. Evidence shows that high glucose (HG) conditions mimicking diabetes can activate the transcription of NF-kappaB-regulated inflammatory genes. However, the underlying in vivo transcription and nuclear chromatin remodeling events are unknown. We therefore carried out chromatin immunoprecipitation (ChIP) assays in monocytes to identify 1) chromatin factors bound to the promoters of tumor necrosis factor-alpha (TNF-alpha) and related NF-kappaB-regulated genes under HG or diabetic conditions, 2) specific lysine (Lys (K)) residues on histone H3 (HH3) and HH4 acetylated in this process. HG treatment of THP-1 monocytes increased the transcriptional activity of NF-kappaB p65, which was augmented by CBP/p300 and p/CAF. ChIP assays showed that HG increased the recruitment of NF-kappaB p65, CPB, and p/CAF to the TNF-alpha and COX-2 promoters. Interestingly, ChIP assays also demonstrated concomitant acetylation of HH3 at Lys(9) and Lys(14), and HH4 at Lys(5), Lys(8), and Lys(12) at the TNF-alpha and COX-2 promoters. Overexpression of histone deacetylase (HDAC) isoforms inhibited p65-mediated TNF-alpha transcription. In contrast, a HDAC inhibitor stimulated gene transcription and histone acetylation. Finally, we demonstrated increased HH3 acetylation at TNF-alpha and COX-2 promoters in human blood monocytes from type 1 and type 2 diabetic subjects relative to nondiabetic. These results show for the first time that diabetic conditions can increase in vivo recruitment of NF-kappaB and HATs, as well as histone acetylation at the promoters of inflammatory genes, leading to chromatin remodeling and transcription.
... The protein blot was then stripped and reprobed for total c-Myc to demonstrate equal expression and loading. proteins, such as Mad1, Mxi-1 (Mad2), Mad3, Mad4, and Mnt [35]. These alternative dimers bind the E-Box and actively repress transcription, and therefore antagonize both the transcriptional and transforming activities of Myc [36]. ...
Human Papillomavirus Viruses (HPVs) are associated with the majority of human cervical and anal cancers and 10-30% of head and neck squamous carcinomas. E6 oncoprotein from high risk HPVs interacts with the p53 tumor suppressor protein to facilitate its degradation and increases telomerase activity for extending the life span of host cells. We published previously that the Myc cellular transcription factor associates with the high-risk HPV E6 protein in vivo and participates in the transactivation of the hTERT promoter. In the present study, we further analyzed the role of E6 and the Myc-Max-Mad network in regulating the hTERT promoter. We confirmed that E6 and Myc interact independently and that Max can also form a complex with E6. However, the E6/Max complex is observed only in the presence of Myc, suggesting that E6 associates with Myc/Max dimers. Consistent with the hypothesis that Myc is required for E6 induction of the hTERT promoter, Myc antagonists (Mad or Mnt) significantly blocked E6-mediated transactivation of the hTERT promoter. Analysis of Myc mutants demonstrated that both the transactivation domain and HLH domain of Myc protein were required for binding E6 and for the consequent transactivation of the hTERT promoter, by either Myc or E6. We also showed that E6 increased phosphorylation of Pol II on the hTERT promoter and induced epigenetic histone modifications of the hTERT promoter. More important, knockdown of Myc expression dramatically decreased engagement of acetyl-histones and Pol II at the hTERT promoter in E6-expressing cells. Thus, E6/Myc interaction triggers the transactivation of the hTERT promoter by modulating both histone modifications, Pol II phosphorylation and promoter engagement, suggesting a novel mechanism for telomerase activation and a new target for HPV- associated human cancer.
... Phosphorylation has been shown as a mechanism for regulating the repression activity of some members of the Mad/Max/Myc network of transcription factors, though this occurs at the level of DNA binding (Berberich and Cole, 1992;Bousset et al., 1993). For example, the C-terminus of Mad1 contains motifs that are targets of casein kinase II (CKII) phosphorylation, which mediates its DNA binding and repression activities (Barrera Hernandez et al., 2000). However, it has not been reported whether the phosphorylation status leads to differential binding of corepressors. ...
A Sin3-interacting domain (SID) originally described in Mad proteins is necessary for both transcriptional repression and growth suppression by these transcription factors. We recently reported that a structurally and functionally related Mad1-like SID is also present in five Sp1-like repressor proteins (TIEG1, TIEG2, BTEB1, BTEB3 and BTEB4), demonstrating that SID-mSin3A interactions have a wider functional impact on transcriptional repression. SID-mSin3A interaction is necessary for the anti-proliferative function of Mad, TIEG and BTEB proteins. It remains to be established, however, whether the SID-mSin3A interaction is constitutive or regulated. Here, we describe that the Mad1-like SID domain of the Sp1-like repressor TIEG2 is inhibited by the epidermal growth factor (EGF)-Ras-MEK1-ERK2 signaling pathway, via phosphorylation of four serine/threonine sites adjacent to the SID. This phenomenon disrupts the SID-mSin3A interaction and thereby inhibits TIEG2's repression activity. Thus, these results show for the first time that the repression of a SID-containing protein is regulated by signaling rather than functioning in a constitutive manner, extending our understanding of how the function of SID-containing repressors may be controlled.
We have constructed a database of alternatively spliced protein forms (ASP), consisting of 13,384 protein isoform sequences of 4422 human genes (www.bioinformatics.ucla.edu/ASP). We identified fifty protein domain types that were selectively removed by alternative splicing at much higher frequencies than average (p-value < 0.01). These include many well-known protein-interaction domains (e.g., KRAB; ankyrin repeats; Kelch) including some that have been previously shown to be regulated functionally by alternative splicing (e.g., collagen domain). We present a number of novel examples (Kruppel transcription factors; Pbx2; Enc1) from the ASP database, illustrating how this pattern of alternative splicing changes the structure of a biological pathway, by redirecting protein interaction networks at key switch points. Our bioinformatics analysis indicates that a major impact of alternative splicing is removal of protein-protein interaction domains that mediate key linkages in protein interaction networks. ASP expands the available dataset of human alternatively spliced protein forms from 1989 human genes (SwissProt release 42) to 5413 (nonredundant set, ASP + SwissProt), a nearly 3-fold increase. ASP will enhance the existing pool of protein sequences that are searched by mass spectroscopy software during the identification of peptide fragments.
The Myc/Max/Mad network of transcription factors regulates cell proliferation, differentiation, and transformation. Similar to other proteins of the network, Mnt forms heterodimers with Max and binds CACGTG E-Box elements. Transcriptional repression by Mnt is mediated through association with mSin3, and deletion of the mSin3-interacting domain (SID) converts Mnt to a transcriptional activator. Mnt is coexpressed with Myc in proliferating cells and has been suggested to be a modulator of Myc function. We report that Mnt is expressed both in growth-arrested and proliferating mouse fibroblasts and is phosphorylated when resting cells are induced to re-enter the cell cycle. Importantly, the interaction between Mnt and mSin3 is disrupted upon serum stimulation resulting in decreased Mnt-associated HDAC activity. Furthermore, we demonstrate that Mnt binds and recruits mSin3 to the Myc target gene cyclin D2 in quiescent mouse fibroblasts. Interference with Mnt expression by RNAi resulted in upregulation of cyclin D2 expression in growth-arrested fibroblasts, supporting the view that Mnt represses cyclin D2 transcription in quiescent cells. Our data suggest a model in which phosphorylation of Mnt at cell cycle entry results in disruption of Mnt-mSin3-HDAC1 interaction, which allows induction of Myc target genes by release of Mnt-mediated transcriptional repression.
A significant body of evidence has been accumulated that demonstrates decisive roles of members of the Myc/Max/Mad network in the control of various aspects of cell behavior, including proliferation, differentiation, and apoptosis. The components of this network serve as transcriptional regulators. Mad family members, including Mad1, Mxi1, Mad3, Mad4, Mnt, and Mga, function in part as antagonists of Myc oncoproteins. At the molecular level this antagonism is reflected by the different cofactor/chromatin remodeling complexes that are recruited by Myc and Mad family members. One important function of the latter is their ability to repress gene transcription. In this review we summarize the current view of how this repression is achieved and what the consequences of Mad action are for cell behavior. In addition, we point out some of the many aspects that have not been clarified and thus leave us with a rather incomplete picture of the functions, both molecular and at the cellular level, of Mad family members.
The basic helix‐loop‐helix‐leucine zipper (bHLHZip) protein Max associates with members of the Myc family, as well as with the related proteins Mad (Mad1) and Mxi1. Whereas both Myc:Max and Mad:Max heterodimers bind related E‐box sequences, Myc:Max activates transcription and promotes proliferation while Mad:Max represses transcription and suppresses Myc dependent transformation. Here we report the identification and characterization of two novel Mad1‐ and Mxi1‐related proteins, Mad3 and Mad4. Mad3 and Mad4 interact with both Max and mSin3 and repress transcription from a promoter containing CACGTG binding sites. Using a rat embryo fibroblast transformation assay, we show that both Mad3 and Mad4 inhibit c‐Myc dependent cell transformation. An examination of the expression patterns of all mad genes during murine embryogenesis reveals that mad1, mad3 and mad4 are expressed primarily in growth‐arrested differentiating cells. mxi1 is also expressed in differentiating cells, but is co‐expressed with either c‐myc, N‐myc, or both in proliferating cells of the developing central nervous system and the epidermis. In the developing central nervous system and epidermis, downregulation of myc genes occurs concomitant with upregulation of mad family genes. These expression patterns, together with the demonstrated ability of Mad family proteins to interfere with the proliferation promoting activities of Myc, suggest that the regulated expression of Myc and Mad family proteins function in a concerted fashion to regulate cell growth in differentiating tissues.
Myn, a novel murine approximately 18 kd basic/helix-loop-helix/"leucine zipper" (B/HLH/LZ) protein, forms a specific DNA-binding complex with the c-Myc oncoprotein through the HLH/LZ motif in both proteins. c-Myc/Myn recognizes a c-Myc-binding site (GACCACGTGGTC) with higher affinity than either protein by itself. CpG methylation of the recognition site greatly inhibits DNA binding, suggesting that DNA methylation may regulate the c-Myc/Myn complex in vivo. In 3T3 fibroblasts, Myn mRNA levels are induced several-fold by serum with delayed early kinetics, suggesting regulation by immediate-early gene products. Coexpression of Myn in a myc/ras rat embryo fibroblast focus formation assay specifically augmented c-myc transforming activity. We suggest that interaction of Myn with c-Myc stabilizes sequence-specific DNA binding in vivo.
The product of the c-myc proto-oncogene is a nuclear phosphoprotein whose normal cellular function has not yet been defined. c-Myc has a number of biochemical properties, however, that suggest that it may function as a potential regulator of gene transcription. Specifically, it is a nuclear DNA-binding protein with a short half-life, a high proline content, segments that are rich in glutamine and acidic residues, and a carboxyl-terminal oligomerization domain containing the leucine zipper and helix-loop-helix motifs that serve as oligomerization domains in known regulators of transcription, such as C/EBP, Jun, Fos, GCN4, MyoD, E12, and E47. In an effort to establish that c-Myc might regulate transcription in vivo, we sought to determine whether regions of the c-Myc protein could activate transcription in an in vitro system. We report here that fusion proteins in which segments of human c-Myc are linked to the DNA-binding domain of the yeast transcriptional activator GAL4 can activate transcription from a reporter gene linked to GAL4-binding sites. Three independent activation regions are located between amino acids 1 and 143, a region that has been shown to be required for neoplastic transformation of primary rat embryo cells in cooperation with a mutated ras gene. These results demonstrate that domains of the c-Myc protein can function to regulate transcription in a model system and suggest that alterations of Myc transcriptional regulatory function may lead to neoplastic transformation.
The positive effects of Myc on cellular growth and gene expression are antagonized by activities of another member of the Myc superfamily, Mad. Characterization of the mouse homolog of human mad on the structural level revealed that domains shown previously to be required in the human protein for anti-Myc repression, sequence-specific DNA-binding activity, and dimerization with its partner Max are highly conserved. Conservation is also evident on the biological level in that both human and mouse mad can antagonize the ability of c-myc to cooperate with ras in the malignant transformation of cultured cells. An analysis of c-myc and mad gene expression in the developing mouse showed contrasting patterns with respect to tissue distribution and developmental stage. Regional differences in expression were more striking on the cellular level, particularly in the mouse and human gastrointestinal system, wherein c-Myc protein was readily detected in immature proliferating cells at the base of the colonic crypts, while Mad protein distribution was restricted to the postmitotic differentiated cells in the apex of the crypts. An increasing gradient of Mad was also evident in the more differentiated subcorneal layers of the stratified squamous epithelium of the skin. Together, these observations support the view that both downregulation of Myc and accumulation of Mad may be necessary for progression of precursor cells to a growth-arrested, terminally differentiated state.
The protein Max binds to c-Myc and the heterodimer c-Myc/Max seems to be the active form in vivo. While the expression of c-myc is extensively regulated, no major changes in max expression have been reported so far with respect to differentiation. We have studied the expression of c-Myc and Max during in vitro differentiation of the bipotent human myeloid leukemia K562 cell line. This cell model system allowed us to compare c-Myc and Max expression during differentiation along erythroid (induced by 1-beta-D-arabinofuranosyl-cytosine) and myelomonocytic lineages (induced by 12-0-tetradecanoylphorbol-13-acetate). We found that c-myc expression decreased as a result of both differentiating treatments. The expression level of max remained unchanged during myelomonocytic differentiation. In contrast, max mRNA and protein were dramatically down-regulated during erythroid differentiation of K562 cells, thus demonstrating that max gene is subjected to regulation during differentiation. We also studied the expression of the other two described members of the c-Myc network: mxi1 and mad. mxi1 expression increased during erythroid differentiation but was strongly down-regulated during myelomonocytic differentiation of K562. mad was constitutively expressed during K562 erythroid differentiation and slightly increased during induction of the myelomonocytic pathway. We have obtained K562 sublines stably transfected with a zinc-inducible c-myc gene. In these clones the overexpression of c-Myc did not interfere with TPA-induced myelomonocytic differentiation. In contrast, erythroid differentiation was significantly inhibited upon c-myc induction despite the down-regulation of endogenous max expression. These results suggest a differential role for c-Myc in the human myeloid cell differentiation depending on the cell lineage.
Mad is a basic region helix-loop-helix leucine zipper transcription factor which can dimerize with the Max protein and antagonize transcriptional activation by the Myc-Max transcription factor heterodimer. While the expression of Myc is necessary for cell proliferation, the expression of Mad is induced upon differentiation of at least some leukemia cell lines. Here, the expression of the mad gene has been explored in developing mouse tissues. During organogenesis in mouse embryos mad mRNA was predominantly expressed in the liver and in the mantle layer of the developing brain. At later stages mad expression was detected in neuroretina, epidermis, and whisker follicles, and in adult mice mad was expressed at variable levels in most organs analyzed. Interestingly, in the skin mad was highly expressed in the differentiating epidermal keratinocytes, but not in the underlying proliferating basal keratinocyte layer. Also, in the gut mad mRNA was abundant in the intestinal villi, where cells cease proliferation and differentiate, but not in the crypts, where the intestinal epithelial cells proliferate. In the testis, mad expression was associated with the completion of meiosis and early development of haploid cells. In cell culture, Mad inhibited colony formation of a mouse keratinocyte cell line and rat embryo fibroblast transformation by Myc and Ras. The pattern of mad expression in tissues and its ability to inhibit cell growth in vitro suggests that Mad can cause the cessation of cell proliferation associated with cell differentiation in vivo.
c-Myc and Max are nuclear phosphoproteins capable of forming DNA-binding, homo- and heteropolymeric complexes in vitro and in vivo. Using a transient cotransfection assay involving c-Myc and Max expression vectors and a reporter gene plasmid containing the Myc/Max binding site, we find that Max represses transcription, whereas a significant stimulation is obtained when Max is coexpressed with c-Myc. Analysis of specific mutants indicates that transcriptional activation requires both the c-Myc and the Max dimerization and DNA-binding domains, as well as the c-Myc transactivation function; transcriptional repression by Max requires both DNA binding and dimerization. Analogously, in stably transfected human B-lymphoblastoid cell lines, overexpressed c-Myc and Max synergize to cause malignant transformation, whereas overexpression of Max alone leads to growth inhibition. These results indicate that the c-Myc and Max are transcriptional regulators with the ability to oppositely regulate target-gene expression and cell proliferation, most likely as the result of the opposite effects of heterodimeric c-Myc-Max (positive) versus homodimeric Max (negative) complexes.
The basic helix-loop-helix-leucine zipper (bHLHZip) protein Max associates with members of the Myc family, as well as with the related proteins Mad (Mad1) and Mxi1. Whereas both Myc:Max and Mad:Max heterodimers bind related E-box sequences, Myc:Max activates transcription and promotes proliferation while Mad:Max represses transcription and suppresses Myc dependent transformation. Here we report the identification and characterization of two novel Mad1- and Mxi1-related proteins, Mad3 and Mad4. Mad3 and Mad4 interact with both Max and mSin3 and repress transcription from a promoter containing CACGTG binding sites. Using a rat embryo fibroblast transformation assay, we show that both Mad3 and Mad4 inhibit c-Myc dependent cell transformation. An examination of the expression patterns of all mad genes during murine embryogenesis reveals that mad1, mad3 and mad4 are expressed primarily in growth-arrested differentiating cells. mxi1 is also expressed in differentiating cells, but is co-expressed with either c-myc, N-myc, or both in proliferating cells of the developing central nervous system and the epidermis. In the developing central nervous system and epidermis, downregulation of myc genes occurs concomitant with upregulation of mad family genes. These expression patterns, together with the demonstrated ability of Mad family proteins to interfere with the proliferation promoting activities of Myc, suggest that the regulated expression of Myc and Mad family proteins function in a concerted fashion to regulate cell growth in differentiating tissues.
The product of the c-myc proto-oncogene is a nuclear phosphoprotein whose normal cellular function has not yet been defined. c-Myc has a number of biochemical properties, however, that suggest that it may function as a potential regulator of gene transcription. Specifically, it is a nuclear DNA-binding protein with a short half-life, a high proline content, segments that are rich in glutamine and acidic residues, and a carboxyl-terminal oligomerization domain containing the leucine zipper and helix-loop-helix motifs that serve as oligomerization domains in known regulators of transcription, such as C/EBP, Jun, Fos, GCN4, MyoD, E12, and E47. In an effort to establish that c-Myc might regulate transcription in vivo, we sought to determine whether regions of the c-Myc protein could activate transcription in an in vitro system. We report here that fusion proteins in which segments of human c-Myc are linked to the DNA-binding domain of the yeast transcriptional activator GAL4 can activate transcription from a reporter gene linked to GAL4-binding sites. Three independent activation regions are located between amino acids 1 and 143, a region that has been shown to be required for neoplastic transformation of primary rat embryo cells in cooperation with a mutated ras gene. These results demonstrate that domains of the c-Myc protein can function to regulate transcription in a model system and suggest that alterations of Myc transcriptional regulatory function may lead to neoplastic transformation.
An in vivo footprint over a potential NF-kappa B site in the first exon of the c-myc gene has been identified on the translocated allele in the Ramos Burkitt's lymphoma cell line. The potential NF-kappa B site in the 5' flanking sequence of c-myc was found to be occupied on the translocated allele in the Raji Burkitt's cell line. Electrophoretic mobility shift assays with each of these sequences demonstrated complexes with mobilities identical to those of the NF-kappa B site from the kappa light-chain gene. A supershift was obtained with anti-p50 antibody with the exon site. The upstream-site shift complex disappeared with the addition of anti-p50 antibody. Binding of NF-kappa B proteins to the c-myc exon and upstream sites was demonstrated by induction of binding upon differentiation of pre-B 70Z/3 cells to B cells. UV cross-linking experiments revealed that a protein with a molecular mass of 50 kDa bound to the exon and upstream sites. Transfection experiments with Raji cells demonstrated that both sites functioned as positive regulatory regions, with a drop in activity level when either site was mutated. Access to these sites is blocked in the silent normal c-myc allele in Burkitt's lymphoma cells, while Rel family proteins bind to these sites in the translocated allele. We conclude that the two NF-kappa B sites function as positive regulatory regions for the translocated c-myc gene in Burkitt's lymphoma.
The c-Myc oncoprotein, which is required for cellular proliferation, resembles in its structure a growing number of transcription factors. However, the mechanism of its action in vivo is not yet clear. The discovery of the specific cognate DNA-binding site for Myc and its specific heterodimerization partner, Max, enabled the use of direct experiments to elucidate how Myc functions in vivo and how this function is modulated by Max. Here we demonstrate that exogenously expressed Myc is capable of activating transcription in vivo through its specific DNA-binding site. Moreover, transcriptional activation by Myc is dependent on the basic region, the integrity of the helix-loop-helix and leucine zipper dimerization motifs located in the carboxy-terminal portion of the protein, and the regions in the amino terminus conserved among Myc family proteins. In contrast to Myc, exogenously expressed Max elicited transcriptional repression and blocked transcriptional activation by Myc through the same DNA-binding site. Our results suggest a functional antagonism between Myc and Max which is mediated by their relative levels in the cells. A model for the activity of Myc and Max in vivo is presented.
A broad base of data has implicated a role for the c-myc proto-oncogene in the control of the cell cycle and cell differentiation. To further define the role of myc in these processes, I examined the effect of enforced myc expression on several events that are thought to be important steps leading to the terminally differentiated state: (i) the ability to arrest growth in G0/G1, (ii) the ability to replicate the genome upon initiation of the differentiation program, and (iii) the ability to lose responsiveness to mitogens and withdraw from the cell cycle. 3T3-L1 preadipocyte cell lines expressing various levels of myc mRNA were established by transfection with a recombinant myc gene under the transcriptional control of the Rous sarcoma virus (RSV) promoter. Cells that expressed high constitutive levels of pRSVmyc mRNA arrested in G0/G1 at densities similar to those of normal cells at confluence. Upon initiation of the differentiation program, such cells traversed the cell cycle with kinetics similar to those of normal cells and subsequently arrested in G0/G1. Thus, enforced expression of myc had no effect on the ability of cells to arrest growth in G0/G1 or to replicate the genome upon initiation of the differentiation program. Cells were then tested for their ability to reenter the cell cycle upon exposure to high concentrations of serum and for their capacity to differentiate. In contrast to normal cells, cells expressing high constitutive levels of myc RNA reentered the cell cycle when challenged with 30% serum and failed to terminally differentiate. The block to differentiation could be reversed by high expression of myc antisense RNA, showing that the induced block was specifically due to enforced expression of pRSVmyc. These findings indicate that 3T3-L1 preadipocytes enter a specific state in G0/G1 after treatment with differentiation inducers, into which cells expressing high constitutive levels of myc RNA are precluded from entering. I propose that myc acts as a molecular switch and directs cells to a pathway that can lead to continued proliferation or to terminal differentiation.
Friend murine erythroleukemia (F-MEL) cells were transfected with a plasmid bearing tandemly arranged mouse c-myc antisense and dihydrofolate reductase transcription units. Sixteen clones were isolated, each containing unrearranged c-myc sequences and expressing high levels of antisense transcripts. All antisense clones examined contained reduced amounts of cytoplasmic endogenous c-myc transcripts. The kinetics of reaccumulation of endogenous c-myc mRNA during a 24-h exposure to dimethyl sulfoxide (DMSO) were also retarded and the ultimate transcript levels attained were less than in control cells. Antisense clones grew as well as control F-MEL cells in medium containing 10% fetal calf serum but at only a half and a quarter of the control rates in media containing 5 and 2% serum, respectively. Antisense clones differentiated faster and to a greater degree than control cells following DMSO exposure. myc antisense transcript expression was increased by growing cells in methotrexate, which resulted in an enhanced response to DMSO. Fluorescence-activated cell sorter (FACS) analysis of cellular DNA content indicated that a greater fraction of antisense nuclei contained a G0/G1 2n DNA content following a 24-h exposure to DMSO. When density-arrested antisense clones were diluted into fresh medium to allow reentry into the cell cycle, they incorporated less [3H]thymidine than control cells. FACS analysis showed that this was because only a portion of the cell population was entering S phase. Whereas control cells did not increase in size following release from density arrested antisense cells contained a subpopulation which were initially smaller and which eventually attained the same size as control cells. Quiescent antisense cells thus comprise two populations, each arrested at a different point in G1. Dilutional replating allowed both populations to reenter the cell cycle. We propose a model which postulates that certain minimal myc levels are necessary for cells to traverse G1. Those with insufficient levels, due, for example, to antisense inhibition, are unable to completely traverse G1 during density arrest and synchronize at an earlier point than do control cells. This earlier point may be along the differentiation pathway and may account for the greater responsiveness of antisense cells to DMSO induction. This model postulates that F-MEL cells overexpressing myc fail to differentiate because myc levels are never sufficiently low enough to allow cells to enter the differentiation pathway.
Protein kinase CK2 (casein kinase II) is a serine-threonine protein kinase with a wide range of substrates, many of which are involved in cell cycle regulation. CK2 activity is elevated in a variety of human tumors and we have used a transgenic mouse model to demonstrate that dysregulated expression of CK2 can induce lymphoma. Thus, CK2 fulfills the definition of an oncogene: A mutated, dysregulated, or mis-expressed gene that contributes to cancer in a dominant fashion. CK2 cooperates in transforming cells with other lymphoid oncogenes such as myc and tal-1, and here we show cooperativity with loss of the tumor suppressor gene p53. To understand more about the physiological and pathological role of CK2, we are cloning the murine CK2α′ cDNA and gene. CK2α′ will be used to generate transgenic and knockout mice and the regulatory elements for gene expression will be analyzed. (Mol Cell Biochem 191: 65–74, 1999)
Mad is a basic region helix-loop-helix leucine zipper transcription factor which can dimerize with the Max protein and antagonize transcriptional activation by the Myc-Max transcription factor heterodimer. While the expression of Myc is necessary for cell proliferation, the expression of Mad is induced upon differentiation of at least some leukemia cell lines. Here, the expression of the mad gene has been explored in developing mouse tissues. During organogenesis in mouse embryos mad mRNA was predominantly expressed in the liver and in the mantle layer of the developing brain. At later stages mad expression was detected in neuroretina, epidermis, and whisker follicles, and in adult mice mad was expressed at variable levels in most organs analyzed. Interestingly, in the skin mad was highly expressed in the differentiating epidermal keratinocytes, but not in the underlying proliferating basal keratinocyte layer. Also, in the gut mad mRNA was abundant in the intestinal villi, where cells cease proliferation and differentiate, but not in the crypts, where the intestinal epithelial cells proliferate. In the testis, mad expression was associated with the completion of meiosis and early development of haploid cells. In cell culture, Mad inhibited colony formation of a mouse keratinocyte cell line and rat embryo fibroblast transformation by Myc and Ras. The pattern of mad expression in tissues and its ability to inhibit cell growth in vitro suggests that Mad can cause the cessation of cell proliferation associated with cell differentiation in vivo.
Normal mammalian growth and development are highly dependent on
the regulation of the expression and activity of the Myc family of
transcription factors. Mxi1-mediated inhibition of Myc activities
requires interaction with mammalian Sln3A or Sin3B proteins, which
have been purported to act as scaffolds for additional co-repressor
factors. The identification of two such Sin3-associated factors, the
nuclear receptor co-repressor (N-CoR) and histone deacetylase (HD1),
provides a basis for Mxi1/Sin3-induced transcriptional repression and
tumour suppression.
The Myc family proteins are thought to be involved in transcription because they have both a carboxy-terminal basic-helix-loop-helix-zipper (bHLH-Z) domain, common to a large class of transcription factors, and an amino-terminal fragment which, for c-Myc, has transactivating function when assayed in chimaeric constructs. In addition, c-, N- and L-Myc proteins heterodimerize, in vitro and in vivo, with the bHLH-Z protein Max. In vitro, Max homodimerizes but preferentially associates with Myc, which homodimerizes poorly. Furthermore Myc-Max heterodimers specifically bind the nucleotide sequence CACGTG with higher affinity than either homodimer alone. The identification of Max and the specific DNA-binding activities of Myc and Max provides an opportunity for directly testing the transcriptional activities of these proteins in mammalian cells. We report here that Myc overexpression activates, whereas Max overexpression represses, transcription of a reporter gene. Max-induced repression is relieved by overexpression of c-Myc. Repression requires the DNA-binding domain of Max, whereas relief of repression requires the dimerization and transcriptional activation activities of Myc. Both effects require Myc-Max-binding sites in the reporter gene.
The Max protein forms a heterodimeric complex with the Myc family of proteins and binds to DNA in a sequence-specific manner. We investigated the role of the helix-loop-helix (HLH), leucine zipper (LZ) and basic domains of Max in protein complex formation, DNA-binding activity and transcriptional regulation. We mutagenized the basic, HLH and LZ domains of Max and studied the ability of the normal and mutant proteins to bind to DNA as both homo- and heterodimers and their ability to heterodimerize with Myc. Helix-1 and helix-2 regions of Max were found to be critical for homodimer formation and subsequent DNA binding, while the LZ was essential for heterodimer formation. In transient transfection assays the Myc protein functioned as a transcriptional activator while Max protein repressed the trans-activation observed with Myc.
We have examined the interactions and DNA-binding activities of the c-Myc oncoprotein and its partner Max. In cell extracts virtually all c-Myc molecules are associated with Max in heterodimeric complexes. Moreover, DNA-binding studies with in vitro-translated protein and cell extracts show that both Max alone and c-Myc/Max bind the same DNA sequence. Conversely, c-Myc is unable to bind this sequence in the absence of Max. These findings suggest that c-Myc may function via obligate complex formation with Max.
The c-, L-, and N-Myc nuclear phosphoproteins share several highly conserved regions that partially overlap putative functional domains of the c-Myc protein. All three myc oncogenes can cooperate with an activated ras gene to transform primary rat embryo cells (REC), and deregulated expression of c- and L-myc can block differentiation of murine erythroleukemia (MEL) cells. In the present study, we demonstrate that N-myc also can block MEL cell differentiation, and we identify regions within the c-Myc protein that are necessary for inhibition of MEL differentiation. C19 MEL cells were transfected with six human c-myc genes which were partially deleted in different areas of the coding region. Four of the genes lack sequences that overlap either the putative transcriptional activation domain, the helix-loop-helix motif, or the leucine zipper motif and were previously shown to have lost REC cotransforming activity (J. Stone, T. DeLange, G. Ramsay, E. Jakobovitz, J.M. Bishop, H. Varmus, and W. Lee, Mol. Cell. Biol., 7: 1697-1709, 1987). In this study, we demonstrate that they also fail to inhibit N,N'-hexamethylene-bis-acetamide-induced differentiation of MEL cells. In contrast, two partially deleted c-myc genes, one lacking a short NH2-terminal region and the other lacking 118 amino acids at the center of the coding region, which were fully active in REC cotransformation, also exhibited full activity associated with the former and only partial activity with the latter in blocking MEL differentiation. We conclude that the mutated genes tested in this study behave similarly in inhibition of MEL cell differentiation and in REC cotransformation.
The myc family of cellular oncogenes encodes three highly related nuclear phosphoproteins (c-Myc, N-Myc, and L-Myc) that are believed to function as sequence-specific transcription factors capable of regulating genes important in cellular growth and differentiation. Current evidence indicates that Myc family proteins exist as biologically active heterodimeric complexes in association with another helix-loop-helix leucine zipper phosphoprotein, Max. We have investigated the common and unique properties among the Myc family, as well as the physiological role of Max in the regulation of Myc family function. We demonstrate that trans-activation-incompetent mutants of one Myc family member can act in trans to dominantly suppress the cotransformation activities of all three Myc oncoproteins, indicating that the Myc family functions through common genetic elements in its cellular transformation pathways. Employing co-immunoprecipitation with either anti-Myc or anti-Max antibodies, we show that the transfected normal c-Myc, N-Myc, and L-Myc oncoproteins associate with the endogenous Max protein in REF transformants, indicating that the Max interaction represents at least one component common to Myc family function. In addition, we observed a striking reduction in Myc cotransformation activity when a Max expression construct was added to myc/ras co-transfections. We discuss these biological findings in the context of a proposed model for Myc/Max function and regulation in which Max serves as either an obligate partner in the Myc/Max transcriptional complex or as a repressor in the form of a transcriptionally inert Max/Max homodimer capable of occupying Myc/Max-responsive gene targets.
Max is a heterodimeric partner of the Myc oncoprotein with sequence-specific DNA-binding activity. We found that the DNA-binding activity of bacterially expressed Max homodimers was inhibited in an ATP-dependent reaction by phosphorylation in vitro with purified bovine casein kinase II (CKII). In contrast, phosphorylation of Max and/or Myc by CKII had no inhibitory or stimulatory effect on the DNA-binding activity of Myc/Max heterodimers. By deletion analysis and site-directed mutagenesis, the inhibitory domain was localized to a CKII phosphorylation site in the amino terminus of Max. Finally, extracts prepared from NIH-3T3 cell lines that overexpress Max contained a phosphorylated Max protein which, following phosphatase treatment or heterodimerization with Myc, was capable of sequence-specific DNA-binding activity. Immunoprecipitation experiments confirmed that Max was also phosphorylated in NIH-3T3 cells, demonstrating that Max phosphorylation may have an important physiological function.
The myc protooncogene family has been implicated in cell proliferation, differentiation, and neoplasia, but its mechanism
of function at the molecular level is unknown. The carboxyl terminus of Myc family proteins contains a basic region helix-loop-helix
leucine zipper motif (bHLH-Zip), which has DNA-binding activity and has been predicted to mediate protein-protein interactions.
The bHLH-Zip region of c-Myc was used to screen a complementary DNA (cDNA) expression library, and a bHLH-Zip protein, termed
Max, was identified. Max specifically associated with c-Myc, N-Myc, and L-Myc proteins, but not with a number of other bHLH,
bZip, or bHLH-Zip proteins. The interaction between Max and c-Myc was dependent on the integrity of the c-Myc HLH-Zip domain,
but not on the basic region or other sequences outside the domain. Furthermore, the Myc-Max complex bound to DNA in a sequence-specific
manner under conditions where neither Max nor Myc exhibited appreciable binding. The DNA-binding activity of the complex was
dependent on both the dimerization domain and the basic region of c-Myc. These results suggest that Myc family proteins undergo
a restricted set of interactions in the cell and may belong to the more general class of eukaryotic DNA-binding transcription
factors.
Small deletions of 7 to 48 amino acids have been generated in the leucine zipper domain of the avian cMyc protein and the mutant cMyc proteins expressed using an avian retroviral vector. Retrovirally encoded cMyc protein transforms primary chick embryo fibroblasts and leads to abnormal regulation of the endogenous c-myc gene. Deletion of the most C-terminal leucine of the zipper motif confers a partial phenotype affecting some but not all parameters of transformation. Complete loss of transforming activity results from deletion of further leucine residues, including one which is not part of the heptad repeat. In cMyc transformed cells endogenous c-myc mRNA is expressed at a low level and is abnormally refractory to induction by serum stimulation. In contrast, a non-transforming cMyc protein which lacks the zipper does not affect normal c-myc expression. These results demonstrate that the leucine zipper domain of avian cMyc is required for both transformation and autoregulation, and suggests that essential leucine residues within the motif may be spaced differently from those in the zippers of Fos and Jun.
A broad base of data has implicated a role for the c-myc proto-oncogene in the control of the cell cycle and cell differentiation. To further define the role of myc in these processes, I examined the effect of enforced myc expression on several events that are thought to be important steps leading to the terminally differentiated state: (i) the ability to arrest growth in G0/G1, (ii) the ability to replicate the genome upon initiation of the differentiation program, and (iii) the ability to lose responsiveness to mitogens and withdraw from the cell cycle. 3T3-L1 preadipocyte cell lines expressing various levels of myc mRNA were established by transfection with a recombinant myc gene under the transcriptional control of the Rous sarcoma virus (RSV) promoter. Cells that expressed high constitutive levels of pRSVmyc mRNA arrested in G0/G1 at densities similar to those of normal cells at confluence. Upon initiation of the differentiation program, such cells traversed the cell cycle with kinetics similar to those of normal cells and subsequently arrested in G0/G1. Thus, enforced expression of myc had no effect on the ability of cells to arrest growth in G0/G1 or to replicate the genome upon initiation of the differentiation program. Cells were then tested for their ability to reenter the cell cycle upon exposure to high concentrations of serum and for their capacity to differentiate. In contrast to normal cells, cells expressing high constitutive levels of myc RNA reentered the cell cycle when challenged with 30% serum and failed to terminally differentiate. The block to differentiation could be reversed by high expression of myc antisense RNA, showing that the induced block was specifically due to enforced expression of pRSVmyc. These findings indicate that 3T3-L1 preadipocytes enter a specific state in G0/G1 after treatment with differentiation inducers, into which cells expressing high constitutive levels of myc RNA are precluded from entering. I propose that myc acts as a molecular switch and directs cells to a pathway that can lead to continued proliferation or to terminal differentiation.
Friend murine erythroleukemia (F-MEL) cells were transfected with a plasmid bearing tandemly arranged mouse c-myc antisense and dihydrofolate reductase transcription units. Sixteen clones were isolated, each containing unrearranged c-myc sequences and expressing high levels of antisense transcripts. All antisense clones examined contained reduced amounts of cytoplasmic endogenous c-myc transcripts. The kinetics of reaccumulation of endogenous c-myc mRNA during a 24-h exposure to dimethyl sulfoxide (DMSO) were also retarded and the ultimate transcript levels attained were less than in control cells. Antisense clones grew as well as control F-MEL cells in medium containing 10% fetal calf serum but at only a half and a quarter of the control rates in media containing 5 and 2% serum, respectively. Antisense clones differentiated faster and to a greater degree than control cells following DMSO exposure. myc antisense transcript expression was increased by growing cells in methotrexate, which resulted in an enhanced response to DMSO. Fluorescence-activated cell sorter (FACS) analysis of cellular DNA content indicated that a greater fraction of antisense nuclei contained a G0/G1 2n DNA content following a 24-h exposure to DMSO. When density-arrested antisense clones were diluted into fresh medium to allow reentry into the cell cycle, they incorporated less [3H]thymidine than control cells. FACS analysis showed that this was because only a portion of the cell population was entering S phase. Whereas control cells did not increase in size following release from density arrested antisense cells contained a subpopulation which were initially smaller and which eventually attained the same size as control cells. Quiescent antisense cells thus comprise two populations, each arrested at a different point in G1. Dilutional replating allowed both populations to reenter the cell cycle. We propose a model which postulates that certain minimal myc levels are necessary for cells to traverse G1. Those with insufficient levels, due, for example, to antisense inhibition, are unable to completely traverse G1 during density arrest and synchronize at an earlier point than do control cells. This earlier point may be along the differentiation pathway and may account for the greater responsiveness of antisense cells to DMSO induction. This model postulates that F-MEL cells overexpressing myc fail to differentiate because myc levels are never sufficiently low enough to allow cells to enter the differentiation pathway.
The Friend-virus-derived mouse erythroleukaemia (MEL) cell lines represent transformed early erythroid precursors that can be induced to differentiate into more mature erythroid cells by a variety of agents including dimethyl sulphoxide (DMSO). There is a latent period of 12 hours after inducer is added, when 80-90% of the cells become irreversibly committed to the differentiation programme, undergoing several rounds of cell division before permanently ceasing to replicate. After DMSO induction, a biphasic decline in steady-state levels of c-myc and c-myb messenger RNAs occurs. Following the initial decrease in c-myc mRNA expression, the subsequent increase occurs in, and is restricted to, the G1 phase of the cell cycle. We sought to determine whether the down-regulation is a necessary step in chemically induced differentiation. Experiments reported here indicate that expression in MEL cells of a transfected human c-myc gene inhibits the terminal differentiation process.
It has recently been reported that c-myc is an inducible gene, regulated directly by growth signals which promote proliferation and expressed in a cell-cycle dependent manner. Because various leukaemic cell lines express high levels of c-myc messenger RNA, it was of interest to discover whether the gene could be down-regulated in these cells by a growth inhibitor such as interferon (IFN). We show here that in Daudi Burkitt's lymphoma cells, IFN-alpha produces a five- to sevenfold reduction in c-myc mRNA through a decreased rate of c-myc gene transcription. By isolating a growth-resistant Daudi cell variant that had escaped from this down-regulation, we provide the first clear link between reduction of c-myc mRNA and the IFN-mediated G0/G1 arrest characteristic of Daudi cells. Furthermore, by screening other cell lines, we demonstrate the heterogeneity of human leukaemic cells with respect to these criteria. Thus, IFN-alpha fails to reduce the c-myc mRNA and to change the cell-cycle distribution in HL-60 and U937 cells, although normal induction of other IFN-regulated activities takes place. The latter group of cells shows a decline in c-myc gene expression when they become arrested in the G0/G1 phase as part of their terminal differentiation.
While a number of oncogenes are expressed in a cell cycle-dependent manner, their role in the control of cell proliferation can only be established by a direct functional assay. The c-myc protein, upon microinjection into nuclei of quiescent Swiss 3T3 cells, cooperated with platelet-poor plasma in the stimulation of cellular DNA synthesis. This suggests that c-myc protein, like platelet-derived growth factor (PDGF), may act as a competence factor in the cell cycle to promote the progression of cells to S phase. The presence in the medium of an antibody against PDGF abolished DNA synthesis induced by microinjected PDGF; however, the microinjected c-myc protein stimulated DNA synthesis even when its own antibody was present in the medium. The c-myc protein may act as an intracellular competence factor, while PDGF expresses its biological activity only from outside the cells.
To investigate how overexpression of MAD, an antagonist of MYC oncogenes influences the malignant phenotype of human cancer cells, an adenovirus vector system was used to transfer the human MAD gene (AdMAD) into human astrocytoma cells. Decreased growth potential of AdMAD-infected cells was evidenced by a decrease in [3H]thymidine incorporation, an increase in cell doubling time and alteration of cell-cycle distribution. Diminished malignant potential of AdMAD-infected cells was manifested by their loss of anchorage-independent growth in soft agar and by their inability, in general, to induce tumorigenesis in a xenograft animal model. These studies indicate that adenovirus constructs encoding MAD dramatically inhibit the proliferation and tumorigenicity of human astrocytoma cells and support the use of MAD for gene therapy of human tumours.
c-Myc is an essential component of the regulatory mechanisms controlling cell growth. Max is the obligatory partner of c-Myc for all its biological functions analysed to date. Recently two Max interacting proteins, Mad and Mxi1, have been identified. It has been suggested that these two proteins modulate c-Myc function, in the simplest model by competing with c-Myc for the interaction with Max. We have analysed different aspects of Mad function in comparison to Max. Native Mad/Max heterodimers bound specifically to a c-Myc/Max consensus DNA binding site. Furthermore Mad inhibited efficiently c-Myc, mutant p53, adenovirus E1a, or human papilloma virus type 16 transformation of rat embryo cells in cooperation with activated Ha-Ras. Myc transformed clones showed an increased cell cycle time and a reduced immortalization frequency after cotransfection with either mad or max. In contrast to Mad, Max did not inhibit E1a/Ha-Ras cotransformation but repressed c-Myc/Ha-Ras transformation efficiently. Mad delta N, an N-terminal deletion mutant of Mad, was as efficient in repressing c-Myc/Ha-Ras cotransformation as full length Mad but showed little inhibitory activity when assayed on E1a/Ha-Ras. Unlike wt Mad, Mad delta N had little effect on cell growth. Our data suggest that Mad affects cell growth at least in part by a c-Myc independent mechanism.
Mad is a bHLH/Zip protein that, as a heterodimer with Max, can repress Myc-induced transcriptional transactivation. Expression of Mad is induced upon terminal differentiation of several cell types, where it has been postulated to down-regulate Myc-induced genes that drive cell proliferation. Here we show that Mad also blocks transformation of primary rat embryo fibroblasts by c-Myc and the activated c-Ha-Ras oncoproteins. Mad mutants lacking either the basic region, the leucine zipper, or an intact NH2-terminal protein interaction domain fail to inhibit Myc-Ras cotransformation. These results indicate that the repression of cotransformation requires DNA-binding and is mediated by multiple protein-protein interactions involving both Max and mSin3, a putative mammalian corepressor protein. With increasing amounts of the cotransfected myc gene, the numbers of transformed foci are reduced and the ability of Mad to inhibit focus formation is attenuated. Moreover, cell lines derived from such foci constitutively express both Myc and Mad proteins. Whereas Bcl-2 can significantly increase the numbers of transformed foci by enhancing the survival of myc-ras-transfected cells, it does not counteract the repressive effects of Mad on transformation, suggesting that Mad affects the growth properties rather than the viability of cells. Taken together, our results demonstrate that Mad is capable of antagonizing the biological effects of Myc and thereby suggest that Mad could function as a tumor suppressor gene.
MAD and MXI1, two recently described members of the basic helix-loop-helix (bHLH) gene family, encode proteins that dimerize with and modulate the DNA binding of max. In turn, mad-max or mxi1-max heterodimers or max homodimers can compete for DNA binding sites with dimers formed between max and myc oncoproteins and antagonize the transcriptional activities of this latter class of proteins. Using a combination of somatic cell mapping and fluorescence in situ hybridization techniques, we have determined the chromosomal locations of the MAD and MXI1 genes. The MAD gene maps to chromosome 2p12-p13, a region involved in translocations and deletions in acute and chronic lymphocytic leukemias as well as non-lymphocytic leukemias and Hodgkin disease. The MXI1 gene localizes to chromosome 10q24-q25, a region involved in translocations and deletions in acute and chronic lymphocytic leukemias and prostatic carcinomas. The availability of genomic clones of MAD and MXI1 will permit an assessment of their involvement in these diseases at the molecular level.
Documented interactions among members of the Myc superfamily support a yin-yang model for the regulation of Myc-responsive genes in which transactivation-competent Myc-Max heterodimers are opposed by repressive Mxi1-Max or Mad-Max complexes. Analysis of mouse mxi1 has led to the identification of two mxi1 transcript forms possessing open reading frames that differ in their capacity to encode a short amino-terminal alpha-helical domain. The presence of this segment dramatically augments the suppressive potential of Mxi1 and allows for association with a mammalian protein that is structurally homologous to the yeast transcriptional repressor SIN3. These findings provide a mechanistic basis for the antagonistic actions of Mxi1 on Myc activity that appears to be mediated in part through the recruitment of a putative transcriptional repressor.
Transformed cells do not necessarily lose their capacity to differentiate. Various agents can induce many types of neoplastic cells to terminal differentiation. Among such inducers, a particularly potent group consists of hybrid polar compounds; hexamethylene bisacetamide (HMBA) is the prototype of this group. With virus-transformed murine erythroleukemia cells as a model, HMBA was shown to cause these cells to arrest in G1 phase and express globin genes. This review focuses on HMBA-induced modulation of factors regulating G1-to-S phase progression, including a decrease in the G1 cyclin-dependent kinase cdk4, associated with inhibition of phosphorylation of the retinoblastoma protein pRB and possibly other related proteins that, in turn, sequester factors required for initiation of DNA synthesis; this provides a possible mechanism for HMBA-induced terminal cell division. Evidence that hybrid polar compounds have therapeutic potential for cancer treatment will also be reviewed.
An in vivo footprint over a potential NF-kappa B site in the first exon of the c-myc gene has been identified on the translocated allele in the Ramos Burkitt's lymphoma cell line. The potential NF-kappa B site in the 5' flanking sequence of c-myc was found to be occupied on the translocated allele in the Raji Burkitt's cell line. Electrophoretic mobility shift assays with each of these sequences demonstrated complexes with mobilities identical to those of the NF-kappa B site from the kappa light-chain gene. A supershift was obtained with anti-p50 antibody with the exon site. The upstream-site shift complex disappeared with the addition of anti-p50 antibody. Binding of NF-kappa B proteins to the c-myc exon and upstream sites was demonstrated by induction of binding upon differentiation of pre-B 70Z/3 cells to B cells. UV cross-linking experiments revealed that a protein with a molecular mass of 50 kDa bound to the exon and upstream sites. Transfection experiments with Raji cells demonstrated that both sites functioned as positive regulatory regions, with a drop in activity level when either site was mutated. Access to these sites is blocked in the silent normal c-myc allele in Burkitt's lymphoma cells, while Rel family proteins bind to these sites in the translocated allele. We conclude that the two NF-kappa B sites function as positive regulatory regions for the translocated c-myc gene in Burkitt's lymphoma.
The Myc proto-oncoprotein family is considered to play an important role in the control of cell growth and differentiation. It appears that the interaction of Myc with its heterodimeric partner Max is essential for Myc function. Recently two other partners of Max, called Mad and Mxi1, have been identified. In an effort to gain insight into the network of these four proteins we have started to analyse the expression of the c-myc, max, mad and mxi1 genes at the mRNA level during hematopoietic cell growth and differentiation. In the human myeloid cell lines U-937, HL-60 and ML-1 c-myc expression was down-regulated as shown previously after induction of differentiation, whereas the expression of max was only slightly affected. In contrast to these two genes the expression of mad was induced upon differentiation in the three cell lines by TPA, retinoic acid, vitamin D3, dimethyl sulfoxide, and interferon-gamma and remained elevated for at least 3 days. A kinetic analysis showed that the induction of mad in U-937 in response to TPA was rapid (15 min) and at least in part transcriptional, reminiscent of immediate early genes. The expression of mxi1 was induced in U-937 by some inducers but not in HL-60 or ML-1. Its induction occurred slowly, peaking around 48 h. These analysis thus suggest that the expression of mad and c-myc is inversely regulated during induced hematopoietic differentiation.
Mad and Mxi1, two members of the Myc-related basic-region helix-loop-helix/leucine-zipper family of proteins, associate directly with Max to form sequence-specific DNA binding heterodimers that are transactivation-incompetent. Mad-Max complexes have been shown to exert a strong repressive effect on Myc-induced transactivation, perhaps through the competitive occupation of common promoter binding sites also recognized by active Myc-Max heterodimers. To place these recent biochemical observations in a biological context, mad and mxi1 expression vectors were tested for their ability to influence Myc transformation activity in the rat embryo fibroblast cooperation assay. Addition of an equimolar amount of mad or mxi1 expression vector to mouse c-myc/ras cotransfections resulted in a dramatic reduction in both the number of foci generated and the severity of the malignant phenotype. Myc-specific suppression by Mad and Mxi1 was demonstrated by their ability to affect c- and N-myc-, but not ela-, induced transformation. In contrast, mad and mxi1 expression constructs bearing deletions in the basic region exerted only mild repressive effects on Myc transformation activity, suggesting that occupation of common DNA binding sites by transactivation-incompetent Mad-Max or Mxi1-Max complexes appears to play a more dominant role in this suppression than titration of limited intracellular pools of Max away from active Myc-Max complexes. Thus, these biological data support a current model for regulation of Myc function in which relative intracellular levels of Mad and Mxi1 in comparison to those of Myc may determine the degree of activation of Myc-responsive growth pathways.
Mad is a basic-helix-loop-helix-zipper protein that heterodimerizes with Max in vitro. Mad:Max heterodimers recognize the same E-box-related DNA-binding sites as Myc:Max heterodimers. However, in transient transfection assays Myc and Mad influence transcription in opposite ways through interaction with Max; Myc activates while Mad represses transcription. Here, we demonstrate that Mad protein is induced rapidly upon differentiation of cells of the myeloid lineage. The Mad protein is synthesized in human cells as a 35-kD nuclear phosphoprotein with an extremely short half-life (t1/2 = 15-30 min) and can be detected in vivo in a complex with Max. In the undifferentiated U937 monocyte cell line Max was found complexed with Myc but not Mad. However, Mad:Max complexes began to accumulate as early as 2 hr after induction of macrophage differentiation with TPA. By 48 hr following TPA treatment only Mad:Max complexes were detectable. These data show that differentiation is accompanied by a change in the composition of Max heterocomplexes. We speculate that this switch in heterocomplexes results in a change in the transcriptional regulation of Myc:Max target genes required for cell proliferation.
Myc proteins have been implicated in the regulation of cell growth and differentiation. The identification of Max, a basic region/helix-loop-helix/leucine zipper protein, as a partner for Myc has provided insights into Myc's molecular function as a transcription factor. Recent evidence indicates that the relative abundance of Myc and Max is important to determine the level of specific gene transcription. In this report we have identified two major in vivo phosphorylation sites in Max (Ser-2 and -11) which can be modified in vitro by casein kinase II (CKII). Phosphorylation of these sites modulates DNA-binding by increasing both the on- and off-rates of Max homo- as well as Myc/Max heterodimers. In addition, our data indicate that the steady state binding of the shorter version of Max (p21) to DNA was similar yet its rate of dissociation faster than that of longer version of Max (p22). These data argue that different Max complexes have different kinetic properties and that these can be modified by CKII phosphorylation. We propose this as an important biological mechanism by which different dimeric complexes can exchange with varying efficiencies on DNA, thereby responding to changes in cell growth conditions.
Both the MAD and the MXI1 genes encode basic-helix-loop-helix-leucine zipper (bHLH-Zip) transcription factors which bind Max in vitro, forming a sequence-specific DNA-binding complex similar to the Myc-Max heterodimer. Mad and Myc compete for binding to Max. In addition, Mad has been shown to act as a transcriptional repressor while Myc appears to function as an activator. Mxi1 also appears to lack a transcriptional activation domain. Therefore, Mxi1 and Mad might antagonize Myc function and are candidate tumor suppressor genes. We report here the mapping of the MAD and MXI1 genes in human and mouse by fluorescence in situ hybridization (FISH) and by recombination mapping. The MAD gene was mapped to human chromosome 2 at band p13 by FISH and to mouse chromosome 6 by meiotic mapping. The MXI1 gene was mapped to human chromosome 10 at band q25 and on mouse chromosome 19 at region D by FISH. There was a second site of hybridization on mouse chromosome 2 at region C, which may represent a pseudogene or a related sequence. The mapping results confirm regions of conservation between human chromosome 2p13 and mouse chromosome 6 and between chromosome 10q25 and mouse chromosome 19D. Human chromosomes 2p13 and 10q25 have been involved in specific tumors where the role of Mad and Mxi1 can now be investigated.
The product of the human c-myc protooncogene (Myc) is a sequence-specific DNA binding protein. Here, we demonstrate that the placement of the specific Myc DNA binding site CACGTG upstream of a luciferase reporter gene conferred Myc-stimulated expression that was inhibited by the overexpression of the basic-helix-loop-helix/leucine zipper protein Max. It was observed that Myc was phosphorylated in vivo within the NH2-terminal domain at Thr-58 and Ser-62. Replacement of these phosphorylation sites with Ala residues caused a marked decrease in Myc-stimulated reporter gene expression. In contrast, the replacement of Thr-58 or Ser-62 with an acidic residue (Glu) caused only a small inhibition of transactivation. Together, these data demonstrate that the NH2-terminal phosphorylation sites Thr-58 and Ser-62 are required for high levels of transactivation of gene expression by Myc.
The c-Myc oncoprotein, which is required for cellular proliferation, resembles in its structure a growing number of transcription
factors. However, the mechanism of its action in vivo is not yet clear. The discovery of the specific cognate DNA-binding
site for Myc and its specific heterodimerization partner, Max, enabled the use of direct experiments to elucidate how Myc
functions in vivo and how this function is modulated by Max. Here we demonstrate that exogenously expressed Myc is capable
of activating transcription in vivo through its specific DNA-binding site. Moreover, transcriptional activation by Myc is
dependent on the basic region, the integrity of the helix-loop-helix and leucine zipper dimerization motifs located in the
carboxy-terminal portion of the protein, and the regions in the amino terminus conserved among Myc family proteins. In contrast
to Myc, exogenously expressed Max elicited transcriptional repression and blocked transcriptional activation by Myc through
the same DNA-binding site. Our results suggest a functional antagonism between Myc and Max which is mediated by their relative
levels in the cells. A model for the activity of Myc and Max in vivo is presented.
c-Myc (Myc) and Max proteins dimerize and bind DNA through basic-helix-loop-helix-leucine zipper motifs (b-HLH-LZ). Using a genetic approach, we demonstrate that binding to Max is essential for Myc transforming activity and that Myc homodimers are inactive. Mutants of Myc and Max that bind efficiently to each other but not to their wild-type partners were generated by either exchanging the HLH-LZ domains or reciprocally modifying LZ dimerization specificities. While transformation defective on their own, complementary mutants restore Myc transforming activity when coexpressed in cells. The HLH-LZ exchange mutants also have dominant negative activity on wild-type Myc function. In addition, wild-type max antagonizes myc function in a dose-dependent manner, presumably through competition of Max-Max and Myc-Max dimers for common target DNA sites. Therefore, Max can function as both suppressor and activator of Myc. A general model for the role of Myc and Max in growth control is discussed.
We used the interaction trap to isolate a novel human protein that specifically interacts with Max. This protein, Mxi1 (for Max interactor 1), contains a bHLH-Zip motif that is similar to that found in Myc family proteins. Mxi1 interacts specifically with Max to form heterodimers that efficiently bind to the Myc-Max consensus recognition site. When bound to DNA by a LexA moiety in yeast, Mxi1 does not stimulate transcription. mxi1 mRNA is expressed in many tissues, and its expression is elevated in U-937 myeloid leukemia cells that have been stimulated to differentiate. These facts are consistent with a model in which Mxi1-Max heterodimers indirectly inhibit Myc function in two ways: first, by sequestering Max, thus preventing the formation of Myc-Max heterodimers, and second, by competing with Myc-Max heterodimers for binding to target sites.
Myc family proteins appear to function through heterodimerization with the stable, constitutively expressed bHLH-Zip protein, Max. To determine whether Max mediates the function of regulatory proteins other than Myc, we screened a lambda gt11 expression library with radiolabeled Max protein. One cDNA identified encodes a new member of the bHLH-Zip protein family, Mad. Human Mad protein homodimerizes poorly but binds Max in vitro, forming a sequence-specific DNA binding complex with properties very similar to those of Myc-Max. Both Myc-Max and Mad-Max heterocomplexes are favored over Max homodimers, and, unlike Max homodimers, the DNA binding activity of the heterodimers is unaffected by CKII phosphorylation. Mad does not associate with Myc or with representative bHLH, bZip, or bHLH-Zip proteins. In vivo transactivation assays suggest that Myc-Max and Mad-Max complexes have opposing functions in transcription and that Max plays a central role in this network of transcription factors.
c-myc, N-myc, and L-myc genes are subject to highly variable degrees of tissue-specific regulation. Their aberrant expression has also been implicated in the pathogenesis of a variety of malignant tumors. The recently identified max protein dimerizes with c-myc to promote its sequence-specific DNA binding. max exists in two forms (long and short) that differ by virtue of a 9-amino acid insertion/deletion at the N terminus. We tested recombinant myc and max proteins for binding to six oligonucleotides containing related c-myc sites. Each myc protein, alone and in association with max proteins, manifested a unique pattern of DNA binding. Phosphorylation of both max proteins was observed when they were incubated in a rabbit reticulocyte lysate. This strongly affected DNA binding by max(long) but not by max(short). Our results point to the existence of specific DNA binding preferences for each of the myc proteins. The 9-amino acid segment that distinguishes max(long) from max(short) appears to serve a regulatory function that provides additional control over DNA sequence recognition.
c-Myc and Mad each form heterodimers with Max that bind the same E-box related DNA sequences. Whereas Myc:Max complexes activate transcription and promote cell proliferation and transformation, Mad:Max complexes repress transcription and block c-Myc-mediated cell transformation. Here we examine these antagonistic transcription factors during epithelial differentiation and neoplastic progression. During differentiation of primary human keratinocytes, Mad is rapidly induced and c-Myc is downregulated, resulting in a switch from c-Myc:Max to Mad:Max heterodimers. In normal epidermis and colonic mucosa c-myc expression is restricted to proliferating cell layers, while mad expression is restricted to differentiating cell layers. Using HPV18 transformed keratinocytes that vary in their ability to differentiate in organotypic cultures, we find that Mad induction occurs only in those cells that retain a differentiation response. In the epidermis of transgenic mice in which expression of the HPV16 E6 and E7 oncogenes are targeted to basal keratinocytes, neoplastic progression occurs and is marked by an expansion of c-myc expressing basal-like cells. Expression of mad is found only in growth-arrested differentiating cells on the outer edges of preneoplastic lesions. The squamous cell carcinomas that arise evidence a variable number of sites within the tumor masses where mad expression and morphological differentiation coincide; increasing malignancy correlates with loss of both mad and capability to differentiate. These results indicate that c-Myc and Mad expression are tightly coupled to the transition from proliferation to differentiation of epithelial cells and that restriction of Mad expression may be associated with loss of normal differentiation capability and with tumorigenesis.