diminutive
The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though
heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding
complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc
antibodies, a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo have been identified.
Four of 27 sites recovered by this procedure correspond to the highest affinity 'canonical' CACGTG
sequence. However, the most common in vivo binding sites belong to the group of 'non-canonical' E
box-related binding sites previously identified by in vitro selection. Several of the genomic fragments
isolated contain transcribed sequences, including one, MrDb, encoding an evolutionarily conserved
RNA helicase of the DEAD box family. The corresponding mRNA is induced following activation
of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor,
consistent with this helicase gene being a direct target of Myc-Max. As for c-Myc, the
expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B
cells and down-regulated during terminal differentiation of HL60 leukemia cells. These results indicate
that (1) Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences, and (2)
that Myc is likely to activate multiple target genes, including a highly conserved DEAD box protein.
Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and
metabolism (Grandori, 1996). MrDb is a homolog of the Drosophila gene pitchoune.
Cad is the trifunctional enzyme
carbamoyl-phosphate synthase/aspartate transcarbamoylase/dihydroorotase, which is required for the first three rate-limiting steps of pyrimidine
biosynthesis. The expression of virtually all proposed c-Myc target genes is unchanged in cells containing a
homozygous null deletion of c-myc. Two noteworthy exceptions are the gene cad, which has reduced log phase
expression and serum induction in c-myc null cells, and the growth arrest gene gadd45, which is derepressed by
c-myc knockout. Thus, cad and gadd45 are the only proposed targets of c-Myc that may contribute to the dramatic
slow growth phenotype of c-myc null cells. These results demonstrate that a loss-of-function approach is critical for the
evaluation of potential c-Myc target genes (Bush, 1998).
The c-Myc protein is a site-specific DNA-binding transcription factor that is up-regulated in a number of different cancers. Binding of Myc correlates with increased transcription of the cad promoter. The mechanism by which Myc mediates
transcriptional activation of the cad gene has been investigated. Using a chromatin immunoprecipitation assay, high levels of RNA polymerase II were found bound to the cad promoter in quiescent NIH 3T3 cells and in differentiated U937 cells, even though the promoter is inactive. However, chromatin immunoprecipitation with an antibody that recognizes the hyperphosphorylated form of the RNA
polymerase II carboxyl-terminal domain (CTD) reveals that phosphorylation of
the CTD does correlate with c-Myc binding and cad transcription. The c-Myc transactivation domain interacts with cdk9 and cyclin T1,
components of the CTD kinase P-TEFb. Furthermore, activator bypass experiments
have shown that direct recruitment of cyclin T1 to the cad promoter can
substitute for c-Myc to activate the promoter. In summary, these results suggest that c-Myc activates transcription of cad by stimulating promoter clearance and elongation, perhaps via recruitment of P-TEFb (Eberhardy, 2001).
Telomere maintenance has been proposed as an essential prerequisite to human tumor development. The telomerase enzyme is itself a marker for tumor cells, but the genetic alterations that activate the enzyme during neoplastic transformation have remained a mystery. Myc induces telomerase in both normal human mammary epithelial cells (HMECs) and normal human diploid fibroblasts. Myc increases expression of hEST2 (hTRT/TP2), the limiting subunit of telomerase, and both Myc and hEST2 can extend the life span of HMECs. The ability of Myc to activate telomerase may contribute to its ability to promote tumor formation (Wang, 1998).
E-cadherin plays a pivotal role in the biogenesis of the first epithelium during development, and its down-regulation is associated with metastasis of carcinomas. Inactivation of RB family proteins by simian virus 40 large T antigen (LT) in MDCK epithelial cells results in a mesenchymal conversion associated with invasiveness and a down-regulation of c-Myc. Reexpression of RB or c-Myc in such cells allows the reexpression of epithelial markers, including E-cadherin. Both RB and c-Myc specifically activate transcription of the E-cadherin promoter in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated in both cases by the transcription factor AP-2. In vitro AP-2 and RB interaction involves the N-terminal domain of AP-2 and the oncoprotein binding domain and C-terminal domain of RB. In vivo physical interaction between RB and AP-2 has been demonstrated in MDCK and HaCat cells. In LT-transformed MDCK cells, LT, RB, and AP-2 were all coimmunoprecipitated by each of the corresponding antibodies, and a mutation of the RB binding domain of the oncoprotein inhibits its binding to both RB and AP-2. Taken together, these results suggest that there is a tripartite complex between LT, RB, and AP-2 and that the physical and functional interactions between LT and AP-2 are mediated by RB. Moreover, they define RB and c-Myc as coactivators of AP-2 in epithelial cells and shed new light on the significance of the LT-RB complex, linking it to the dedifferentiation processes occurring during tumor progression. These data confirm the important role for RB and c-Myc in the maintenance of the epithelial phenotype and reveal a novel mechanism of gene activation by c-Myc (Batsche, 1998).
A set of c-Myc-responsive genes has been identified in the Rat1a fibroblast through the application of cDNA representational difference analysis (RDA) to cDNAs isolated from nonadherent Rat1a and Rat1a-myc cells. In this system, c-Myc overexpression is sufficient to induce the transformed phenotype of anchorage-independent growth. Twenty differentially expressed cDNAs have been identified, several of which represent novel cDNA sequences. One of the novel cDNAs identified in this screen, termed rcl, is (1) directly stimulated by c-Myc; (2) stimulated in the in vivo growth system of regenerating rat liver, as is c-myc, and (3) elevated in human lymphoid cells that overexpress c-myc. The Rcl protein was found to be a 23-kDa nuclear protein. Ectopic expression of the protein encoded by the rcl cDNA induces anchorage-independent growth in Rat1a fibroblasts, albeit to a diminished extent compared to ectopic c-Myc expression. These data suggest a role for rcl during cellular proliferation and c-Myc-mediated transformation (Lewis, 1997).
The c-Myc protein activates transcription as part of a heteromeric complex with Max. However, Myc-transformed cells are characterized by loss of expression of several genes, suggesting that Myc may also repress gene expression. Two-hybrid cloning identifies a novel POZ domain Zn finger protein (Miz-1; Myc-interacting Zn finger protein-1) that specifically interacts with Myc, but not with Max or USF. Miz-1 binds to start sites of both the adenovirus major late promoter and the cyclin D1 promoter; it activates transcription from both promoters. Miz-1 has a potent growth arrest function. Binding of Myc to Miz-1 requires the helix-loop-helix domain of Myc and a short amphipathic helix located in the carboxy-terminus of Miz-1. Expression of Myc inhibits transactivation, overcomes Miz-1-induced growth arrest and renders Miz-1 insoluble in vivo. These processes depend on the association of Myc and Miz-1, and on the integrity of the POZ domain of Miz-1, suggesting that Myc binding activates a latent inhibitory function for this domain. Fusion of a nuclear localization signal induces efficient nuclear transport of Miz-1 and impairs the ability of Myc to overcome transcriptional activation and growth arrest by Miz-1. These data suggest a model for how gene repression by Myc may occur in vivo (Peukert, 1997).
Cell proliferation is regulated by the induction of growth promoting genes and the suppression of growth inhibitory genes. Malignant growth can result from the altered balance of expression of these genes in favor of cell proliferation. Induction of the transcription factor, c-Myc, promotes cell proliferation and transformation by activating growth promoting genes, including the ODC and cdc25A genes. c-Myc transcriptionally represses the expression of a growth arrest gene, gas1. A conserved Myc structure, Myc box 2, is required for repression of gas1, and for Myc induction of proliferation and transformation, but not for activation of ODC. Activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen is sufficient to repress gas1 gene transcription. These findings suggest that transcriptional repression of growth arrest genes, including gas1, is one step in the promotion of cell growth by Myc (Lee, 1997).
A problem common to many investigators is uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a
characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion
proteins, the timing and functional consequences of the binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular
genes has been analyzed. The endogenous cad promoter (cad is carbamoyl-phosphate
synthase/aspartate carbamoyltransferase/dihydroorotase, a growth responsive gene) can be immunoprecipitated with antibodies against Myc and USF1. Although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. The amount
of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, these data on both DNA binding and promoter activity in intact
cells suggest that cad is a Myc target gene. In addition, Myc binding can occur at many sites in vivo but the position of the binding site
determines the functional consequences of this binding. These data indicate that a post-DNA-binding mechanism determines Myc target gene specificity.
Importantly, the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes has been demonstrated (Boyd, 1998).
The lactate dehydrogenase A (LDH-A) gene, whose product participates in normal anerobic glycolysis and is frequently increased in human cancers, has been identified as a c-Myc-responsive gene. It was of interest, therefore, to compare the effect of glucose deprivation in c-Myc-transformed and nontransformed cells. Glucose deprivation or treatment with the glucose antimetabolite 2-deoxyglucose causes nontransformed cells to arrest in the G0/G1 phase of the cell cycle. In contrast, c-Myc-transformed fibroblasts, lymphoblastoid, or lung carcinoma cells undergo extensive apoptosis. Ectopic expression of LDH-A alone in Rat1a fibroblasts is sufficient to induce apoptosis with glucose deprivation but not with serum withdrawal, suggesting that LDH-A mediates the unique apoptotic effect of c-Myc when glycolysis is blocked. The apoptosis caused by glucose deprivation is blocked by Bcl-2 expression but appears to be independent of wild-type p53 activity. These studies provide insights on the coupling of glucose metabolism and the cell cycle in c-Myc-transformed cells and may in the future be exploited for cancer therapeutics (Shim, 1998).
To identify genes regulated by N-myc, subtraction of whole embryo cDNA was carried out between wild type and N-myc-deficient mutant mice. Six cDNA clones were isolated as representing genes expressed at higher levels in the mutant embryos and two as those expressed at lower levels. One of them, Ndr1, coding for 43 kDa cytoplasmic protein, was studied in detail. The Ndr1 gene is augmented 20-fold in the mutant embryos at 10.5 days of development, which is indicative of repression by N-myc. An inverse relationship exists between the expression of N-myc and Ndr1 in various developing tissues of the wild type embryos. In the early stage of differentiation of these tissues, when N-myc expression is high, Ndr1 expression is low or undetectable, and later when N-myc activity diminishes, Ndr1 expression is augmented concomitantly with the occurrence of terminal differentiation. To establish the direct link between N-myc activity and the Ndr1 regulation, the Ndr1 gene was cloned and analyzed. The Ndr1 promoter activity is down-regulated by N-myc, and more strongly by the combination of N-myc and Max in the cotransfection assay. This repressive effect is mediated by the promoter region within 52 base pairs from the transcription start site but direct binding of N-myc:Max to the promoter sequence has not been demonstrated. This failure is analogous to other cases reported for transcriptional repression by c-myc. c-myc also represses Ndr1 promoter activity similar to N-myc. The effect of N-myc:Max is sensitive to Trichostatin A, indicating involvement of histone deacetylase activity in repression of the Ndr1 promoter. The strategy adopted in identifying target genes should prove widely applicable when animals mutant for given transcription factors are available (Shimono, 1999).
Cell number is regulated by maintaining a balance between cell proliferation and cell death through apoptosis. Key regulators of this balance include the oncogene product c-Myc, which promotes either entry into the cell cycle or apoptosis. Although the mechanism of c-Myc-induced apoptosis remains unclear, it is susceptible to regulation by survival factors and can proceed through the interaction of Fas ligand (FasL) with its receptor, Fas. Activated T lymphocytes are eliminated by
an apoptotic process known as activation-induced cell death (AICD), which requires the transcriptional induction of FasL expression and sustained levels of c-Myc. The FasL promoter can be driven by c-Myc overexpression, and functional inhibitors of Myc and its binding partner, Max, inhibit the transcriptional activity of the FasL promoter. A non-canonical binding site (ATTCTCT) was identified for c-Myc-Max heterodimers in the FasL promoter, which, when mutated, abolishes activity in response to c-Myc. Exchange of the canonical c-Myc responsive elements (CACGTG) in the ornithine decarboxylase (ODC) promoter with the non-canonical sequence in the FasL promoter generates an ODC-FasL promoter that is significantly more responsive to c-Myc than the wild-type ODC promoter. These findings identify a precise physiological role for c-Myc in the induction of apoptosis as a transcriptional regulator of the FasL gene (Kasibhatla, 2000).
Why should FasL expression in lymphocytes be linked to the expression and function of c-Myc? Only activated, proliferating cells, and not resting lymphocytes, express c-Myc. Since clonal expansion forms the basis of immune responses, it is only the proliferating cells that may represent a threat to the body should they happen to be auto-reactive (or even hyper-reactive, since these will also cause extensive
bystander damage). Re-stimulation of activated cells therefore induces FasL, which in turn serves to check cellular expansion by induction of apoptosis. Alternatively (and non-exclusively), activated, proliferating lymphocytes that take on effector functions do so, in part, through the expression of FasL (except in this case they are resistant to Fas-mediated death), which functions to kill Fas-expressing
cells with which they come in contact. By limiting this expression to proliferating cells, it serves as a fail-safe mechanism to ensure that once the cells cease to express c-Myc (for example, at the cessation of the response) expression of this lethal molecule will also cease (Kasibhatla, 2000).
c-Myc plays a key role in the cell cycle dependent control
of the PDGF ß-receptor mRNA. The mouse platelet-derived
growth factor (PDGF) ß-receptor promoter
contains a CCAAT motif, and NF-Y plays an essential role
in its transcription. NF-Y is a trimeric CCAAT-binding factor with histone fold subunits (NF-YB/NF-YC) and bipartite activation domains located on
NF-YA and NF-YC. Coexpression of c-Myc represses PDGF
ß-receptor luciferase reporter activity, and the CCAAT
motif in the promoter is indispensable for this repression.
c-Myc binds NF-Y subunits, YB and
YC. The in vitro-translated c-Myc also binds the
glutathione S-transferase (GST)-NF-YB fusion protein and
GST-NF-YC, but not GST-NF-YA. The most C-terminal
region of HAP domains of NF-YB and NF-YC, and the Myc
homology boxes, but not the C-terminal bHLHZip domain,
are indispensable for the coimmunoprecipitation, and also
for the repression of PDGF ß-receptor. c-Myc binds NF-Y
complex without affecting the efficiency of NF-Y binding
to DNA. However, the expression of Myc represses the
transcriptional activation of NF-YC when fused to the
GAL4 DNA binding domain. Furthermore, this repression
is seen only when Myc homology boxes are present, and
NF-YC contains the c-Myc binding region (Izumi, 2001).
Myc transcription factor induces transcription of the E2F1, E2F2, and E2F3 genes. Using primary mouse embryo fibroblasts deleted for individual E2F genes, it has now been shown that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase is impaired in the absence of either E2F2 or E2F3 but not E2F1 or E2F4. In contrast, the ability of Myc to induce apoptosis is markedly reduced in cells deleted for E2F1 but not E2F2 or E2F3. From this data, it is proposed that the induction of specific E2F activities is an essential component in the Myc pathways that control cell proliferation and cell fate decisions (Leone, 2001).
Deregulated Myc expression results in both the induction of S phase and the induction of apoptosis if survival factors are limiting. How these processes are linked is not well understood. A variety of possible Myc target
genes have been identified, including the recent description of Id2. Like E2F2 and E2F3, Id2 appears to be essential for Myc-induced cell proliferation but not for Myc-induced apoptosis. In addition, these studies have also identified a role for Id2 in the control of Rb function, since the loss of Id2
function can partially suppress the phenotype resulting from loss of Rb. As such, it has been proposed that one role for Myc in the stimulation of cell growth is the induction of Id2, leading to inactivation of Rb. Nevertheless,
since the loss of Id2 does not fully suppress an Rb null phenotype (mice die at birth), and previous work has shown a suppression of Rb phenotype as a result of loss of either E2F1, E2F2, or E2F3 function, it seems likely that the control of E2F proteins and interactions with Id2 are both important for Rb function. Moreover, given the ability of Myc to induce both Id2 and E2Fs, it is concluded that the induction of both groups of activities is likely to be an important function of Myc (Leone, 2001).
In addition to results showing the requirement for distinct E2Fs to mediate Myc-induced S phase versus apoptosis, other work has also suggested that distinct downstream events mediate these two functions of Myc. In particular, cdk activation has been shown to be necessary for the Myc-mediated induction of proliferation but not apoptosis. Although the lack of a cdk requirement for Myc-induced apoptosis might suggest an E2F-independent event, since E2F accumulation is normally regulated by Rb through cdk-mediated phosphorylation of Rb, previous work has demonstrated an ability of Myc to induce E2F1 accumulation in the absence of cdk activity, presumably by bypassing the normal Rb control. Thus, Myc-induced apoptosis could bypass the need for the cell cycle machinery by directly activating E2F1 (Leone, 2001).
Myc oncoproteins promote cell cycle progression in part through the transcriptional up-regulation of the cyclin D2 gene. Myc is bound to the cyclin D2 promoter in vivo. Binding of Myc induces cyclin D2 expression and histone acetylation at a single nucleosome in a MycBoxII/TRRAP-dependent manner.
TRRAP is a component of TIP60 and PCAF/GCN5 histone acetyl transferase (HAT) complexes (see Drosophila Pcaf). Down-regulation of cyclin D2 mRNA expression in differentiating HL60 cells is preceded by a switch of promoter occupancy from Myc/Max to Mad/Max complexes, loss of TRRAP binding, increased HDAC1 binding, and histone deacetylation. Thus, recruitment of TRRAP and regulation of histone acetylation are critical for transcriptional activation by Myc (Bouchard, 2001).
The aim of this study was to resolve the role of MBII (an effector domain of Myc that binds TRRAP) and TRRAP in gene activation by Myc, using an endogenous target gene of Myc, cyclin D2, as model. Upon binding to the cyclin D2 promoter, Myc recruits TRRAP and induces the preferential
acetylation of histone H4 at a single nucleosome. Conversely, loss of endogenous Myc binding correlates with histone deacetylation and
loss of TRRAP binding during the TPA-induced differentiation of a human promyelocytic cell line, HL60. The integrity of MBII is
required for TRRAP recruitment, histone acetylation, and transcriptional activation at the cyclin D2 locus. Therefore, previous
suggestions that MBII has no role in transcriptional activation based on transient reporter assays need to be reevaluated. Deletion of the entire N terminus of Myc up to MBII (s-Myc) renders Myc unable to induce cell cycle progression and expression of either cyclin A or cyclin D2 in 3T3 fibroblasts, consistent with recent results that the N terminus of Myc is required for regulation of proliferation and induction of gene expression in a cell-type-dependent manner. Most likely, this is because stable association with TRRAP requires sequences in the N terminus of Myc in addition to MBII (Bouchard, 2001 and references therein).
Mad proteins are thought to antagonize the function of Myc by recruiting a repressor complex that contains histone deacetylase activity. Observations suggest that this model applies to the cyclin D2 promoter: (1) repression of the cyclin D2 promoter by Mad1 requires the integrity of an N-terminal domain, which mediates recruitment of histone deacetylase complexes
through interaction with Sin3A; (2) during HL60 differentiation, Mad1 and HDAC1 are corecruited to the cyclin D2 promoter, correlating with histone deacetylation of both histones H3 and H4 at the cyclin D2 promoter. Taken together, these data strongly support a model in which endogenous Myc/Max and Mad/Max complexes contribute to the regulation of transcription of the cyclin D2 gene through their antagonistic effects on histone acetylation. In addition, these findings show the functional relevance of the switch between Myc/Max and Mad/Max complexes during differentiation of hematopoietic cells. Recent work on the gene encoding the catalytic subunit of telomerase, htert, suggests that this model also may apply to this promoter (Bouchard, 2001 and references therein).
Up-regulation of the CAD (carbamoyl phosphate synthase, aspartate transcarbamylase, dihydroorotase) gene by Myc does not involve changes in histone acetylation. Instead, high levels of histone acetylation at the promoter were found in both quiescent and proliferating cells, showing that Myc can control at least one step in addition to histone acetylation to promote active
transcription. Additional proteins have been identified that bind to different domains of Myc and that are candidates for such an activity: for example, the C terminus of Myc binds to Ini1, a component of the Swi/Snf family of chromatin-remodeling complexes. Clearly, a detailed analysis of the role of Myc in
activation of individual promoters will be required before the role of each interaction in Myc biology can be resolved fully (Bouchard, 2001 and references therein).
The Period2 gene plays a key role in controlling circadian rhythm in mice. Mice deficient in the mPer2 gene are cancer prone. After gamma radiation, these mice show a marked increase in tumor development and reduced apoptosis in thymocytes. The core circadian genes are induced by gamma radiation in wild-type mice but not in mPer2 mutant mice. Temporal expression of genes involved in cell cycle regulation and tumor suppression, such as Cyclin D1, Cyclin A, Mdm-2, and Gadd45alpha, is deregulated in mPer2 mutant mice. In particular, the transcription of c-myc is controlled directly by circadian regulators and is deregulated in the mPer2 mutant. BMAL1/NPAS2 or BMAL1/CLOCK heterodimers likely repress transcription of c-myc through E box-mediated reactions in the P1 promoter, and mPer2 can suppress c-myc expression indirectly through stimulating Bmal1 transcription. Deregulation of Bmal1 in mPer2m/m cells, therefore, results in c-myc overexpression.
These studies suggest that the mPer2 gene functions in tumor suppression by regulating DNA damage-responsive pathways (Fu, 2002).
Based on recent discoveries from c-myc studies and the results of this study, a model is proposed for the role of mPer2 in tumor suppression. In this model, the loss of mPer2 function results in dampened Bmal1 expression and decreased intracellular levels of BMAL1/NPAS2 or BMAL1/CLOCK heterodimers, leading to the derepression of c-myc throughout 24 hr L/D cycles. Overexpression of c-myc causes genomic DNA damage and eventually leads to hyperplasia and tumor development. Following gamma radiation, the loss of mPer2 function partially impairs p53-mediated apoptosis, leading to accumulation of damaged cells. However, the mPer2m/m cells, expressing c-myc at elevated levels, can still progress through cell cycle in the presence of genomic DNA damage, resulting in the high incidence of tumor development after gamma radiation (Fu, 2002).
Activation of the tumor suppressor p53 by DNA damage induces either cell cycle arrest or apoptotic cell death. The cytostatic effect of p53 is mediated by transcriptional activation of the cyclin-dependent kinase (CDK) inhibitor p21Cip1, whereas the apoptotic effect is mediated by transcriptional activation of mediators including PUMA and PIG3. What determines the choice between cytostasis and apoptosis is not clear. The transcription factor Myc is shown to be a principal determinant of this choice. Myc is directly recruited to the p21Cip1 promoter by the DNA-binding protein Miz-1. This interaction blocks p21Cip1 induction by p53 and other activators. As a result Myc switches, from cytostatic to apoptotic, the p53-dependent response of colon cancer cells to DNA damage. Myc does not modify the ability of p53 to bind to the p21Cip1 or PUMA promoters, but selectively inhibits bound p53 from activating p21Cip1 transcription. By inhibiting p21Cip1 expression Myc favors the initiation of apoptosis, thereby influencing the outcome of a p53 response in favor of cell death (Seoane, 2002).
Several conclusions can be drawn from these results. Myc selectively targets p21Cip1 in the p53 transcriptional program, sparing the ability of p53 to induce the expression of PUMA or PIG3. Myc does not alter the ability of p53 to bind to the p21Cip1 promoter but inhibits p21Cip1 transcriptional activation by promoter-bound p53. In the presence of p21, p53 can still bind to the PUMA promoter and induce the accumulation of its product, but apoptosis is not achieved. Thus, the p21-dependent block in apoptosis maps to a step downstream of the DNA damage-p53-PUMA pathway. The mechanism for this provocative observation is not obvious. These results suggest a model in which Myc selectively prevents p53-dependent transcriptional activation of p21Cip1, enabling pro-apoptotic factors such as PUMA to execute a cell death program. Thus, these results define, in mechanistic terms, how one element of the cellular context, that is, the level of Myc activity, can determine the outcome of the p53 response. Although it remains to be seen whether repression of p21Cip1 would be beneficial in cancer treatment, the mechanism proposed here suggests ways to influence the cell's response to stresses that result in activation of p53 (Seoane, 2002).
The c-myc proto-oncogene encodes a ubiquitous transcription factor involved in the control of cell growth and implicated in inducing tumorigenesis. Understanding the function of c-Myc and its role in cancer depends upon the identification of c-Myc target genes. Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, and chromosomal instability. The NBS gene product, NBS1 (p95 or nibrin: Drosophila homolog Nbs), is a part of the hMre11 complex, a central player associated with double-strand break (DSB) repair. NBS1 contains domains characteristic for proteins involved in DNA repair, recombination, and replication. This study shows that c-Myc directly activates NBS1. c-Myc-mediated induction of NBS1 gene transcription occurs in different tissues, is independent of cell proliferation, and is mediated by a c-Myc binding site in the intron 1 region of NBS1 gene. Overexpression of NBS1 in Rat1a cells increased cell proliferation. These results indicate that NBS1 is a direct transcriptional target of c-Myc and links the function of c-Myc to the regulation of DNA DSB repair pathway operating during DNA replication (Chiang, 2003).
In hypoxic cells, HIF-1alpha escapes from oxygen-dependent proteolysis and binds to the hypoxia-responsive element (HRE) for transcriptional activation of target genes involved in angiogenesis and glycolysis. The G1 checkpoint gene p21(cip1)is activated by HIF-1alpha with a novel mechanism that involves the HIF-1alpha PAS domains to displace Myc binding from p21(cip1) promoter. This HIF-1alpha-Myc pathway may account for up- and down-regulation of other hypoxia-responsive genes that lack the HRE. Moreover, the role of HIF-1alpha in cell cycle control indicates a dual, yet seemingly conflicting, nature of HIF-1alpha: promoting cell growth and arrest in concomitance. It is speculated that a dynamic balance between the two processes is achieved by a 'stop-and-go' strategy to maintain cell growth and survival. Tumor cells may adopt such a scheme to evade the killing by chemotherapeutic agents (Koshiji, 2004).
The glutamate transporter gene, EAAT2/GLT-1, is induced by epidermal growth
factor (EGF) and downregulated by tumor necrosis factor alpha
(TNFalpha). While TNFalpha is
generally recognized as a positive regulator of NF-kappaB-dependent
gene expression, its ability to control transcriptional repression
is not well characterized. Additionally, the regulation of NF-kappaB by
EGF is poorly understood. Both TNFalpha-mediated repression and
EGF-mediated activation of EAAT2 expression require
NF-kappaB. EGF activates NF-kappaB independently of signaling to IkappaB. Furthermore, TNFalpha can abrogate IKKbeta- and
p65-mediated activation of EAAT2. These results suggest that NF-kappaB can
intrinsically activate EAAT2 and that TNFalpha
mediates repression through a distinct pathway also requiring NF-kappaB.
Consistently, it was found that N-myc is recruited to the EAAT2 promoter with TNFalpha
and that N-myc-binding sites are required for TNFalpha-mediated
repression. Moreover, N-myc overexpression inhibits both basal and
p65-induced activation of EAAT2. These data highlight the remarkable specificity
of NF-kappaB activity to regulate gene expression in response to diverse
cellular signals and have implications for glutamate homeostasis and
neurodegenerative disease (Sitcheran, 2005).
The ability of NF-kappaB to regulate EAAT2 expression has important
implications for the regulation of glutamate homeostasis in the CNS. To prevent
the overstimulation of neuronal glutamate receptors that can trigger excitotoxic
mechanisms and cell death, extracellular concentrations of excitatory amino
acids are tightly controlled by transport systems on both neurons and glial
cells. EAAT2 is critical for rapid clearance of synaptically
released glutamate for proper neurotransmission.
Accumulation of excessive glutamate levels in neuronal synapses can lead to
excitotoxic neuronal death, which has been implicated in the pathogenesis of
numerous neurodegenerative diseases, as well as CNS injury resulting from stroke
and ischemia. Notably, these conditions have been associated with
increased NF-kappaB activity, and reduced EAAT2
expression is observed after brain injury and in patients with Alzheimer's
disease, Huntington's disease, amyotrophic lateral sclerosis and multiple
sclerosis. Interestingly, EAAT2 may also
have a role in development, as work in Drosophila demonstrates that this
transporter is involved in terminal glial cell differentiation.
Moreover, reduced glial cell populations are observed in mice lacking the
EGF receptor suggesting that EGF signaling is also
important for glial cell differentiation. Based on the work in this study, it is
proposed that positive regulation of EAAT2 by NF-kappaB in response to EGF may
promote glial cell differentiation and uptake of synaptic glutamate by glial
cells, whereas TNF-mediated inhibition of EAAT2 by NF-kappaB may contribute to
glutamate toxicity and cell death in neuroinflammation and disease (Sitcheran, 2005).
The protein encoded by the c-MYC proto-oncogene is a transcription factor that can both activate and
repress the expression of target genes, but few of its transcriptional targets have been identified.
c-MYC is shown to repress the expression of the heavy subunit of the protein ferritin (H-ferritin),
which sequesters intracellular iron, and to stimulate the expression of the iron regulatory protein-2
(IRP2), which increases the intracellular iron pool. Down-regulation of the expression of the H-ferritin
gene is required for cell transformation by c-MYC. These results indicate that c-MYC coordinately
regulates genes controlling intracellular iron concentrations and that this function is essential for the
control of cell proliferation and transformation by c-MYC. The finding that c-MYC controls iron metabolism is consistent with observations that iron chelation leads to growth arrest and decreased synthesis of the cell cycle regulators cdc2 and cyclin A, whereas increased iron availability up-regulates the activity of ribonucleotide reductase (Wu, 1999a).
The MYC proto-oncogene encodes a ubiquitous transcription factor (c-MYC) involved in the control of
cell proliferation and differentiation. Deregulated expression of c-MYC caused by gene amplification,
retroviral insertion, or chromosomal translocation is associated with tumorigenesis. The function of
c-MYC and its role in tumorigenesis are poorly understood because few c-MYC targets have been
identified. c-MYC has a direct role in induction of the activity of telomerase, the
ribonucleoprotein complex expressed in proliferating and transformed cells, in which it preserves
chromosome integrity by maintaining telomere length. c-MYC activates telomerase by inducing
expression of its catalytic subunit, telomerase reverse transcriptase (TERT). Telomerase complex
activity is dependent on TERT, a specialized type of reverse transcriptase. TERT and c-MYC are
expressed in normal and transformed proliferating cells, downregulated in quiescent and terminally
differentiated cells, and both can induce immortalization when constitutively expressed in transfected
cells. Consistent with the recently reported association between MYC overexpression and induction of
telomerase activity, the TERT promoter contains numerous c-MYC-binding sites that
mediate TERT transcriptional activation. c-MYC-induced TERT expression is rapid and independent
of cell proliferation and additional protein synthesis, consistent with direct transcriptional activation of
TERT. These results indicate that TERT is a target of c-MYC activity and identify a pathway linking cell
proliferation and chromosome integrity in normal and neoplastic cells (Wu, 1999b).
Transcription repression by the basic region-helix-loop-helix-zipper (bHLHZip) protein Mad1 requires DNA binding as a ternary (three protein) complex with Max and mSin3A or mSin3B, the mammalian orthologs of the S. cerevisiae transcriptional corepressor SIN3. The interaction between Mad1 and mSin3 is mediated by three potential amphipathic alpha-helices: one in the N terminus of Mad (mSin interaction domain, or SID) and two within the second paired amphipathic helix domain (PAH2) of mSin3A. Mutations that alter the structure of the SID inhibit in vitro interaction between Mad and mSin3 and inactivate Mad's transcriptional repression activity. A 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region. A fusion protein comprising the Mad1 SID linked to a Ga14 DNA binding domain mediates repression of minimal as well as complex promoters dependent on Ga14 DNA binding sites. In addition, the SID represses the transcriptional activity of linked VP16 and c-Myc transactivation domains. When fused to a full-length c-Myc protein, the Mad1 SID specifically represses both c-Myc's transcriptional and transforming activities. Fusions between the GAL DNA binding domain and full-length mSin3 are also capable of repression. The association between Mad1 and mSin3 is not only dependent on the helical SID but is also dependent on both putative helices of the mSin3 PAH2 region, suggesting that stable interaction requires all three helices. These data indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad protein family and that SID repression is dominant over several distinct transcriptional activators (Ayer, 1996).
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 (Schreiber-Agus, 1995).
The bHLH-ZIP protein Mad heterodimerizes with Max as a sequence-specific transcriptional repressor. Mad is rapidly induced upon differentiation, and the associated switch from Myc-Max to Mad-Max heterocomplexes seem to repress genes normally activated by Myc-Max. Two related mammalian cDNAs have been identified that encode Mad-binding proteins. Both possess sequence homology with the yeast transcription repressor Sin3, including four conserved paired amphipathic helix (PAH) domains. mSin3A and mSin3B bind specifically to Mad and the related protein Mxi1. Mad-Max and mSin3 form ternary complexes in solution that specifically recognize the Mad-Max E box-binding site. Mad-mSin3 association requires PAH2 of mSin3A/mSin3B and the first 25 residues of Mad, which contains a putative amphipathic alpha-helical region. Point mutations in this region eliminate interaction with mSin3 proteins and block Mad transcriptional repression. It is suggested that Mad-Max represses transcription by tethering mSin3 to DNA as corepressors and that a transcriptional repression mechanism is conserved from yeast to mammals (Ayer, 1995).
mSin3A, a corepressor that binds to the transcription factor Mad and appears to tether MAD to histone deacetylase, in contrast to other repressors, is not detected in the complex between Retinoblastoma protein and histone deacetylase. Interaction between domain A and B in the Rb pocket forms a site for association with histone deacetylase. Recruitment of histone deacetylase by either Rb or Mad results in a decrease in acetylated histone H3 associated with the promoter in vivo, consistent with the idea that this recruitment indeed results in deacetylation of histones bound to the promoter. This Rb-mediated recruitment of histone deacetylase can only repress a subset of promoters and transcription factors. Repression of the adenovirus major late promoter by Rb and Mad is dependent on histone deacetylase activity, while repression of the tyrosine kinase promoter and the SV40 enhancer by Rb is independent of histone deacetylase activity (Luo, 1998).
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 activity requires interaction with mammalian Sin3A or Sin3B proteins, which are 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 involvement of histone deacetylase suggests that the silencing function of Mxi1 involves a modification of chromatin involving deacetylation, converting chromatin into a form that impedes the interaction of the transcriptional apparatus with promoter regions (Alland, 1997).
Members of the Mad family of bHLH-Zip proteins heterodimerize with Max to repress transcription in a sequence-specific manner. Transcriptional repression by Mad:Max heterodimers is mediated by ternary complex formation with either of the corepressors mSin3A or mSin3B. mSin3A is an in vivo component of large, heterogeneous multiprotein complexes and is tightly and specifically associated with at least seven polypeptides. Two of the mSin3A-associated proteins, p50 and p55, are highly related to the histone deacetylase HDAC1. The mSin3A immunocomplexes possess histone deacetylase activity that is sensitive to the specific deacetylase inhibitor trapoxin. mSin3A-targeted repression is reduced by trapoxin treatment, suggesting that histone deacetylation mediates transcriptional repression through Mad-Max-mSin3A multimeric complexes (Hassig, 1997).
Transcriptional repression by mammalian nuclear receptors has been correlated with the binding of the putative co-repressor, N-CoR to nuclear receptors. A complex has been identified that contains N-CoR, the Mad presumptive co-repressor mSin3, and the histone deacetylase mRPD3, and which is required for both nuclear receptor- and Mad-dependent repression, but not for repression by transcription factors of the ets-domain family. mSin3 and mRPD3 are required to mediate thyroid-hormone receptor mediated repression. These data predict that the ligand-induced switch of heterodimeric nuclear receptors from repressor to activator functions involves the exchange of complexes containing histone deacetylases with those that have histone acetylase activity. This work provides a molecular mechanism for integrating chromatin remodelling and interactions with the core transcriptional machinery. A model is presented for the reversal of repression. Upon binding of activating ligands, the co-repressor complex dissociates from nuclear receptors and is replaced by a co-activator complex containing Creb binding protein (CBP, a factor with intrinsic histone acetylation activity), the histone chaperone P/CAF, NCoA-1/SRC-1 (a steroid receptor coactivator), and a factor termed p/CIP (Heinzel, 1997).
Transcriptional repression by Mad-Max heterodimers requires interaction of Mad with the corepressors mSin3A/B. Sin3p, the S. cerevisiae homolog of mSin3, functions in the same pathway as Rpd3p, a protein related to two recently identified mammalian histone deacetylases, HDAC1 and HDAC2. mSin3A and HDAC1/2 are associated in vivo. HDAC2 binding requires a conserved region of mSin3A capable of mediating transcriptional repression. Mad1 forms a complex with mSin3 and HDAC2 that contains histone deacetylase activity. Trichostatin A, an inhibitor of histone deacetylases, abolishes Mad repression. It is proposed that Mad-Max functions by recruiting the mSin3-HDAC corepressor complex that deacetylates nucleosomal histones, producing alterations in chromatin structure that block transcription (Laherty, 1997). The protooncogene MYC plays an important role in the regulation of cellular proliferation, differentiation, and apoptosis and has been implicated in a variety of human tumors. MYC and the closely related MYCN encode highly conserved nuclear phosphoproteins (Myc and NMyc) that apparently function as transcription factors in the cell. A large and highly conserved nuclear protein has been identified that interacts directly with the transcriptional activating domain of Myc (designated "protein associated with Myc" or Pam). Pam contains an extended amino acid sequence with similarities to a protein known as regulator of chromosome condensation (RCC1), which may play a role in the function of chromatin. RCC1 contains a motif of 50-60 aa that is repeated seven times in tandem. These repeats form a seven-bladed propeller structure as determined recently by X-ray crystallography. A similar 7-fold repeat is present in Pam, but is divided into two elements (RHD-1 and RHD-2) by an insertion of 134 aa after the fourth repeat. The insertion between RHD-1 and RHD-2 contains a C-terminal region of 55 aa that is rich in basic amino acids. Such a short basic region (40-50 aa) is also present in RCC1 proteins at their N termini. These N termini are important for chromatin binding. Although most RCC1 proteins end with a repeated element, the Drosophila RCC1 protein BJ1 has a substantial C-terminal extension, which has limited homology to chromatin proteins such as Xenopus histone-binding protein N1/N2. Pam contains a similar region, situated in the midst of a serine-rich domain and in the vicinity of the Myc-binding domain, but relatively distant from RHD-1/2. The gene encoding Pam (PAM) is expressed in all of the human tissue examined, but expression is exceptionally abundant in brain and thymus. Pam binds specifically to Myc, but not NMyc. The region in Myc required for binding to Pam includes a domain that is essential for the function of Myc and that is frequently mutated in Burkitt's lymphomas. PAM is located within a 300-kb region on chromosome 13q22 (Guo, 1998).
The Myc protein binds DNA and activates transcription by mechanisms that are still unclear. Chromatin immunoprecipitation
(ChIP) was used to evaluate Myc-dependent changes in histone acetylation at seven target loci. Upon serum stimulation of Rat1 fibroblasts, Myc
associates with chromatin, histone H4 becomes locally hyperacetylated, and gene expression is induced. These responses are lost or severely impaired in Myc-deficient cells, but are restored by adenoviral delivery of Myc simultaneous with mitogenic stimulation. When targeted to chromatin in the absence of mitogens, Myc directly induces H4 acetylation. In addition, Myc recruits TRRAP to chromatin,
consistent with a role for this cofactor in histone acetylation. Finally, unlike serum, Myc alone is very inefficient in inducing expression of most target genes. Myc therefore governs a step, most likely H4 acetylation, that is required but not sufficient for transcriptional activation. It is proposed that Myc acts as a permissive factor, allowing additional signals to activate target promoters (Frank, 2001).
A new quantitative proteomics technology has been applied to the analysis of the function of the Myc oncoprotein in mammalian cells. Employing isotope-coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry, the global pattern of protein expression in rat myc-null cells has been compared with that of myc-plus cells (myc-null cells in which myc has been introduced) to generate a differential protein expression catalog. Expression differences among many functionally related proteins were identified, including reduction of proteases, induction of protein synthesis pathways and upregulation of anabolic enzymes in myc-plus cells, which are predicted to lead to increased cell mass (cell growth). In addition, reduction in the levels of adhesion molecules, actin network proteins and Rho pathway proteins were observed in myc-plus cells, leading to reduced focal adhesions and actin stress fibers as well as altered morphology. These effects are dependent on the highly conserved Myc Box II region. These results reveal a novel cytoskeletal function for Myc and indicate the feasibility of quantitative whole-proteome analysis in mammalian cells (Shiio, 2002).
Accumulating evidence suggests that Myc influences cell growth (defined as an increase in cell mass). Myc overexpression was shown to increase cell size both in Drosophila and mammalian cells. Using global protein expression analysis, a more comprehensive view of the mode of action of Myc on cell growth has been obtained. In Myc(+) cells there is an increase in many proteins implicated in protein biosynthesis, a decrease in different proteases and an increase in several anabolic enzymes. Consistent with the observed augmentation in the levels of many ribosomal protein subunits, proteins implicated in rRNA processing and assembly (fibrillarin, Nop56, Nop58, Bop1, DDX5, DDX17 and DDX21), and a translation initiation factor (eIF2B), the rate of protein synthesis was increased by nearly 3-fold in Myc(+) cells when compared with Myc(-) cells. The increased levels of anabolic enzymes (such as fatty acid synthase, adenylate kinase and cad) should result in increased synthesis of fatty acids, nucleotides, amino acids and ATP. Collectively, the increase in these biomolecules may account for the growth stimulatory effects of Myc. The Rho pathway has also been shown to negatively regulate cell and organism size, raising the possibility that downregulation of the Rho pathway by Myc plays some role in cell growth in addition to the well established role in cytoskeletal organization (Shiio, 2002).
The Myc transcription factor is an essential mediator of cell growth and
proliferation through its ability to both positively and negatively regulate
transcription. The mechanisms by which Myc silences gene expression are not well
understood. The current model is that Myc represses transcription through
functional interference with transcriptional activators. Myc is shown to
bind the corepressor DNA CpG methyltransferase Dnmt3a and associate with DNA methyltransferase activity
in vivo. In cells with reduced Dnmt3a levels, specific
reactivation of the Myc-repressed p21Cip1 gene is seen, whereas the expression of
Myc-activated E-boxes genes is unchanged. In addition, it was found that Myc can
target Dnmt3a selectively to the promoter of p21Cip1. Myc is known to be
recruited to the p21Cip1 promoter by the DNA-binding factor Miz-1.
Consistent with this, Myc and Dnmt3a form a ternary complex with
Miz-1 and this complex can corepress the p21Cip1 promoter. Finally,
it is shown that DNA methylation is required for Myc-mediated repression of
p21Cip1. These data identify a new mechanism by which Myc can silence gene
expression not only by passive functional interference but also by active
recruitment of corepressor proteins. Furthermore, these findings suggest that
targeting of DNA methyltransferases by transcription factors is a wide and
general mechanism for the generation of specific DNA methylation patterns within
a cell (Brenner, 2005).
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