diminutive

EVOLUTIONARY HOMOLOGS

Table of contents

Myc interaction with Miz-1 and Myc destruction

The human proto-oncogene c-myc encodes a highly unstable transcription factor that promotes cell proliferation. Although the extreme instability of Myc plays an important role in preventing its accumulation in normal cells, little is known about how Myc is targeted for rapid destruction. An investigation has been carried out of mechanisms regulating the stability of Myc. Myc is destroyed by ubiquitin-mediated proteolysis; two elements are defined in Myc that oppositely regulate its stability: a transcriptional activation domain that promotes Myc destruction, and a region required for association with the POZ domain protein Miz-1, that stabilizes Myc. Myc is stabilized by cancer-associated and transforming mutations within its transcriptional activation domain. What is the significance of Myc stabilization by Miz-1? Miz-1 is a zinc finger/POZ domain protein that, alone, has been shown to bind to and transactivate the cyclin D1 promoter. When Miz-1 is complexed with Myc, however, a latent Miz-1 repressor function is revealed, and cyclin D1 promoter activity is attenuated. Because of this behavior, Miz-1 has been proposed as the partner protein through which Myc represses transcription of cyclin D1 and other genes in vivo. It is hypothesized that stabilization of Myc by Miz-1 relates to the function of Miz-1-Myc complexes as transcriptional repressors. Perhaps, therefore, enhanced protein stability is a common feature of transcriptional repressors, permitting the formation of recalcitrant complexes that stably attenuate gene transcription. In conclusion data reveals a complex network of interactions regulating Myc destruction, and imply that enhanced protein stability contributes to oncogenic transformation by mutant Myc proteins (Salghetti, 1999).

Deregulated expression of c-myc can induce cell proliferation in established cell lines and in primary mouse embryonic fibroblasts (MEFs), through a combination of both transcriptional activation and repression by Myc. Miz-1, a Myc-associated zinc finger transcription factor, arrests cells in G1 phase and inhibits cyclin D-associated kinase activity. Miz-1 upregulates expression of the cyclin-dependent kinases (CDK) inhibitor p15INK4b by binding to the initiator element of the p15INK4b promoter. Myc and Max form a complex with Miz-1 at the p15 initiator and inhibit transcriptional activation by Miz-1. Expression of Myc in primary cells inhibits the accumulation of p15INK4b that is associated with cellular senescence; conversely, deletion of c-myc in an established cell line activates p15INK4b expression. Alleles of c-myc that are unable to bind to Miz-1 fail to inhibit accumulation of p15INK4b messenger RNA in primary cells and are, as a consequence, deficient in immortalization (Staller, 2001).

The Myc oncoprotein represses initiator-dependent transcription through the POZ domain transcription factor Miz-1. Transactivation by Miz-1 is negatively regulated by association with topoisomerase II binding protein (TopBP1); UV irradiation downregulates expression of TopBP1 and releases Miz-1. Miz-1 binds to the p21Cip1 core promoter in vivo and is required for upregulation of p21Cip1 upon UV irradiation. Using both c-myc-/- cells and a point mutant of Myc that is deficient in Miz-1 dependent repression, it has been shown that Myc negatively regulates transcription of p21Cip1 upon UV irradiation and facilitates recovery from UV-induced cell cycle arrest through binding to Miz-1. These data implicate Miz-1 in a pathway that regulates cell proliferation in response to UV irradiation (Herold, 2002).

One conserved biological function of Myc proteins that is critical for their tumorigenic effects is their ability to upregulate transcription of E box-dependent target genes. Myc uses a conserved surface of the helix-loop-helix/leucine zipper domain for interaction with Max. A second evolutionary conserved surface on the helix-loop-helix domain of Myc proteins has now been identified that binds to Miz-1 but not to Max. Extrapolating from the crystal structure of the Max homodimer, amino acids of Myc involved in binding to Miz-1 point away from the interface with Max, consistent with data that demonstrate the presence of a trimeric Max/Myc/Miz-1 complex at the p15Ink4b initiator. It is suggested, therefore, that Myc proteins have integrated and conserved at least two distinct biochemical properties: the ability to activate through Max and the ability to repress through Miz-1. E box-dependent transactivation by Myc proteins is antagonized by Mad proteins, which also complex with Max. The residues of Myc that bind to Miz-1 are not present in Mad proteins, and Mad proteins do not signal through Miz-1. These data therefore also suggest that loss of Mad proteins is not equivalent to deregulation of Myc during tumorigenesis. The precise importance of the complex formation between Myc and Miz-1 in tumorigenesis can now be addressed with appropriate transgenic and knockin models (Herold, 2002).

In response to UV irradiation, Myc inhibits UV-induced cell cycle arrest by p53 through Miz-1. In this regard, the function of c-myc is very similar to c-jun, a well-characterized negative regulator of the mammalian UV response. Like c-jun, expression of c-myc itself can be induced by UV irradiation in fibroblasts. Furthermore, c-jun is required for the recovery from UV-induced cell cycle arrest, and c-myc-/- cells also recover very poorly from UV irradiation. Both c-jun and c-myc enhance p53-dependent apoptosis, and thus the balance between cell cycle arrest and apoptosis in response to UV irradiation is affected by the status of either gene. It is suggested therefore, that c-myc and c-jun regulate cell fate in response to UV irradiation and switch the response from cell cycle arrest to apoptosis. Recent work shows that Myc overrides G1 arrest not only in response to UV irradiation but also in response to gamma-irradiation and to DNA damage inflicted by reactive oxygen species. In both cases, the DNA damage causes a p53-dependent G1 arrest, and the damage response is overridden by Myc. It is therefore possible that inhibition of Miz-1-dependent transactivation is a general mechanism through which Myc overrides p53-dependent checkpoint functions (Herold, 2002).

Myc interaction with INI1/hSNF5

Chromatin organization plays a key role in the regulation of gene expression. The evolutionarily conserved SWI/SNF complex is one of several multiprotein complexes that activate transcription by remodeling chromatin in an ATP-dependent manner. SWI2/SNF2 is an ATPase whose homologs, BRG1 and hBRM, mediate cell-cycle arrest; the SNF5 homolog, INI1/hSNF5, appears to be a tumor suppressor. A search for INI1-interacting proteins using the two-hybrid system led to the isolation of c-MYC, a transactivator. The c-MYC-INI1 interaction has been observed both in vitro and in vivo. The c-MYC basic helix-loop-helix (bHLH) and leucine zipper (Zip) domains and the INI1 repeat 1 (Rpt1) region are required for this interaction. c-MYC-mediated transactivation is inhibited by a deletion fragment of INI1 and the ATPase mutant of BRG1/hSNF2 in a dominant-negative manner contingent upon the presence of the c-MYC bHLH-Zip domain. These results suggest that the SWI/SNF complex is necessary for c-MYC-mediated transactivation and that the c-MYC-INI1 interaction helps recruit the complex. Recruitment of the SWI/SNF complex, mediated by the interaction of INI1 with c-MYC, may facilitate the transcription of a discrete subset of c-MYC target genes, especially those involved in apoptosis, which might explain the tumor-suppressor activity of INI1 (Cheng, 1999).

The c-myc oncogene product (c-Myc) is a transcription factor that forms a complex with Max and recognizes the E-box sequence. c-Myc plays key functions in cell proliferation, differentiation and apoptosis. As for its activity towards cell proliferation, it is generally thought that c-Myc transactivates the E-box-containing genes that encode proteins essential to cell-cycle progression. Despite the characterization of candidate genes regulated by c-Myc in culture cells, these have still not been firmly recognized as real target genes for c-Myc. c-Myc has been found to directly bind to the N-terminal region of origin recognition complex-1 (ORC1), a region that is responsible for gene silencing, in a state of complex containing other ORC subunits and Max in vivo and in vitro. Furthermore, ORC1 inhibits E-box-dependent transcription activity of c-Myc by competitive binding to the C-terminal region of c-Myc with SNF5, a component of chromatin remodelling complex SNF/Swi1. These results suggest that ORC1 suppresses the transcription activity of c-Myc by its recruitment into an inactive form of chromatin during some stage of the cell cycle (Takayama, 2000).

Phosphorylation of Myc

Myc family transcription factors are destabilized by phosphorylation of a conserved amino-terminal GSK-3ß motif. In proliferating cerebellar granule neuron precursors (CGNPs), Sonic hedgehog signaling induces N-myc expression, and N-myc protein is stabilized by insulin-like growth factor-mediated suppression of GSK-3ß. N-myc phosphorylation-mediated degradation is a prerequisite for CGNP growth arrest and differentiation. This study investigated whether N-myc phosphorylation and turnover are thus linked to cell cycle exit in primary mouse CGNP cultures and the developing cerebellum. Phosphorylation-induced turnover of endogenous N-myc protein in CGNPs increases during mitosis, due to increased priming phosphorylation of N-myc for GSK-3ß. The priming phosphorylation requires the Cdk1 complex, whose cyclin subunits are indirect Sonic hedgehog targets. These findings provide a mechanism for promoting growth arrest in the final cycle of neural precursor proliferation competency, or for resetting the cell cycle in the G1 phase, by destabilizing N-myc in mitosis (Sjostrom, 2005).

Increased S54 phosphorylation is strongly associated with N-myc destabilization. Further, the mitotic kinase Cdk1, in complex with cyclins A and B1, mediates N-myc S54 phosphorylation in primary CGNPs. N-myc is thus primed for GSK-3ß-mediated phosphorylation, which promotes degradation of c-myc and other cell cycle regulatory proteins. This model is consistent with permitting primary neural precursor exit from the cell cycle before G1 is reentered. This allows differentiation to begin, in accordance with intrinsic programs (Sjostrom, 2005).

Shh directly induces N-myc expression and indirectly affects N-myc posttranslational modification, mediated by its indirect targets, cyclins A and/or B. Mitotic degradation of N-myc permits neuronal precursor cell cycle exit in the absence of Shh signaling or in the case of an intrinsic program-directed shift toward differentiation (Sjostrom, 2005).

The data indicate an increase in phosphorylation-associated N-myc degradation during mitosis, which occurs over an interval of less than 1 hr. N-myc is a short-lived protein with a half-life of approximately 40 min, while the cell cycle in mouse CGNPs at PN4 lasts 16-18 hr. How is N-myc disposed of during interphase in proliferating CGNPs? There is growing evidence that myc protein stability regulation may also involve regions outside of the amino-terminal myc box 1 domain containing T50 and S54. Interactions between the myc box 2 domain and the F box protein Skp2 promote c-myc proteolysis during the G1-to-S phase transition, indicating that myc metabolism may be regulated in a cell cycle-dependent manner. With regard to N-myc, these mechanisms have yet to be verified in primary neural precursors (Sjostrom, 2005).

GSK-3ß-mediated phosphorylation targets c-myc for degradation through a mechanism involving the F box protein Fbw7 in a variety of cell lines. A similar mechanism for N-myc degradation was suggested. Whether Fbw7, or another F box protein with an appropriate spatiotemporal expression pattern, plays a role in N-myc proteolysis in primary cells and during cerebellar development in vivo remains to be determined. To act upon N-myc at T50, GSK-3ß must first be primed by phosphorylation at S54. Basal levels of N-myc S54 phosphorylation could be mediated by nonmitotic kinases, such as Erk, outside mitosis. N-myc was also identified as a substrate for the neural-specific kinase Cdk5. However, neither Erk nor Cdk5 activity was specific for S54. It was found that the Cdk1 complex contains a potent, specific N-myc S54 kinase (Sjostrom, 2005).

Cdk1 heterodimerizes with cyclin A and cyclin B1. Cell-free in vitro assays have shown that cyclin A in complex with Cdk1 and p107 can phosphorylate GST-c-myc fusion proteins. Both cyclin A and cyclin B1 immunoprecipitate specific activity toward N-myc, and both cyclins are expressed in Shh-treated CGNPs during mitosis. Cyclin A has also been found in interphase CGNPs, consistent with its participation in Cdk2 complexes during S phase. The lack of S54 phosphorylation in G1/S-arrested cells indicates that cyclin A:Cdk2 does not phosphorylate N-myc. Although cyclin A:Cdk1 complexes may have some substrates distinct from those of cyclin B:Cdk1 complexes, it has been shown that many Cdk substrates are indifferent as to the cyclin subunit of the cyclin:Cdk1 complex. These analyses suggest that, in primary CGNPs, N-myc S54 can be targeted by either cyclin A:Cdk1 or cyclin B1:Cdk1 (Sjostrom, 2005).

Early studies reported that c-myc protein synthesis and modification is not altered during mitosis. Later studies demonstrated mitosis-specific c-myc phosphorylation. These studies were carried out in cell lines with flexible cell cycle exit and reentry capacity. This work with N-myc has been conducted in primary neuronal precursors with a defined intrinsic program for irreversible cell cycle exit and subsequent differentiation. Many previous myc turnover studies have relied on overexpression, while this study focused on regulation of endogenous N-myc stability, in primary cultures and in vivo. The finding that N-myc is largely degraded at the conclusion of the primary neural precursor cell cycle provides insight as to how enhanced stability of N-myc protein can contribute to brain tumorigenesis, by enhancing CGNPs' capacity for ongoing division. Increased activity of IGF2, which is predicted to stabilize N-myc, is associated with increased proliferation in primary neural precursors and in mouse and human medulloblastomas. Thus, future analysis of how N-myc turnover is regulated during CGNP expansion in vivo will yield greater understanding of normal brain development and brain tumor biology (Sjostrom, 2005).

Other Myc interactions

The Myc protein binds to and transactivates the expression of genes via E-box elements containing a central CAC(G/A)TG sequence. The transcriptional activation function of Myc is required for its ability to induce cell cycle progression, cellular transformation and apoptosis. Transactivation by Myc is under negative control by the transcription factor AP-2 (see Drosophila AP-2). AP-2 inhibits transactivation by Myc via two distinct mechanisms. First, high affinity binding sites for AP-2 overlap Myc-response elements in two bona fide target genes of Myc, prothymosin-alpha and ornithine decarboxylase. On these sites, AP-2 competes for binding of either Myc/Max heterodimers or Max/Max homodimers. The second mechanism involves a specific interaction between C-terminal domains of AP-2 and the BR/HLH/LZ domain of Myc, but not Max or Mad. Binding of AP-2 to Myc does not preclude association of Myc with Max, but impairs DNA binding of the Myc/Max complex and inhibits transactivation by Myc even in the absence of an overlapping AP-2 binding site. Taken together, these data suggest that AP-2 acts as a negative regulator of transactivation by Myc (Gaubatz, 1995).

TIP49 was originally identified as a TBP interacting protein using TBP as an in vitro affinity matrix for rat liver nuclear extracts. However, there is no observable association between TIP49 and TBP in vivo. No TBP is detected in the affinity-purified proteins that bind to the Myc N terminus. TIP49 has also been cloned in a two-hybrid screen using the replication protein 3 as bait and termed RUVBL1, due to the limited homology with RuvB. However, as with TBP, no in vivo association of TIP49 with RPA3 has been demonstrated, and there are no data implicating TIP49 for a role in DNA replication. Of relevance to the present study is the observation that a portion of the cellular TIP49 could be isolated in chromatographic fractions containing RNA polymerase II. TIP49 associates with the nuclear matrix (Wood, 2000 and references therein).

The finding that TIP49 can also bind to beta-catenin and LEF-1/TCF supports a role for TIP49 as a cofactor that is likely to function with diverse transcription factors. Despite the identification of TIP49 through binding studies with different nuclear components, no functional role for TIP49 in these systems has previously been established. The observation that the TIP49D302N allele inhibits Myc oncogenic activity demonstrates that TIP49 is an essential nuclear cofactor in at least the Myc transcription factor pathway. Since the analogous mutation is nonviable in yeast and it targets the conserved Walker B motif, the data imply that Myc-mediated oncogenesis requires the ATPase activity of TIP49. Even though the predicted ATPase-deficient TIP49 inhibits oncogenic transformation, it is not overtly toxic since there is no inhibition of drug-resistant colony formation in several cell types. It is likely that ectopic expression of the TIP49D302N protein creates only a partial loss of function within a cell and that this partial loss of function is only rate limiting when high levels of Myc activity are demanded. The inviability of the yeast strain with the analogous TIP49D311N mutation argues that the exclusive expression of the TIP49D302N protein in mammalian cells would also be incompatible with cell growth. Nevertheless, a direct role for TIP49 in the transcriptional activation of any specific Myc target gene is only inferred at this stage, but not yet readily assayed. Coexpression of TIP49wt or TIP49D302N with Myc neither augments nor inhibits the activation or repression of several reporter constructs. A similar finding has been reported for TIP49 in the beta-catenin/LEF-1 system. The activity of TIP49 may only be apparent with currently uncharacterized promoters or with chromosomal target sites that are not adequately recapitulated by DNA transfection (Wood, 2000).

The c-Myc and E2F transcription factors are among the most potent regulators of cell cycle progression in higher eukaryotes. This report describes the isolation of a novel, highly conserved 434 kDa protein, designated TRRAP, which interacts specifically with the c-Myc N terminus and has homology to the ATM/PI3-kinase family. TRRAP also interacts specifically with the E2F-1 transactivation domain. Expression of transdominant mutants of the TRRAP protein or antisense RNA blocks c-Myc- and E1A-mediated oncogenic transformation. These data suggest that TRRAP is an essential cofactor for both the c-Myc and E1A/E2F oncogenic transcription factor pathways (McMahon, 1998).

The c-Myc protein functions as a transcription factor to facilitate oncogenic transformation; however, the biochemical and genetic pathways leading to transformation remain undefined. The recently described c-Myc cofactor TRRAP recruits histone acetylase activity, which is catalyzed by the human GCN5 protein (see Drosophila Pcaf). Since c-Myc function is inhibited by recruitment of histone deacetylase activity through Mad family proteins, these opposing biochemical activities are likely to be responsible for the antagonistic biological effects of c-Myc and Mad on target genes and ultimately on cellular transformation (McMahon, 2000).

The c-Myc oncogene has been implicated in the genesis of diverse human tumors. Ectopic expression of the c-Myc gene in cultured epithelial cells causes resistance to the antiproliferative effects of TGF-ß. However, little is known about the precise mechanisms of c-Myc-mediated TGF-ß resistance. In this study, it has been revealed that c-Myc physically interacts with Smad2 and Smad3, two specific signal transducers involved in TGF-ß signaling. Through its direct interaction with Smads, c-Myc binds to the Sp1-Smad complex on the promoter of the p15Ink4B gene, thereby inhibiting the TGF-ß-induced transcriptional activity of Sp1 and Smad/Sp1-dependent transcription of the p15Ink4B gene. These results suggest that oncogenic c-Myc promotes cell growth and cancer development partly by inhibiting the growth inhibitory functions of Smads (Feng, 2002).

The cellular Bcr protein consists of an N-terminal serine/threonine kinase domain, a central guanine nucleotide exchange factor homology region and a C-terminal GTPase-activating protein domain. Bcr is a multifunctional protein that is the fusion partner for Abl (p210 Bcr-Abl) in Philadelphia chromosome positive leukemias. c-Myc has been identified as a binding partner for Bcr in both yeast and mammalian cells. Interactions between natively expressed c-Myc and Bcr have been observed in leukemic cell lines. The interaction between c-Myc and Bcr is detected in a fragment containing residues 871-910 that encompasses the small region between the PH domain and the C2 domain of Bcr. The smallest fragment of c-Myc that retains its ability to interact with the full-length Bcr clone consists of the carboxy-terminal B/HLH/Z domain. Although Bcr and Max have overlapping binding sites on c-Myc, Bcr cannot interact with Max, or with the c-Myc/Max heterodimer. Bcr expression blocks activation of c-Myc-responsive genes, as well as the transformed phenotype induced by coexpression of c-Myc and H-Ras, and this finding suggests that one function of Bcr is to limit the activity of c-Myc. However, Bcr does not block c-Myc function by preventing its nuclear localization. Interestingly, increased Bcr dosage in COS-7 and K-562 cells correlates with a reduction in c-Myc protein levels, suggesting that Bcr may in fact be limiting c-Myc activity by regulating its stability. These data indicate that Bcr is a novel regulator of c-Myc function whose disrupted expression may contribute to the high level of c-Myc protein that is observed in Bcr-Abl transformed cells (Mahon, 2003).

c-Myc promotes cellular proliferation, sensitizes cells to apoptosis and prevents differentiation. It binds cyclin T1 structurally and functionally from the positive transcription elongation factor b (P-TEFb). The cyclin-dependent kinase 9 (Cdk9) in P-TEFb then phosporylates the C-terminal domain of RNA polymerase II, which is required for the transition from initiation to elongation of eukaryotic transcription. Inhibiting P-TEFb blocks the transcription of its target genes as well as cellular proliferation and apoptosis induced by c-Myc (Kanazawa, 2003).

The c-Myc oncoprotein (Myc) controls cell fate by regulating gene transcription in association with a DNA-binding partner, Max. While Max lacks a transcription regulatory domain, the N terminus of Myc contains a transcription activation domain (TAD) that recruits cofactor complexes containing the histone acetyltransferases (HATs) GCN5 and Tip60. This study reports a novel functional interaction between Myc TAD and the p300 coactivator-acetyltransferase. p300 associates with Myc in mammalian cells and in vitro through direct interactions with Myc TAD residues 1 to 110 and acetylates Myc in a TAD-dependent manner in vivo at several lysine residues located between the TAD and DNA-binding domain. Moreover, the Myc:Max complex is differentially acetylated by p300 and GCN5 and is not acetylated by Tip60 in vitro, suggesting distinct functions for these acetyltransferases. Whereas p300 and CBP can stabilize Myc independent of acetylation, p300-mediated acetylation results in increased Myc turnover. In addition, p300 functions as a coactivator that is recruited by Myc to the promoter of the human telomerase reverse transcriptase gene; also, p300/CBP stimulates Myc TAD-dependent transcription in a HAT domain-dependent manner. These results suggest dual roles for p300/CBP in Myc regulation: as a Myc coactivator that stabilizes Myc and as an inducer of Myc instability via direct Myc acetylation (Faiola, 2005).

The c-MYC oncoprotein functions as a sequence-specific transcription factor. The ability of c-MYC to activate transcription relies in part on the recruitment of cofactor complexes containing the histone acetyltransferases mammalian GCN5 (mGCN5)/PCAF and TIP60. In addition to acetylating histones, these enzymes have been shown to acetylate other proteins involved in transcription, including sequence-specific transcription factors. This study was initiated in order to determine whether c-MYC is a direct substrate of mGCN5 and TIP60. mGCN5/PCAF and TIP60 are shown to acetylate c-MYC in vivo. By using nanoelectrospray tandem mass spectrometry to examine c-MYC purified from human cells, the major mGCN5-induced acetylation sites have been mapped. Acetylation of c-MYC by either mGCN5/PCAF or TIP60 results in a dramatic increase in protein stability. The data reported here suggest a conserved mechanism by which acetyltransferases regulate c-MYC function by altering its rate of degradation (Patel, 2005).

Mad-Sin3 complex and the modification of chromatin

Members of the Mad family of bHLHZip proteins heterodimerize with Max and function to repress the transcriptional and transforming activities of the Myc proto-oncogene. Mad:Max heterodimers repress transcription by recruiting a large multi-protein complex containing the histone deacetylases, HDAC1 and HDAC2, to DNA. The interaction between Mad proteins and HDAC1/2 is mediated by the corepressor mSin3A and requires sequences at the amino terminus of the Mad proteins, termed the SID, for Sin3 interaction domain, and the second of four paired amphipathic alpha-helices (PAH2) in mSin3A. To better understand the requirements for the interaction between the SID and PAH2, mutagenesis and structural studies on the SID have been performed. These studies show that amino acids 8-20 of Mad1 are sufficient for SID:PAH2 interaction. Further, this minimal 13-residue SID peptide forms an amphipathic alpha-helix in solution, and residues on the hydrophobic face of the SID helix are required for interaction with PAH2. Finally, the minimal SID can function as an autonomous and portable repression domain, demonstrating that it is sufficient to target a functional mSin3A/HDAC corepressor complex (Eilers, 1999).

Gene-specific targeting of the Sin3 corepressor complex by DNA-bound repressors is an important mechanism of gene silencing in eukaryotes. The Sin3 corepressor specifically associates with a diverse group of transcriptional repressors, including members of the Mad family, that play crucial roles in development. The NMR structure of the complex formed by the PAH2 domain of mammalian Sin3A with the transrepression domain (SID) of human bHLHZip protein Mad1 reveals that both domains undergo mutual folding transitions upon complex formation generating an unusual left-handed four-helix bundle structure and an amphipathic alpha helix, respectively. The SID helix is wedged within a deep hydrophobic pocket defined by two PAH2 helices. Structure-function analyses of the Mad-Sin3 complex provide a basis for understanding the underlying mechanism(s) that lead to gene silencing (Brubaker, 2000).

Sin3 appears to function as a large protein scaffold capable of multiple protein-protein interactions. While Sin3 interacts with class I histone deacetylases (HDAC1 and HDAC2) and presumed accessory proteins such as RbAp48, SAP30, and SAP18 it also associates with a surprisingly wide range of DNA binding transcription factors, including the nuclear hormone receptors (through the N-CoR and SMRT corepressors), MeCP2, Ski, p53, Ikaros and Aiolos, REST/NRSF, MNF-beta, and the Mad family of Max binding bHLH-Zip transcriptional repressors. The activities of these proteins and their ability to interact with Sin3 are thought to be crucial for cell proliferation and differentiation (Brubaker, 2000 and references therein).

The nature and possible regulation of the specific interaction between transcription factors and Sin3 is of great interest. For nuclear hormone receptors, the interaction with N-CoR/SMRT is hormone regulated, while for 'dedicated' repressors such as the Mad protein family, the association appears to be constitutive. In the case of the Mad proteins, all four family members (Mad1, Mxi1, Mad3, and Mad4) and the related repressor, Mnt (or Rox) contain an ~30-residue, N-terminally located segment known as the Sin3 interaction domain, or SID, which is both necessary and sufficient for Sin3 association and for transcriptional repression. Deletion or specific mutation of the SID abrogates Mad repression as well as its growth inhibitory functions. Furthermore, the Mad SID is capable of conferring repression activity when fused to a heterologous DNA binding domain. Helical wheel analysis and circular dichroism (CD) studies of the Mad SID suggest that it has the potential to form an amphipathic alpha helix. Mutational analyses further demonstrate that a cluster of residues on the apolar face of the helix is essential for interaction with mammalian Sin3A (mSin3A) (Brubaker, 2000 and references therein).

Sin3 interacts with many proteins in the complex through four imperfect repeats of ~100 residues known as paired amphipathic helix (PAH) domains. The PAH domains, which were each suggested to be organized into two alpha helices separated by a flexible spacer region, are among the most evolutionarily conserved regions of the large Sin3 proteins (100-170 kDa). Indeed, these domains are important for Sin3 function as a corepressor, most likely through their independent associations with various repressors and other associated proteins. For example, PAH2 is both necessary and sufficient for interaction with the Mad proteins as well as with a newly discovered Sin3-interacting protein, Pf1. However, PAH1 associates with N-CoR and PLZF, while PAH3 binds the SAP30 protein (Brubaker, 2000 and references therein).

While previous work on repressor-Sin3 corepressor interactions has localized functionally important regions and provides hints regarding their structure, details of these important interactions have remained largely unknown. In this study, a high-resolution structure is described for the Mad1 SID bound to the PAH2 domain of mSin3A determined by NMR (nuclear magnetic resonance) methods. Mutational studies of mSin3A are presented that confirm many of the specific interactions predicted from the NMR structure. Finally, it is shown that an unrelated Sin3-interacting protein, Pf1, with an interaction domain distinct from the Mad family SID, is likely to interact with PAH2 in a manner closely resembling Mad1 SID (Brubaker, 2000).

MYC associates with TIP60 complex

The c-Myc transactivation domain was used to affinity purify tightly associated nuclear proteins. Two of these proteins were identified as TIP49 and a novel related protein called TIP48, both of which are highly conserved in evolution and contain ATPase/helicase motifs. TIP49 and TIP48 are complexed with c-Myc in vivo, and binding is dependent on a c-Myc domain essential for oncogenic activity. A missense mutation in the TIP49 ATPase motif acts as a dominant inhibitor of c-Myc oncogenic activity but does not inhibit normal cell growth, indicating that functional TIP49 protein is an essential mediator of c-Myc oncogenic transformation. The TIP49 and TIP48 ATPase/helicase proteins represent a novel class of cofactors recruited by transcriptional activation domains that function in diverse pathways (Wood, 2000).

The c-Myc oncoprotein functions as a transcription factor that can transform normal cells into tumor cells, as well as playing a direct role in normal cell proliferation. The c-Myc protein transactivates cellular promoters by recruiting nuclear cofactors to chromosomal sites through an N-terminal transactivation domain. Four different c-Myc cofactors: TRRAP, hGCN5, TIP49, and TIP48 have been identified and functionally characterized. This study presents the identification and characterization of the actin-related protein BAF53 as a c-Myc-interacting nuclear cofactor that forms distinct nuclear complexes. In addition to the human SWI/SNF-related BAF complex, BAF53 forms a complex with TIP49 and TIP48 and a separate biochemically distinct complex containing TRRAP and a histone acetyltransferase which does not contain TIP60. Using deletion mutants of BAF53, it is shown that BAF53 is critical for c-Myc oncogenic activity. These results indicate that BAF53 plays a functional role in c-Myc-interacting nuclear complexes (Park, 2002).

The transcription factor MYC binds specific DNA sites in cellular chromatin and induces the acetylation of histones H3 and H4. However, the histone acetyltransferases (HATs) that are responsible for these modifications have not yet been identified. MYC associates with TRRAP, a subunit of distinct macromolecular complexes that contain the HATs GCN5/PCAF or TIP60. Although the association of MYC with GCN5 has been shown, its interaction with TIP60 has never been analysed. This study shows that MYC associates with TIP60 and recruits it to chromatin in vivo with four other components of the TIP60 complex: TRRAP, p400, TIP48 and TIP49. Overexpression of enzymatically inactive TIP60 delays the MYC-induced acetylation of histone H4, and also reduces the level of MYC binding to chromatin. Thus, the TIP60 HAT complex is recruited to MYC-target genes and, probably with other other HATs, contributes to histone acetylation in response to mitogenic signals (Frank, 2003).

Pontin (Tip49) and Reptin (Tip48; see Drosophila Reptin) are highly conserved components of multimeric protein complexes important for chromatin remodelling and transcription. They interact with many different proteins including TATA box binding protein (TBP), beta-catenin and c-Myc and thus, potentially modulate different pathways. As antagonistic regulators of Wnt-signalling, they control wing development in Drosophila and heart growth in zebrafish. This study shows that the Xenopus xPontin and xReptin in conjunction with c-Myc regulate cell proliferation in early development. Overexpression of xPontin or xReptin results in increased mitoses and bending of embryos, which is mimicked by c-Myc overexpression. Furthermore, the knockdown of either xPontin or xReptin resulted in embryonic lethality at late gastrula stage, which is abrogated by the injection of c-Myc-RNA. The N-termini of xPontin and xReptin, which mediate the mitogenic effect were mapped to contain c-Myc interaction domains. c-Myc protein promotes cell cycle progression either by transcriptional activation through the c-Myc/Max complex or by repression of cyclin dependent kinase inhibitors (p21, p15) through c-Myc/Miz-1 interaction. Importantly, xPontin and xReptin exert their mitogenic effect through the c-Myc/Miz-1 pathway as dominant negative Miz-1 and wild-type c-Myc but not a c-Myc mutant deficient in Miz-1 binding could rescue embryonic lethality. Finally, promoter reporter studies revealed that xPontin and xReptin but not the N-terminal deletion mutants enhance p21 repression by c-Myc. It is concluded that xPontin and xReptin are essential genes regulating cell proliferation in early Xenopus embryogenesis through interaction with c-Myc. A novel function of xPontin and xReptin is proposed as co-repressors in the c-Myc/Miz-1 pathway (Etard, 2005).

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