25 years of the c-Myc oncogene.
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Animals↗
Biomedical subjects
Michael D Cole
Publications and source records attributed to Michael D Cole.
Mechanism of transcriptional activation by the Myc oncoproteins.
The Myc family proteins are potent oncogenes that can activate and repress a very large number of cellular target genes. The amino terminus of Myc contains a transactivation domain that can recruit a number of nuclear cofactors with diverse activities. Functional studies link transactivation to the ability of Myc to promote normal cell proliferation and for oncogenic transformation. The biochemical mechanism of Myc-mediated transactivation has revealed a wide range of effects on chromatin and basal transcription. This review summarizes recent advances in understanding the function of Myc as a transcriptional activator and the role of this activity in Myc biological activities.
Adaptor Proteins, Signal Transducing↗
A conserved Myc protein domain, MBIV, regulates DNA binding, apoptosis, transformation, and G2 arrest.
The myc family of oncogenes is well conserved throughout evolution. Here we present the characterization of a domain conserved in c-, N-, and L-Myc from fish to humans, N-Myc317-337, designated Myc box IV (MBIV). A deletion of this domain leads to a defect in Myc-induced apoptosis and in some transformation assays but not in cell proliferation. Unlike other Myc mutants, MycDeltaMBIV is not a simple loss-of-function mutant because it is hyperactive for G2 arrest in primary cells. Microarray analysis of genes regulated by N-MycDeltaMBIV reveals that it is weakened for transactivation and repression but not nearly as defective as N-MycDeltaMBII. Although the mutated region is not part of the previously defined DNA binding domain, we find that N-MycDeltaMBIV has a significantly lower affinity for DNA than the wild-type protein in vitro. Furthermore, chromatin immunoprecipitation shows reduced binding of N-MycDeltaMBIV to some target genes in vivo, which correlates with the defect in transactivation. Thus, this conserved domain has an unexpected role in Myc DNA binding activity. These data also provide a novel separation of Myc functions linked to the modulation of DNA binding activity.
Amino Acid Sequence↗
Polycomb mediates Myc autorepression and its transcriptional control of many loci in Drosophila.
Aberrant accumulation of the Myc oncoprotein propels proliferation and induces carcinogenesis. In normal cells, however, an abundance of Myc protein represses transcription at the c-myc locus. Cancer cells often lose this autorepression. We examined the control of myc in Drosophila and show here that the Drosophila ortholog, dmyc, also undergoes autorepression. We find that the developmental repressor Polycomb (Pc) is required for dmyc autorepression, and that this Pc-dMyc-mediated repression spreads across an 875-kb region encompassing the dmyc gene. To further investigate the relationship between Myc and Polycomb, we used microarrays to identify genes regulated by each, and identify a striking relationship between the two: A large set of dMyc activation targets is normally repressed by Pc, and 73% of dMyc repression targets require Pc for this repression. Chromatin immunoprecipitation confirmed that many dMyc-Pc-repressed loci have an epigenetic mark recognized by Pc. Our results suggest a novel relationship between Myc and Polycomb, wherein Myc enhances Polycomb repression in order to repress targets, and Myc suppresses Polycomb repression in order to activate targets.
Animals↗
Identification of RecQL1 as a Holliday junction processing enzyme in human cell lines.
Homologous recombination provides an effective way to repair DNA double-strand breaks (DSBs) and is required for genetic recombination. During the process of homologous recombination, a heteroduplex DNA structure, or a 'Holliday junction' (HJ), is formed. The movement, or branch migration, of this junction is necessary for recombination to proceed correctly. In prokaryotes, the RecQ protein or the RuvA/RuvB protein complex can promote ATP-dependent branch migration of Holliday junctions. Much less is known about the processing of Holliday junctions in eukaryotes. Here, we identify RecQL1 as a predominant ATP-dependent, HJ branch migrator present in human nuclear extracts. A reduction in the level of RecQL1 induced by RNA interference in HeLa cells leads to an increase in sister chromatid exchange. We propose that RecQL1 is involved in the processing of Holliday junctions in human cells.
Adenosine Triphosphatases↗
Development of a harmonized method for the profiling of amphetamines. II. Stability of impurities in organic solvents.
The present study focused on the stability of 22 amphetamine impurities dissolved in six organic solvents: isooctane, toluene, ethanol, dichloromethane, ethyl acetate, and diethyl ether. The aim was to find the most inert, and thereby most suitable, solvent for amphetamine profiling. Mixtures of the impurities were prepared in the different solvents, and changes in the concentrations of the individual compounds over-time were monitored by gas chromatographic analysis after 0, 4, 12, 24, 48, and 96 h. Isooctane and toluene provided the most inert conditions, although, a few of the impurities were insufficiently stable in these two solvents. The present experiments were performed as a part of the development of a harmonized method for profiling of amphetamine. The results can be used to support the choice of organic solvents for sample preparation. They also provide information about the stability of the impurities that are found in profiles of illicit amphetamine. This is essential due to the fact, that unstable compounds can have a negative influence on the comparison of profiles.
Journal Article↗
Development of a harmonized method for the profiling of amphetamines. I. Synthesis of standards and compilation of analytical data.
Reference material was synthesised for 21 substances that are frequently present as synthetic impurities, i.e. by-products, in illicitly produced amphetamine. Each of these substances is a typical by-product for at least one of the three approaches most often used to synthesise amphetamine, namely, the Leuckart, the reductive amination of benzyl methyl ketone, and the nitrostyrene routes. A large body of data on the substances was recorded, including the following: mass spectra, ultraviolet spectra, Fourier transform infrared spectra, infrared spectra in gas phase, and 1H NMR and 13C NMR spectra.
Journal Article↗
A human T-cell lymphotropic virus type 1 enhancer of Myc transforming potential stabilizes Myc-TIP60 transcriptional interactions.
The human T-cell lymphotropic virus type 1 (HTLV-1) infects and transforms CD4+ lymphocytes and causes adult T-cell leukemia/lymphoma (ATLL), an aggressive lymphoproliferative disease that is often fatal. Here, we demonstrate that the HTLV-1 pX splice-variant p30II markedly enhances the transforming potential of Myc and transcriptionally activates the human cyclin D2 promoter, dependent upon its conserved Myc-responsive E-box enhancer elements, which are associated with increased S-phase entry and multinucleation. Enhancement of c-Myc transforming activity by HTLV-1 p30II is dependent upon the transcriptional coactivators, transforming transcriptional activator protein/p434 and TIP60, and it requires TIP60 histone acetyltransferase (HAT) activity and correlates with the stabilization of HTLV-1 p30II/Myc-TIP60 chromatin-remodeling complexes. The p30II oncoprotein colocalizes and coimmunoprecipitates with Myc-TIP60 complexes in cultured HTLV-1-infected ATLL patient lymphocytes. Amino acid residues 99 to 154 within HTLV-1 p30II interact with the TIP60 HAT, and p30II transcriptionally activates numerous cellular genes in a TIP60-dependent or TIP60-independent manner, as determined by microarray gene expression analyses. Importantly, these results suggest that p30II functions as a novel retroviral modulator of Myc-TIP60-transforming interactions that may contribute to adult T-cell leukemogenesis.
Acetyltransferases↗
The Mad and Myc basic domains are functionally equivalent.
The Myc/Max/Mad family of transcription factors plays a fundamental role in the regulation of cell proliferation, oncogenic transformation, and cell differentiation. However, it remains unclear whether different heterodimers, such as Myc/Max and Mad/Max, recognize the same or different target genes in vivo. We show by chromatin immunoprecipitation that Myc target genes are also recognized by Mad1 in differentiated HL60 cells. We also substituted the complete basic region of Myc for the corresponding region of Mad. Wild-type c-Myc was then compared with c-Myc(Mad-BR) in oncogenic transformation, regulation of cell proliferation, induction of apoptosis, activation of chromosomal gene expression, and direct binding to chromosomal sites by chromatin immunoprecipitation. We find that the wild-type c-Myc and c-Myc/MadBR proteins have indistinguishable biological activity and target gene recognition in vivo. These data are consistent with a model in which Myc and Mad regulate a common set of target genes.
Amino Acid Sequence↗
Nmyc upregulation by sonic hedgehog signaling promotes proliferation in developing cerebellar granule neuron precursors.
Hedgehog pathway activation is required for expansion of specific neuronal precursor populations during development and is etiologic in the human cerebellar tumor, medulloblastoma. We report that sonic hedgehog (Shh) signaling upregulates expression of the proto-oncogene Nmyc in cultured cerebellar granule neuron precursors (CGNPs) in the absence of new protein synthesis. The temporal-spatial expression pattern of Nmyc, but not other Myc family members, precisely coincides with regions of hedgehog proliferative activity in the developing cerebellum and is observed in medulloblastomas of Patched (Ptch) heterozygous mice. Overexpression of Nmyc promotes cell-autonomous G(1) cyclin upregulation and CGNP proliferation independent of Shh signaling. Furthermore, Myc antagonism in vitro significantly decreases proliferative effects of Shh in cultured CGNPs. Together, these findings identify Nmyc as a direct target of the Shh pathway that functions to regulate cell cycle progression in cerebellar granule neuron precursors.
Animals↗
TIP49, but not TRRAP, modulates c-Myc and E2F1 dependent apoptosis.
We previously described two nuclear cofactors, TRRAP and TIP49, that have functional roles in Myc-mediated oncogenesis. We have now expanded the analysis of these Myc-associated cofactors to investigate their roles in apoptosis and cell proliferation. Although TRRAP and TIP49 are both essential for transformation, TIP49 modulates c-Myc-mediated apoptosis whereas disruption of TRRAP activity has no apparent effect on apoptosis. We extended our analysis of TIP49 to show that it also binds to the E2F1 transactivation domain and modulates both transforming and apoptotic activities. These results indicate that individual cofactors differentially potentiate c-Myc and E2F1 functions.
ATPases Associated with Diverse Cellular Activitie↗
The proto-oncogene c-myc acts through the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) to facilitate the activation of Cdk4/6 and early G(1) phase progression.
Progression through the early G(1) phase of the cell cycle requires mitogenic stimulation, which ultimately leads to the activation of cyclin-dependent kinases 4 and 6 (Cdk4/6). Cdk4/6 activity is promoted by D-type cyclins and opposed by Cdk inhibitor proteins. Loss of c-myc proto-oncogene function results in a defect in the activation of Cdk4/6. c-myc(-/-) cells express elevated levels of the Cdk inhibitor p27(Kip1) and reduced levels of Cdk7, the catalytic subunit of Cdk-activating kinase. We show here that in normal (c-myc(+/+)) cells, the majority of cyclin D-Cdk4/6 complexes are assembled with p27 and remain inactive during cell cycle progression; their function is presumably to sequester p27 from Cdk2 complexes. A small fraction of Cdk4/6 protein was found in lower molecular mass catalytically active complexes. Conditional overexpression of p27 in c-myc(+/+) cells caused inhibition of Cdk4/6 activity and elicited defects in G(0)-to-S phase progression very similar to those seen in c-myc(-/-) cells. Overexpression of cyclin D1 in c-myc(-/-) cells rescued the defect in Cdk4/6 activity, indicating that the limiting factor is the number of cyclin D-Cdk4/6 complexes. Cdk-activating kinase did not rescue Cdk4/6 activity. We propose that the defect in Cdk4/6 activity in c-myc(-/-) cells is caused by the elevated levels of p27, which convert the low abundance activable cyclin D-Cdk4/6 complexes into unactivable complexes containing higher stoichiometries of p27. These observations establish p27 as a physiologically relevant regulator of cyclin D-Cdk4/6 activity as well as mechanistically a target of c-Myc action and provide a model by which c-Myc influences the early-to-mid G(1) phase transition.
Animals↗
Human ING1 proteins differentially regulate histone acetylation.
ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33(ING1b), but not of p47(ING1a), induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47(ING1a) inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33(ING1b) affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT.ING1.PCNA protein complexes.
Acetylation↗
Adenovirus E1A requires the yeast SAGA histone acetyltransferase complex and associates with SAGA components Gcn5 and Tra1.
The budding yeast Saccharomyces cerevisiae was used as a model system to study the function of the adenovirus E1A oncoprotein. Previously we demonstrated that expression of the N-terminal 82 amino acids of E1A in yeast causes pronounced growth inhibition and specifically interferes with SWI/SNF-dependent transcriptional activation. Further genetic analysis identified the yeast transcription factor Adr1 as a high copy suppressor of E1A function. Transcriptional activation by Adr1 requires interaction with co-activator proteins Ada2 and Gcn5, components of histone acetyltransferase complexes including ADA and SAGA. Analysis of mutant alleles revealed that several components of the SAGA complex, including proteins from the Ada, Spt, and Taf classes were required for E1A-induced growth inhibition. Growth inhibition also depended on the Gcn5 histone acetyltransferase, and point mutations within the Gcn5 HAT domain rendered cells E1A-resistant. Also required was SAGA component Tra1, a homologue of the mammalian TRRAP protein which is required for c-myc and E1A induced cellular transformation. Additionally, Gcn5 protein could associate with E1A in vitro in a manner that depended on the N-terminal domain of E1A, and Tra1 protein was co-immunoprecipitated with E1A in vivo. These results indicate a strong requirement for intact SAGA complex for E1A to function in yeast, and suggest a role for SAGA-like complexes in mammalian cell transformation.
Acetyltransferases↗
Nuclear receptor function requires a TFTC-type histone acetyl transferase complex.
Nuclear receptors (NRs) regulate transcription in a ligand-dependent way through two types of coactivator complexes: the p160/CBP histone acetyl transferase (HAT) complex and the DRIP/TRAP/SMCC complex without HAT activity. Here we identified a large human (h) coactivator complex necessary for the estrogen receptor alpha (ERalpha) transactivation. This complex contains the GCN5 HAT, the c-Myc interacting protein TRRAP/PAF400, TAF(II)30, and other subunits. Similarly to known TFTC (TBP-free TAF(II)-containing)-type HAT complexes (hTFTC, hPCAF, and hSTAGA), TRRP directly interacted with liganded ER alpha, or other NRs. ER alpha transactivation was enhanced by the purified complex in vitro. Antisense TRRAP RNA inhibited estrogen-dependent cell growth of breast cancer cells. Thus, the isolated TFTC-type HAT complex acts as a third class of coactivator complex for NR function.
Acetyltransferases↗
TRRAP-dependent and TRRAP-independent transcriptional activation by Myc family oncoproteins.
We demonstrate that transformation-transactivation domain-associated protein (TRRAP) binding and the recruitment of histone H3 and H4 acetyltransferase activities are required for the transactivation of a silent telomerase reverse transcriptase (TERT) gene in exponentially growing human fibroblasts by c-Myc or N-Myc protein. However, recruitment of TRRAP by c- or N-Myc is dispensable for the partial induction of several basally expressed genes in exponentially growing primary and immortalized fibroblasts. Furthermore, recruitment of TRRAP is required for c-Myc- or N-Myc-mediated oncogenic transformation but not for the partial restoration of the growth defect in myc-null fibroblasts. A segment of the adenovirus E1A protein fused to a transformation-defective N-Myc protein carrying a small deletion in the transactivation domain specifically restores interaction with TRRAP, activates the silent TERT gene, induces acetylation of histones H3 and H4 at the TERT promoter, and transforms primary cells. Accordingly, wild-type L-Myc is much less efficient in TRRAP binding, activation of the silent TERT gene, and transformation of primary fibroblasts. Nevertheless, L-Myc is a potent activator of several basally expressed genes and can fully restore the growth defect of myc-null cells. These results suggest a differential requirement for TRRAP for several Myc-mediated activities.
Acetyltransferases↗
A functional screen for Myc-responsive genes reveals serine hydroxymethyltransferase, a major source of the one-carbon unit for cell metabolism.
A cDNA library enriched with Myc-responsive cDNAs but depleted of myc cDNAs was used in a functional screen for growth enhancement in c-myc-null cells. A cDNA clone for mitochondrial serine hydroxymethyltransferase (mSHMT) that was capable of partial complementation of the growth defects of c-myc-null cells was identified. Expression analysis and chromatin immunoprecipitation demonstrated that mSHMT is a direct Myc target gene. Furthermore, a separate gene encoding the cytoplasmic isoform of the same enzyme is also a direct target of Myc regulation. SHMT enzymes are the major source of the one-carbon unit required for folate metabolism and for the biosynthesis of nucleotides and amino acids. Our data establish a novel functional link between Myc and the regulation of cellular metabolism.
Animals↗
BAF53 forms distinct nuclear complexes and functions as a critical c-Myc-interacting nuclear cofactor for oncogenic transformation.
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. We have previously reported the identification and functional characterization of four different c-Myc cofactors: TRRAP, hGCN5, TIP49, and TIP48. Here we present 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, we show that BAF53 is critical for c-Myc oncogenic activity. Our results indicate that BAF53 plays a functional role in c-Myc-interacting nuclear complexes.
ATPases Associated with Diverse Cellular Activitie↗