RESUMEN
The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.
Asunto(s)
Complejo Antígeno-Anticuerpo , Fragmentos Fab de Inmunoglobulinas , Humanos , Cristalización/métodos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/química , Complejo Antígeno-Anticuerpo/química , Antígenos/química , Cristalografía por Rayos X , Conformación ProteicaRESUMEN
The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.
Asunto(s)
Proteínas Arqueales/química , Methanocaldococcus/metabolismo , Complejos Multiproteicos/química , ARN de Transferencia/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Cristalografía por Rayos X , Methanocaldococcus/genética , Modelos Moleculares , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Conformación de Ácido Nucleico , Unión Proteica , Dominios Proteicos , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN de Transferencia de Lisina/química , ARN de Transferencia de Lisina/genética , ARN de Transferencia de Lisina/metabolismoRESUMEN
The human NDR family kinases control diverse aspects of cell growth, and are regulated through phosphorylation and association with scaffolds such as MOB1. Here, we report the crystal structure of the human NDR1 kinase domain in its non-phosphorylated state, revealing a fully resolved atypically long activation segment that blocks substrate binding and stabilizes a non-productive position of helix αC. Consistent with an auto-inhibitory function, mutations within the activation segment of NDR1 dramatically enhance in vitro kinase activity. Interestingly, NDR1 catalytic activity is further potentiated by MOB1 binding, suggesting that regulation through modulation of the activation segment and by MOB1 binding are mechanistically distinct. Lastly, deleting the auto-inhibitory activation segment of NDR1 causes a marked increase in the association with upstream Hippo pathway components and the Furry scaffold. These findings provide a point of departure for future efforts to explore the cellular functions and the mechanism of NDR1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Células Epiteliales/enzimología , Factor de Crecimiento de Hepatocito/química , Proteínas Asociadas a Microtúbulos/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular , Línea Celular Tumoral , Clonación Molecular , Cristalografía por Rayos X , Células Epiteliales/citología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Cinética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina-Treonina Quinasa 3 , Transducción de Señal , Especificidad por SustratoRESUMEN
RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,'5'-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon-regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small-angle X-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the nonhydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain, together with nucleotide binding, imposes a rigid intertwined dimer configuration that is essential for RNase catalytic and antiviral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold.
Asunto(s)
Nucleótidos de Adenina/química , Endorribonucleasas/química , Oligorribonucleótidos/química , Adenosina Difosfato/química , Adenilil Imidodifosfato/química , Animales , Repetición de Anquirina , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Virus de la Encefalomiocarditis , Endorribonucleasas/genética , Endorribonucleasas/fisiología , Células HeLa , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Picornaviridae , Estructura Terciaria de Proteína , Dispersión de Radiación , Relación Estructura-Actividad , Sus scrofaRESUMEN
RAF kinases have a prominent role in cancer. Their mode of activation is complex but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and, as a result, undesirably stimulate RAS/ERK pathway-mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe bioluminescence resonance energy transfer-based biosensors for the extended RAF family that enable the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization and for probing structural determinants of RAF dimerization in vivo. Our findings, which seem generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-raf/química , Técnicas Biosensibles , Cristalización , ADN Complementario/metabolismo , Dimerización , Transferencia de Energía , Células HEK293 , Humanos , Luminiscencia , Mutación , Neoplasias/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-raf/metabolismo , Factores de Tiempo , UltracentrifugaciónRESUMEN
The universally conserved Kae1/Qri7/YgjD and Sua5/YrdC protein families have been implicated in growth, telomere homeostasis, transcription and the N6-threonylcarbamoylation (t(6)A) of tRNA, an essential modification required for translational fidelity by the ribosome. In bacteria, YgjD orthologues operate in concert with the bacterial-specific proteins YeaZ and YjeE, whereas in archaeal and eukaryotic systems, Kae1 operates as part of a larger macromolecular assembly called KEOPS with Bud32, Cgi121, Gon7 and Pcc1 subunits. Qri7 orthologues function in the mitochondria and may represent the most primitive member of the Kae1/Qri7/YgjD protein family. In accordance with previous findings, we confirm that Qri7 complements Kae1 function and uncover that Qri7 complements the function of all KEOPS subunits in growth, t(6)A biosynthesis and, to a partial degree, telomere maintenance. These observations suggest that Kae1 provides a core essential function that other subunits within KEOPS have evolved to support. Consistent with this inference, Qri7 alone is sufficient for t(6)A biosynthesis with Sua5 in vitro. In addition, the 2.9 Å crystal structure of Qri7 reveals a simple homodimer arrangement that is supplanted by the heterodimerization of YgjD with YeaZ in bacteria and heterodimerization of Kae1 with Pcc1 in KEOPS. The partial complementation of telomere maintenance by Qri7 hints that KEOPS has evolved novel functions in higher organisms.
Asunto(s)
Adenosina/análogos & derivados , Proteínas de Unión al ADN/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Adenosina/biosíntesis , Adenosina/metabolismo , Dimerización , Metaloendopeptidasas/fisiología , Proteínas Mitocondriales/fisiología , Modelos Moleculares , Subunidades de Proteína/fisiología , ARN de Transferencia/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/fisiología , Homeostasis del TelómeroRESUMEN
Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas Ubiquitinadas/metabolismo , Sustitución de Aminoácidos , Línea Celular , Cristalografía por Rayos X , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Enzimas Desubicuitinizantes , Humanos , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Organismos Modificados Genéticamente , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitinación , Levaduras/genética , Levaduras/crecimiento & desarrolloRESUMEN
Kae1 is a universally conserved ATPase and part of the essential gene set in bacteria. In archaea and eukaryotes, Kae1 is embedded within the protein kinase-containing KEOPS complex. Mutation of KEOPS subunits in yeast leads to striking telomere and transcription defects, but the exact biochemical function of KEOPS is not known. As a first step to elucidating its function, we solved the atomic structure of archaea-derived KEOPS complexes involving Kae1, Bud32, Pcc1, and Cgi121 subunits. Our studies suggest that Kae1 is regulated at two levels by the primordial protein kinase Bud32, which is itself regulated by Cgi121. Moreover, Pcc1 appears to function as a dimerization module, perhaps suggesting that KEOPS may be a processive molecular machine. Lastly, as Bud32 lacks the conventional substrate-recognition infrastructure of eukaryotic protein kinases including an activation segment, Bud32 may provide a glimpse of the evolutionary history of the protein kinase family.
Asunto(s)
Proteínas Arqueales/química , Complejos Multiproteicos/química , Proteínas Quinasas/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Portadoras/química , Cristalografía por Rayos X , Escherichia coli/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Methanococcus/genética , Methanococcus/metabolismo , Modelos Moleculares , Complejos Multiproteicos/fisiología , Resonancia Magnética Nuclear Biomolecular , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Homología de Secuencia de Aminoácido , Telómero/metabolismo , Thermoplasma/genética , Thermoplasma/metabolismo , Transcripción GenéticaRESUMEN
In this issue of Molecular Cell, Ngo et al. (2008) describe the crystal structure of the SRPK1 protein kinase in complex with its substrate, the spliceosome factor ASF/SF2, providing an unprecedented view of multiple targeting mechanisms in action on a single substrate.
Asunto(s)
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Animales , Humanos , Ratones , Proteínas Nucleares/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Unión al ARN , Factores de Empalme Serina-Arginina , Especificidad por SustratoRESUMEN
Using an expression cloning approach, we identify CUL7, a member of the cullin family, as a functional inhibitor of Myc-induced apoptosis. Deregulated expression of the Myc oncogene drives cellular proliferation yet also sensitizes cells to undergo p53-dependent and p53-independent apoptosis. Here, we report that CUL7 exerts its antiapoptotic function through p53. CUL7 binds directly to p53, and small interfering RNA-mediated knockdown of CUL7 results in the elevation of p53 protein levels. This antiapoptotic role of CUL7 enables this novel oncogene to cooperate with Myc to drive transformation. Deregulated ectopic expression of c-Myc and CUL7 promotes Rat1a cell growth in soft agar, and knockdown of CUL7 significantly blocks human neuroblastoma SHEP cell growth in an anchorage-independent manner. Furthermore, using public microarray data sets, we show that CUL7 mRNA is significantly overexpressed in non-small cell lung carcinoma and is associated with poor patient prognosis. We provide experimental evidence to show CUL7 is a new oncogene that cooperates with Myc in transformation by blocking Myc-induced apoptosis in a p53-dependent manner.
Asunto(s)
Apoptosis/genética , Proteínas Cullin/genética , Oncogenes , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Proteínas Cullin/metabolismo , Humanos , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Proteínas Proto-Oncogénicas c-myc/genética , ARN Interferente Pequeño/genética , Ratas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The ability of p53 to promote apoptosis and cell cycle arrest is believed to be important for its tumor suppression function. Besides activating the expression of cell cycle arrest and proapoptotic genes, p53 also represses a number of genes. Previous studies have shown an association between p53 activation and down-regulation of c-myc expression. However, the mechanism and physiological significance of p53-mediated c-myc repression remain unclear. Here, we show that c-myc is repressed in a p53-dependent manner in various mouse and human cell lines and mouse tissues. Furthermore, c-myc repression is not dependent on the expression of p21(WAF1). Abrogating the repression of c-myc by ectopic c-myc expression interferes with the ability of p53 to induce G(1) cell cycle arrest and differentiation but enhances the ability of p53 to promote apoptosis. We propose that p53-dependent cell cycle arrest is dependent not only on the transactivation of cell cycle arrest genes but also on the transrepression of c-myc. Chromatin immunoprecipitation assays indicate that p53 is bound to the c-myc promoter in vivo. We report that trichostatin A, an inhibitor of histone deacetylases, abrogates the ability of p53 to repress c-myc transcription. We also show that p53-mediated transcriptional repression of c-myc is accompanied by a decrease in the level of acetylated histone H4 at the c-myc promoter and by recruitment of the corepressor mSin3a. These data suggest that p53 represses c-myc transcription through a mechanism that involves histone deacetylation.
Asunto(s)
Fase G1 , Proteínas Proto-Oncogénicas c-myc/genética , Transcripción Genética/genética , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Regulación de la Expresión Génica , Histonas/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
The N terminus of the c-Myc oncoprotein interacts with Bin1, a ubiquitously expressed nucleocytoplasmic protein with features of a tumor suppressor. The c-Myc/Bin1 interaction is dependent on the highly conserved Myc Box 1 (MB1) sequence of c-Myc. The c-Myc/Bin1 interaction has potential regulatory significance as c-Myc-mediated transformation and apoptosis can be modulated by the expression of Bin1. Multiple splicing of the Bin1 transcript results in ubiquitous, tissue-specific and tumor-specific populations of Bin1 proteins in vivo. We report on the structural features of the interaction between c-Myc and Bin1, and describe two mechanisms by which the binding of different Bin1 isoforms to c-Myc may be regulated in cells. Our findings identify a consensus class II SH3-binding motif in c-Myc and the C-terminal SH3 domain of Bin1 as the primary structure determinants of their interaction. We present biochemical and structural evidence that tumor-specific isoforms of Bin1 are precluded from interaction with c-Myc through an intramolecular polyproline-SH3 domain interaction that inhibits the Bin1 SH3 domain from binding to c-Myc. Furthermore, c-Myc/Bin1 interaction can be inhibited by phosphorylation of c-Myc at Ser62, a functionally important residue found within the c-Myc SH3-binding motif. Our data provide a structure-based model of the c-Myc/Bin1 interaction and suggest a mode of regulation that may be important for c-Myc function as a regulator of gene transcription.
Asunto(s)
Empalme Alternativo , Proteínas Portadoras/metabolismo , Modelos Moleculares , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilación , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Dominios Homologos srcRESUMEN
The c-Myc transcription factor represses the mRNA expression of the platelet-derived growth factor receptor beta gene (PDGFRB). Using chromatin immunoprecipitation, we show that c-Myc binds to the proximal promoter of the PDGFRB gene in proliferating rat fibroblasts. Interestingly, mutant c-Myc proteins that are unable to repress PDGFRB gene expression, c-Myc(dBR) and c-Myc(d106-143), are still able to bind to the promoter in vivo. Hence, promoter-binding and repression of PDGFRB by c-Myc are separable activities. We also show that Myc repression of PDGFRB is not dependent on previously described or known transactivator-binding regions, suggesting Myc may be recruited to the promoter by multiple or yet unidentified transcription factors. In the presence of intact promoter-binding by Myc, trichostatin A (TSA) can block Myc repression of PDGFRB in vivo, again demonstrating that promoter-binding and repression are separable. Taken together, we hypothesize that Myc repression of PDGFRB expression occurs by a multi-step mechanism in which repression is initiated after Myc is recruited to the promoter.
Asunto(s)
Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Represoras/metabolismo , Animales , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Ácidos Hidroxámicos/farmacología , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , ARN Polimerasa II/metabolismo , Ratas , Proteínas Represoras/antagonistas & inhibidores , Transactivadores/metabolismoRESUMEN
The c-Myc cellular oncogene has diverse activities, including transformation, proliferation, and apoptosis. These activities are dependent on the ability of c-Myc to regulate gene transcription. c-Myc downregulates the GADD45a and GADD153 (DDTI3) genes that are induced in response to genotoxic stresses and that encode protein products with antiproliferative activities. We show that c-Myc represses the expression of GADD45a and GADD153 in response to thapsigargin, a nongenotoxic stress, as well as other endoplasmic reticulum (ER) stress agents. c-Myc represses both the basal expression and the magnitude of ER stress induction of GADD gene transcription. This repression requires the minimal promoter region of GADD45a and GADD153 and is not dependent on the ER stress element or p53-binding sites in the regulatory regions of these genes. Further analysis by chromatin immunoprecipitation shows that c-Myc binds to the minimal promoter region of GADD45a and GADD153 in vivo. c-Myc-associated protein X (Max) is also bound to both GADD gene promoters, whereas c-Myc interacting zinc-finger protein 1 (Miz-1) is bound to the GADD153, but not GADD45a, promoter. RNA polymerase II (RNAPII) is recruited to the GADD gene promoters in the presence and absence of c-Myc, which suggests that c-Myc represses these genes through a post-RNAPII recruitment mechanism.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas de Ciclo Celular/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/fisiología , ARN Polimerasa II/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/genética , Retículo Endoplásmico/fisiología , Humanos , Factor de Transcripción CHOPRESUMEN
The c-myc proto-oncogene encodes a transcription factor, c-Myc, which is deregulated and/or overexpressed in many human cancers. Despite c-Myc's importance, the identity of Myc-regulated genes and the mechanism by which Myc regulates these genes remain unclear. By combining chromatin immunoprecipitation with CpG island arrays, we identified 177 human genomic loci that are bound by Myc in vivo. Analyzing a cohort of known and novel Myc target genes showed that Myc-associated protein X, Max, also bound to these regulatory regions. Indeed, Max is bound to these loci in the presence or absence of Myc. The Myc:Max interaction is essential for Myc-dependent transcriptional activation; however, we show that Max bound targets also include Myc-repressed genes. Moreover, we show that the interaction between Myc and Max is essential for gene repression to occur. Taken together, the identification and analysis of Myc bound target genes supports a model whereby Max plays an essential and universal role in the mechanism of Myc-dependent transcriptional regulation.
Asunto(s)
Islas de CpG/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Genes myc/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Genes Reguladores/genética , Células HL-60 , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proto-Oncogenes Mas , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Myc is a multifunctional nuclear phosphoprotein that can drive cell cycle progression, apoptosis and cellular transformation. Myc orchestrates these activities at the molecular level by functioning as a regulator of gene transcription to activate or repress specific target genes. Previous studies have shown that both the Myc N-terminal domain (NTD) and the C-terminal domain (CTD) are essential for Myc functions. The role of the CTD is relatively well understood as it encodes a basic helix-loop-helix leucine zipper motif important for DNA binding and protein-protein interactions. By contrast, the role of the NTD and the specific domains responsible for different Myc activities are not as well defined. To investigate the regions of the NTD necessary for Myc function and to determine whether these activities are overlapping or independent of one another, we have conducted a detailed structure-function analysis of the Myc NTD. We assessed the ability of a number of deletion and point mutants within the highly conserved regions of the Myc NTD to induce cell cycle progression, apoptosis and transformation as well as repress and activate expression of endogenous target genes. Our analyses highlight the complexity of the Myc NTD and extend previous studies. For example, we show most Myc mutants that were compromised as repressors of gene transcription retained the ability to activate gene transcription, reinforcing the concept that these activities can be uncoupled. Repression of two different target genes could be distinguished by specific mutants, further supporting the notion of at least two different Myc repression mechanisms. Mutants disabled at both inducing and repressing gene transcription could not maximally drive the biological activities of Myc, indicating these functions are tightly linked. Indeed, a close association of Myc repression and apoptosis was also observed.