Clinical studies with the Abl tyrosine kinase inhibitor STI-571 in chronic myeloid leukemia demonstrate that many patients with advanced stage disease respond initially but then relapse. Through biochemical and molecular analysis of clinical material, we find that drug resistance is associated with the reactivation of BCR-ABL signal transduction in all cases examined. In six of nine patients, resistance was associated with a single amino acid substitution in a threonine residue of the Abl kinase domain known to form a critical hydrogen bond with the drug. This substitution of threonine with isoleucine was sufficient to confer STI-571 resistance in a reconstitution experiment. In three patients, resistance was associated with progressive BCR-ABL gene amplification. These studies provide evidence that genetically complex cancers retain dependence on an initial oncogenic event and suggest a strategy for identifying inhibitors of STI-571 resistance.
One of the important regulatory concepts to emerge from studies of eukaryotic gene expression is that RNA polymerase II promoters and their upstream activators are composed of functional modules whose synergistic action regulates the transcriptional activity of a nearby gene. Biochemical analysis of synergy by ZEBRA, a non-acidic activator of the Epstein-Barr virus (EBV) lytic cycle, showed that the synergistic transcriptional effect of promoter sites and activation modules correlates with assembly of the TFIID:TFIIA (DA) complex in DNase I footprinting and gel shift assays. The activator-dependent DA complex differs from a basal DA complex by its ability to bind TFIIB stably in an interaction regulated by TATA-binding protein-associated factors (TAFs). TFIIB enhances the degree of synergism by increasing complex stability. Similar findings were made with the acidic activator GAL4-VP16. Our data suggest a unifying mechanism for gene activation and synergy by acidic and non-acidic activators, and indicate that synergy is manifested at the earliest stage of preinitiation complex assembly.
Cell transformation by nuclear oncogenes such as c-myc presumably involves the transcriptional activation of a set of target genes that participate in the control of cell division. The function of a small evolutionarily conserved domain of the c-myc gene encompassing amino acids 129 to 145 was analyzed to explore the relationship between cell transformation and transcriptional activation. Deletion of this domain inactivated the c-myc oncogene for cell transformation while retaining the ability to activate transcription of either myc consensus binding sites or a GAL4-dependent promoter when the c-myc N-terminus was fused to the GAL4 DNA-binding domain. Point mutations that altered a conserved tryptophan (amino acid 136) within this domain had similar effects. Expression of the wt c-Myc N terminus (amino acids 1 to 262) as a GAL4 fusion was a dominant inhibitor of cell transformation by the c-myc oncogene, and this same domain also inhibited transformation by the adenovirus E1A gene. Surprisingly, deletion of amino acids 129 to 145 eliminated the dominant negative activity of GAL4-Myc on both c-myc and E1A transformation. Expression of the GAL4-Myc protein in Cos cells led to the formation of a specific complex between the Myc N terminus and a nuclear factor, and this complex was absent with the dl129-145 mutant. These results suggest that an essential domain of the c-Myc protein interacts with a specific nuclear factor that is also required for E1A transformation.
Assembly of enhanceosomes requires architectural proteins to facilitate the DNA conformational changes accompanying cooperative binding of activators to a regulatory sequence. The architectural protein HMG-1 has been proposed to bind DNA in a sequence-independent manner, yet, paradoxically, it facilitates specific DNA binding reactions in vitro. To investigate the mechanism of specificity we explored the effect of HMG-1 on binding of the Epstein-Barr virus activator ZEBRA to a natural responsive promoter in vitro. DNase I footprinting, mutagenesis, and electrophoretic mobility shift assay reveal that HMG-1 binds cooperatively with ZEBRA to a specific DNA sequence between two adjacent ZEBRA recognition sites. This binding requires a strict alignment between two adjacent ZEBRA sites and both HMG boxes of HMG-1. Our study provides the first demonstration of sequence-dependent binding by a nonspecific HMG-box protein. We hypothesize how a ubiquitous, nonspecific architectural protein can function in a specific context through the use of rudimentary sequence recognition coupled with cooperativity. The observation that an abundant architectural protein can bind DNA cooperatively and specifically has implications towards understanding HMG-1's role in mediating DNA transactions in a variety of enzymological systems.An emerging theme in eukaryotic gene expression is that promoter-and cell-specific transcription is achieved through regulated assembly of activators into nucleoprotein structures termed enhanceosomes (6,22,37). Enhanceosome assembly is mediated by cooperative protein-protein interactions dictated by the positioning of activator binding sites on a regulatory sequence and the concentration of relevant activators in a cell (6,43). Because interactions between activators generate energetically unfavorable DNA bends, architectural proteins that bend and twist the DNA are necessary to facilitate cooperative binding. An important issue in the field is how such flexure can be provided on a global level for the thousands of combinatorial activator arrays bound to genes in a eukaryotic nucleus (6,22,32,37,38,42).Both sequence-specific and nonspecific DNA architectural proteins have been identified. The largest family of eukaryotic architectural proteins contains the conserved HMG box, a 75-amino-acid sequence of known structure. Numerous examples exist of HMG-box proteins that bind DNA either specifically (e.g., LEF-1) or nonspecifically (e.g., HMG-1 and -2) (4). The function and mechanism of some sequence-specific architectural proteins have been established, while the nonspecific proteins have remained enigmatic. In this paper we examine how the abundant and relatively nonspecific HMG-1 and -2 proteins can function in a specific context.To provide a framework for the problem, consider the action of LEF-1 on the T-cell receptor alpha (TCR-␣) enhanceosome. LEF-1 or TCF-1 binds to an 8-bp sequence within the 75-bp TCR-␣ enhancer, bends the DNA, and stimulates cooperative binding of the flanking activators, PEBP2␣-Ets-1...
Two coordinate forms of transcriptional synergy mediate eukaryotic gene regulation: the greater-thanadditive transcriptional response to multiple promoter-bound activators, and the sigmoidal response to increasing activator concentration. The mechanism underlying the sigmoidal response has not been elucidated but is almost certainly founded on the cooperative binding of activators and the general machinery to DNA. Here we explore that mechanism by using highly purified transcription factor preparations and a strong Epstein-Barr virus promoter, BHLF-1, regulated by the virally encoded activator ZEBRA. We demonstrate that two layers of cooperative binding govern transcription complex assembly. First, the architectural proteins HMG-1 and -2 mediate cooperative formation of an enhanceosome containing ZEBRA and cellular Sp1. This enhanceosome then recruits transcription factor IIA (TFIIA) and TFIID to the promoter to form the DA complex. The DA complex, however, stimulates assembly of the enhanceosome itself such that the entire reaction can occur in a highly concerted manner. The data reveal the importance of reciprocal cooperative interactions among activators and the general machinery in eukaryotic gene regulation.
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