Biomedical subjects
Publications and source records attributed to E M Blackwood.
In eukaryotes, transcription of genes by RNA polymerase II yields messenger RNA intermediates from which protein products are synthesized. Transcriptional enhancers are discrete DNA elements that contain specific sequence motifs with which DNA-binding proteins interact and transmit molecular signals to genes. Here, current models regarding the role of enhancers in the regulation of transcription by RNA polymerase II are presented.
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Activation of the c-myc proto-oncogene by chromosomal translocation or proviral insertion frequently results in the separation of the c-myc coding region from its normal regulatory elements. Such rearrangements are often accompanied by loss or mutation of c-myc exon 1 sequences. These genetic alterations do not affect synthesis of the major c-myc protein, p64, which is initiated from the first AUG codon in exon 2. However they can result in mutation or loss of the CUG codon located in exon 1 that normally serves as an alternative translational initiation codon for synthesis of an N-terminally extended form of c-Myc (p67). It has been hypothesized that p67 is a functionally distinct form of c-Myc whose specific loss during c-myc rearrangements confers a selective growth advantage. Here we describe experiments designed to test the functional properties of the two c-Myc protein forms. We introduced mutations within the translational initiation codons of a normal human c-myc cDNA that alter the pattern of Myc protein synthesis (p64 vs. p67). The functions of each of these proteins were experimentally addressed using co-transformation and transcriptional activation assays. Both the p64 and p67 c-Myc proteins were independently able to collaborate with bcr-abl in the transformation of Rat-1 fibroblasts. In addition, both the exon 1- and exon 2-initiated forms of the c-Myc protein stimulated transcription of a Myc/Max-responsive reporter construct to a similar level. Given the apparent absence of functional differences between p64 and p67, we conclude that the basis for c-Myc oncogenic activation lies primarily in the overall deregulation of its expression and not in alterations in the protein. The existence of the CUG translational initiator may reflect a mechanism for the continued synthesis of c-Myc protein under conditions where AUG initiation is inhibited.
The Max protein belongs to the basic region-helix-loop-helix-leucine zipper family of transcriptional regulators. Max heterodimerizes with Myc family proteins to form sequence-specific DNA-binding complexes. In order to elucidate the potential role of Myc and Max during amphibian embryogenesis, we have isolated and analyzed the expression of two Xenopus Max complementary DNAs: XMax1 and XMax2. Comparison of XMax1 and XMax2 with their mammalian counterparts demonstrates a strikingly high degree of conservation at both the nucleotide and amino acid levels, with the exception of a 24-residue deletion in both XMax proteins within their COOH termini. In addition, the two Xenopus Max proteins differ in that XMax2 contains a unique 27-amino acid insertion that interrupts the COOH-terminal end of the zipper domain; XMax1 lacks this insertion. Despite these differences, both XMax1 and XMax2 can form complexes with either Xenopus or human c-Myc proteins. Analysis of XMax expression during embryogenesis reveals that both mRNA and protein are expressed throughout early development, including the egg, 32-cell stage, and midblastula transition. Although the expression of XMax1 RNA appears to predominate at all stages examined, the ratios of XMax1 to XMax2 protein vary during development as well as between different tissue culture cell lines, suggesting a potential for cell type-specific regulation. Our results demonstrate the presence of Xenopus Max throughout frog development, raising the possibility that Myc and Max could function as a complex even during early embryogenesis.
The Myc family proteins are thought to be involved in transcription because they have both a carboxy-terminal basic-helix-loop-helix-zipper (bHLH-Z) domain, common to a large class of transcription factors, and an amino-terminal fragment which, for c-Myc, has transactivating function when assayed in chimaeric constructs. In addition, c-, N- and L-Myc proteins heterodimerize, in vitro and in vivo, with the bHLH-Z protein Max. In vitro, Max homodimerizes but preferentially associates with Myc, which homodimerizes poorly. Furthermore Myc-Max heterodimers specifically bind the nucleotide sequence CACGTG with higher affinity than either homodimer alone. The identification of Max and the specific DNA-binding activities of Myc and Max provides an opportunity for directly testing the transcriptional activities of these proteins in mammalian cells. We report here that Myc overexpression activates, whereas Max overexpression represses, transcription of a reporter gene. Max-induced repression is relieved by overexpression of c-Myc. Repression requires the DNA-binding domain of Max, whereas relief of repression requires the dimerization and transcriptional activation activities of Myc. Both effects require Myc-Max-binding sites in the reporter gene.
The myc family of oncogenes exhibit deregulated expression in a host of neoplasias. Though the molecular function of the Myc protein in both normal and tumorigenic cells has remained uncertain, it has been postulated to play a role in gene transcription on the basis of amino acid homologies with known transcription factors such as MyoD (Lüscher & Eisenman, 1990). We report here the direct testing of full-length Myc and its dimerization partner, Max, on the transcriptional activity of reporter genes bearing Myc/Max binding sites. Such reporter constructs display an endogenous level of activity in transient transfections which is dependent on the presence of the CACGTG sequence. Exogenous expression of myc results in modest activation of reporter gene transcription. Similar overexpression of max results in a repression of reporter gene activity, an effect which is reversed by co-expression with c-myc. Max repression is dependent on an intact DNA binding region, while Myc activation depends on both the N-terminal activation and the C-terminal dimerization domains. These results suggest a model in which Max homodimers can act as as repressors, and Myc-Max heterodimers as activators, of potential target genes.
The Myc family of oncoproteins are thought to regulate proliferation and differentiation in a wide variety of cell types. Recent studies show that Myc proteins form sequence-specific DNA-binding complexes with Max, a new member of the helix-loop-helix leucine zipper protein class. The properties of the Myc-Max complex suggest a mechanism for Myc's function in both normal and neoplastic cell behavior.
Max is a helix-loop-helix zipper protein that associates in vitro with Myc family proteins to form a sequence-specific DNA-binding complex. We show here, by means of a coimmunoprecipitation assay with anti-Myc and anti-Max antibodies, that Myc and Max are associated in vivo and essentially all of the newly synthesized Myc can be detected in a complex with Max. This complex possesses specific DNA-binding activity for CACGTG-containing oligonucleotides. Although Max itself is a highly stable protein, Myc is rapidly degraded during or after its association with Max. In vivo Max is shown to be a nuclear protein phosphorylated by casein kinase II, and alternatively spliced forms of Max are expressed in cells. Furthermore, the levels of Max expression are equivalent in quiescent, mitogen-stimulated, and cycling cells. We conclude that the highly regulated rate of Myc biosynthesis is likely to be a limiting step in the formation of Myc:Max complexes.
The myc family of proto-oncogenes encodes short-lived nuclear phosphoproteins (Myc) involved in the control of cell proliferation and differentiation. Here we discuss the evidence for Myc's involvement in normal and abnormal cell proliferation and review recent information on Max, a novel protein that forms a sequence-specific DNA-binding complex with Myc. The properties of the Myc: Max heterodimeric complex suggest a model for how Myc may function in the cell.
The protein encoded by the MAX gene is a member of the class of basic region-helix-loop-helix-zipper proteins and has been demonstrated to associate with N-, L-, and c-Myc proteins both in vitro and in vivo. Heterodimers formed between c-Myc and Max proteins have been shown to possess sequence-specific DNA-binding activity. Here we report the mapping of the MAX gene to a single region on human chromosome 14 (bands q22-q24) and to mouse chromosome 12 (region D). Chromosome abnormalities linked to several neoplasms have been previously associated with this region on human chromosome 14. The mapping results also confirm a region of homology between human chromosome 14q22-24 and mouse chromosome 12 region D.
The myc protooncogene family has been implicated in cell proliferation, differentiation, and neoplasia, but its mechanism of function at the molecular level is unknown. The carboxyl terminus of Myc family proteins contains a basic region helix-loop-helix leucine zipper motif (bHLH-Zip), which has DNA-binding activity and has been predicted to mediate protein-protein interactions. The bHLH-Zip region of c-Myc was used to screen a complementary DNA (cDNA) expression library, and a bHLH-Zip protein, termed Max, was identified. Max specifically associated with c-Myc, N-Myc, and L-Myc proteins, but not with a number of other bHLH, bZip, or bHLH-Zip proteins. The interaction between Max and c-Myc was dependent on the integrity of the c-Myc HLH-Zip domain, but not on the basic region or other sequences outside the domain. Furthermore, the Myc-Max complex bound to DNA in a sequence-specific manner under conditions where neither Max nor Myc exhibited appreciable binding. The DNA-binding activity of the complex was dependent on both the dimerization domain and the basic region of c-Myc. These results suggest that Myc family proteins undergo a restricted set of interactions in the cell and may belong to the more general class of eukaryotic DNA-binding transcription factors.
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While it has been known for some time that the c-Myc protein binds to random DNA sequences, no sequence-specific binding activity has been detected. At its carboxyl terminus, c-Myc contains a basic--helix-loop-helix (bHLH) motif, which is important for dimerization and specific DNA binding, as demonstrated for other bHLH protein family members. Of those studied, most bHLH proteins bind to sites that contain a CA- -TG consensus. In this study, the technique of selected and amplified binding-sequence (SAAB) imprinting was used to identify a DNA sequence that was recognized by c-Myc. A purified carboxyl-terminal fragment of human c-Myc that contained the bHLH domain bound in vitro in a sequence-specific manner to the sequence, CACGTG. These results suggest that some of the biological functions of Myc family proteins are accomplished by sequence-specific DNA binding that is mediated by the carboxyl-terminal region of the protein.