Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6162-6166, July 1991 Genetics
Determination of the c-MYC DNA-binding site (oncogene/transcription factor TFEB/helixloop-helix motif)
THANOS D. HALAZONETIS AND AMANY N. KANDIL Department of Cancer Research, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486
Communicated by Philip Leder, March 29, 1991
ABSTRACT The carboxyl terminus of the protein encoded by the c-MYC protooncogene has similarity to the helix-oophelix family of DNA-binding proteins and recognizes a sixnucleotide-long DNA sequence. We have used in vitrotranslated c-MYC protein to further define its DNA-binding specificity. The hexanucleotide originally identified is necessary for DNA binding by c-MYC, but not sufficient; the c-MYC target site is a 12-nucleotide-long palindrome. This site is present within regulatory regions of genes that are expressed during cell growth. Point mutations within the helix-loop-helix motif of c-MYC abolish DNA-binding and transforming activities, indicating that c-MYC acts as a DNA-binding protein to transform cells. c-MYC may transform cells by activating transcription of genes required for cell division.
provide a protein translation initiation codon preceded by a consensus translation initiation sequence (15). The c-MYC coding sequence was cloned as two separate fragments. The BamHI-Cla I fragment was derived from plasmid pOTS-myc (16), whereas the Cla I-Xba I fragment was from M13mpllmycRsa. The latter is an M13mpll vector with a 980-base-pair (bp) genomic Rsa I fragment containing the third exon of c-MYC subcloned into the Sma I site of the polylinker. The BamHI site of the M13mpll polylinker was previously destroyed. Plasmid pGEMmyc3X encodes the carboxyl-terminal 178 amino acids of c-MYC. It was derived from pGEMmycB by substituting the BamHI-Cla I c-MYC fragment with adaptor oligonucleotides (GATCCTGCTCGAGGAAT, upper strand; CGATTCCTCGAGCAG, lower strand). Plasmid pGEMmycPp encodes the carboxyl-terminal 94 amino acids of c-MYC. This plasmid was derived from pGEMmycB by substituting the BamHI-PpuMI c-MYC fragment with adaptor oligonucleotides (GATCCTCTAGACAG, upper strand; GACCTGTCTAGAG, lower strand). Site-directed mutagenesis was performed as described (17) using M13mpllmycRsa as the template. The mutant c-MYC fragments were subcloned into either pGEMmyc3X or pSV7hummyc as Cla I-Bcl I fragments. Plasmid pGEMtfebPX encodes a fragment of the human transcription factor TFEB protein that contains the HLH motif and the adjacent leucine zipper. A Pst I-Xho I fragment of the TFEB gene was isolated from plasmid pUC1-4tfeb and cloned into pGEM4, which had the EcoRI-BamHII fragment of the polylinker replaced by the oligonucleotides that provide an initiation codon, as described above for pGEMmycB. Adaptor oligonucleotides were used to bridge the BamHI site of the vector to the Pst I site of the insert (15). The Xho I site of the TFEB insert was ligated to the Sal I site of the pGEM4 vector. DNA-Binding Assay. This assay was done as described (15), except that poly(dIdC) was substituted with 100 ng per reaction of the following single-stranded oligonucleotide: CAGGAAGCAGGTCATGTGGCAAGGCTATT. Rat Embryo Fibroblast Transformation Assay. This assay was done as described (18), except that the cells were transfected as described by Chen and Okayama (19).
Constitutive expression of the c-MYC oncogene leads to cellular transformation in vitro and the development of tumors in experimental animals (1, 2). Furthermore human tumors are often associated with mutations that activate this gene (3-6). Within the 90 carboxyl-terminal amino acids of c-MYC, the protein product of the c-MYC oncogene, two motifs can be recognized; the helix-loop-helix (HLH) motif, which is preceded by a region rich in basic amino acids, and the leucine zipper motif, which immediately follows the HLH motif (7, 8). Similar motifs are found in sequence-specific DNAbinding proteins, where they serve to mediate protein dimerization and subsequent DNA binding (7-11). The presence of these motifs in c-MYC suggests that c-MYC has sequence-specific DNA-binding activity. Indeed Blackwell et al. (12) expressed in Escherichia coli a fusion protein of glutathione S-transferase with the carboxyl terminus of c-MYC, and Prendergast and Ziff (13) in vitro translated an E12 HLH protein, which had 11 amino acids of the basic region substituted by the corresponding amino acids of c-MYC. Both the above fusion proteins recognize the DNA sequence CACGTG. To address whether the DNA-binding site of c-MYC extends beyond these six nucleotides, we incubated in vitro-translated c-MYC protein with a repertoire of DNA sequences and identified a 12-nucleotide-long sequence that is both necessary and sufficient for DNA binding by c-MYC. Furthermore, point mutations within the c-MYC DNA-binding domain abolish both DNA binding and transforming activities, indicating that c-MYC acts as a sequencespecific DNA-binding protein to transform cells.
RESULTS In Vitro Translation of c-MYC. Several DNA-binding proteins, including members of the HLH family, bind DNA with specificity when expressed in an in vitro translation system (7, 9, 20). The coding sequence of human c-MYC was therefore cloned into the pGEM4 vector (Promega) to generate in vitro-transcribed mRNA, which was then translated by using a rabbit reticulocyte lysate (Fig. LA). Truncated versions were also generated. One of these, Myc3X, contained the amino acid sequences encoded by the third exon of c-MYC, whereas a further truncated protein, MycPp,
MATERIALS AND METHODS Recombinant Plasmids. Classical recombinant technology was used (14). Plasmid pGEMmycB encodes a full-length human c-MYC protein (Fig. LA). It is based on the pGEM4 vector (Promega). First the EcoRI-BamHI fragment of the polylinker was replaced by synthetic oligonucleotides that The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: HLH, helix-loop-helix. 6162
Proc. Natl. Acad. Sci. USA 88 (1991)
Genetics: Halazonetis and Kandil
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Determination of the c-MYC DNA-binding site (oncogene/transcription factor TFEB/helixloop-helix motif)
THANOS D. HALAZONETIS AND AMANY N. KANDIL Department of Cancer Research, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486
Communicated by Philip Leder, March 29, 1991
ABSTRACT The carboxyl terminus of the protein encoded by the c-MYC protooncogene has similarity to the helix-oophelix family of DNA-binding proteins and recognizes a sixnucleotide-long DNA sequence. We have used in vitrotranslated c-MYC protein to further define its DNA-binding specificity. The hexanucleotide originally identified is necessary for DNA binding by c-MYC, but not sufficient; the c-MYC target site is a 12-nucleotide-long palindrome. This site is present within regulatory regions of genes that are expressed during cell growth. Point mutations within the helix-loop-helix motif of c-MYC abolish DNA-binding and transforming activities, indicating that c-MYC acts as a DNA-binding protein to transform cells. c-MYC may transform cells by activating transcription of genes required for cell division.
provide a protein translation initiation codon preceded by a consensus translation initiation sequence (15). The c-MYC coding sequence was cloned as two separate fragments. The BamHI-Cla I fragment was derived from plasmid pOTS-myc (16), whereas the Cla I-Xba I fragment was from M13mpllmycRsa. The latter is an M13mpll vector with a 980-base-pair (bp) genomic Rsa I fragment containing the third exon of c-MYC subcloned into the Sma I site of the polylinker. The BamHI site of the M13mpll polylinker was previously destroyed. Plasmid pGEMmyc3X encodes the carboxyl-terminal 178 amino acids of c-MYC. It was derived from pGEMmycB by substituting the BamHI-Cla I c-MYC fragment with adaptor oligonucleotides (GATCCTGCTCGAGGAAT, upper strand; CGATTCCTCGAGCAG, lower strand). Plasmid pGEMmycPp encodes the carboxyl-terminal 94 amino acids of c-MYC. This plasmid was derived from pGEMmycB by substituting the BamHI-PpuMI c-MYC fragment with adaptor oligonucleotides (GATCCTCTAGACAG, upper strand; GACCTGTCTAGAG, lower strand). Site-directed mutagenesis was performed as described (17) using M13mpllmycRsa as the template. The mutant c-MYC fragments were subcloned into either pGEMmyc3X or pSV7hummyc as Cla I-Bcl I fragments. Plasmid pGEMtfebPX encodes a fragment of the human transcription factor TFEB protein that contains the HLH motif and the adjacent leucine zipper. A Pst I-Xho I fragment of the TFEB gene was isolated from plasmid pUC1-4tfeb and cloned into pGEM4, which had the EcoRI-BamHII fragment of the polylinker replaced by the oligonucleotides that provide an initiation codon, as described above for pGEMmycB. Adaptor oligonucleotides were used to bridge the BamHI site of the vector to the Pst I site of the insert (15). The Xho I site of the TFEB insert was ligated to the Sal I site of the pGEM4 vector. DNA-Binding Assay. This assay was done as described (15), except that poly(dIdC) was substituted with 100 ng per reaction of the following single-stranded oligonucleotide: CAGGAAGCAGGTCATGTGGCAAGGCTATT. Rat Embryo Fibroblast Transformation Assay. This assay was done as described (18), except that the cells were transfected as described by Chen and Okayama (19).
Constitutive expression of the c-MYC oncogene leads to cellular transformation in vitro and the development of tumors in experimental animals (1, 2). Furthermore human tumors are often associated with mutations that activate this gene (3-6). Within the 90 carboxyl-terminal amino acids of c-MYC, the protein product of the c-MYC oncogene, two motifs can be recognized; the helix-loop-helix (HLH) motif, which is preceded by a region rich in basic amino acids, and the leucine zipper motif, which immediately follows the HLH motif (7, 8). Similar motifs are found in sequence-specific DNAbinding proteins, where they serve to mediate protein dimerization and subsequent DNA binding (7-11). The presence of these motifs in c-MYC suggests that c-MYC has sequence-specific DNA-binding activity. Indeed Blackwell et al. (12) expressed in Escherichia coli a fusion protein of glutathione S-transferase with the carboxyl terminus of c-MYC, and Prendergast and Ziff (13) in vitro translated an E12 HLH protein, which had 11 amino acids of the basic region substituted by the corresponding amino acids of c-MYC. Both the above fusion proteins recognize the DNA sequence CACGTG. To address whether the DNA-binding site of c-MYC extends beyond these six nucleotides, we incubated in vitro-translated c-MYC protein with a repertoire of DNA sequences and identified a 12-nucleotide-long sequence that is both necessary and sufficient for DNA binding by c-MYC. Furthermore, point mutations within the c-MYC DNA-binding domain abolish both DNA binding and transforming activities, indicating that c-MYC acts as a sequencespecific DNA-binding protein to transform cells.
RESULTS In Vitro Translation of c-MYC. Several DNA-binding proteins, including members of the HLH family, bind DNA with specificity when expressed in an in vitro translation system (7, 9, 20). The coding sequence of human c-MYC was therefore cloned into the pGEM4 vector (Promega) to generate in vitro-transcribed mRNA, which was then translated by using a rabbit reticulocyte lysate (Fig. LA). Truncated versions were also generated. One of these, Myc3X, contained the amino acid sequences encoded by the third exon of c-MYC, whereas a further truncated protein, MycPp,
MATERIALS AND METHODS Recombinant Plasmids. Classical recombinant technology was used (14). Plasmid pGEMmycB encodes a full-length human c-MYC protein (Fig. LA). It is based on the pGEM4 vector (Promega). First the EcoRI-BamHI fragment of the polylinker was replaced by synthetic oligonucleotides that The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: HLH, helix-loop-helix. 6162
Proc. Natl. Acad. Sci. USA 88 (1991)
Genetics: Halazonetis and Kandil
A
pGEMmycB
IL..
RB R 8
C
PD
Rs;Sm H
Bc
IpGEMmyc3X
I< R B
C
Pp
Bc
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pGEMmycPp
It. . Bc
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1
