The Role of Phosphorylation of DNA-Binding Proteins in Regulation of Transcription of the Human c-myc Gene

L. R. Imamova,¹ B. K. Chernov,¹ and A. V. Itkes^2,3,4

¹Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 117984 Russia; E-mail: itkes@genom-ii.eimb.rssi.ru

²School of Fundamental Medicine, Lomonosov Moscow State University, Moscow, 119899 Russia.

³School of Medicine, Russian University of People’s Friendship, ul. Miklukho-Maklaya 8, Moscow, 117198 Russia; E-mail: itkes@med.pfu.edu.ru

Submitted June 18, 1997; revision submitted July 7, 1997.

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The goal of the present study was to identify transcriptional factors, to determine their specificity toward nucleotide sequences of binding sites, and to elucidate their regulatory effects on transcription of the human c-myc oncogene. Three novel phosphorylated proteins (transcriptional factors) participating in the regulation of transcription of the c-myc gene have been identified, and the following data have been obtained. A) The binding of the regulatory phosphoproteins activates in vitro transcription of the human c-myc gene. B) The recognition sites for the three discovered DNA-binding proteins, i.e., p70, p35, and p23, have been mapped in the c-myc promoter. C) The site-specific mutagenesis of p70 binding sites of the c-myc gene has been performed and the recognition sequence has been identified. The mutagenesis was carried out using the PCR technique using oligonucleotide primers with mismatches in the p70 binding site. The wild-type sequence GGGAAAAGAAAAAA showed the highest affinity toward p70, while the mutant sequence GAAAAtGAcAct exhibited a lack of affinity for the protein. The participation of p70 and other phosphorylated transcriptional factors in regulation of the human c-myc gene expression is discussed.

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KEY WORDS: transcriptional factors, phosphorylation, c-myc gene.

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