Cloning and Characterization of a Human c-myc Promoter-Binding Protein

Abstract

A human cDNA clone encoding a c-myc promoter-binding protein was detected by screening a HeLa cell λ. phage expression cDNA library. The library was screened by using an XhoI-NaeI human c-myc P₂ promoter fragment as a probe. The recombinant phage encoded a fusion protein, myc-binding protein 1 (MBP-1), which had an apparent molecular size of 40 kDa. A corresponding protein with a molecular size of 35 kDa was present in a HeLa cell extract. Sequence analysis of the cloned gene reveals an open reading frame of 1,038 bp with a 3′ untranslated region of 378 bp. The predicted protein sequence contains a proline-rich region in the amino terminus but does not demonstrate a known DNA-binding domain. DNase I footprint analysis demonstrates that MBP-1 binds to the sequence just 5′ of the TATA box sequence of the human c-myc P₂ promoter. MBP-1 cDNA hybridizes to a 1.4-kb mRNA from HeLa and HL-60 cells, indicating that the cDNA insert (1,416 bp) is a full-length clone. Coexpression of the MBP-1 protein represses transcription from the human c-myc promoter, suggesting that MBP-1 may act as a negative regulatory factor for the human c-myc gene.