CArG binding factor A (CBF-A) is involved in transcriptional regulation of the rat Ha-ras promoter

• Published in: Nucleic acids research 2000-10, Vol.28 (19), p.3762-3770
• Main Authors: Mikheev, A M, Mikheev, S A, Zhang, Y, Aebersold, R, Zarbl, H
• Format: Article
• Language: English
• Subjects: Animals; Antineoplastic Agents - pharmacology; Base Sequence; Blotting, Western; CarG box-binding factor A; Cell Cycle Proteins; Chromatography, Affinity; Chromatography, High Pressure Liquid; DNA - genetics; DNA - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; ets protein; Gene Expression Regulation, Neoplastic - drug effects; Genes, ras - genetics; Genes, Reporter - genetics; Ha-ras gene; Heterogeneous-Nuclear Ribonucleoprotein Group A-B; Humans; Mass Spectrometry; Mimosine - pharmacology; Molecular Sequence Data; Molecular Weight; Mutation - genetics; Promoter Regions, Genetic - genetics; Protein Binding - drug effects; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-ets; Rats; Repressor Proteins - chemistry; Repressor Proteins - isolation & purification; Repressor Proteins - metabolism; Response Elements - genetics; Ribonucleoproteins; RNA, Messenger - genetics; RNA, Messenger - metabolism; Thermodynamics; Transcription Factors - metabolism; Transfection; Tumor Cells, Cultured; Ultraviolet Rays
• ISSN: 1362-4962; 0305-1048; 1362-4962
• DOI: 10.1093/nar/28.19.3762

In the present study we identified a positive transcriptional element within the rat Ha-ras promoter previously known as Ha-ras response element (HRE) and identified a trans-acting factor that binds HRE sequences in rat mammary cells. To identify the binding protein we employed sequence specific DNA affinity chromatography. Amino acid sequence analysis of the affinity-purified proteins was performed by tandem mass spectroscopy. The results unexpectedly demonstrated that in rat mammary cells CArG box-binding factor A (CBF-A) is the major protein species that bind specifically to the rat and human HRE sequences with high affinity. The affinity of CBF-A binding to HRE was significantly higher than to the CArG box described as a recognition sequence for CBF-A protein. Transient transfection assays using reporter plasmids verified that mutations within the HRE that disrupt binding of CBF-A also reduced the activity of the rat Ha-ras promoter. Despite the fact that the HRE within the Ha-ras promoter resembles a binding site for Ets transcription factors, we did not detect the binding of Ets-related proteins to the rat HRE in BICR-M1Rk cells. We further demonstrated a correlation between the presence of HRE binding activity and induction of Ha-ras mRNA expression following serum stimulation in the mammary carcinoma cell line. Taken together, our results suggest that CBF-A may play an important role in transcriptional regulation of Ha-ras promoter activity during normal mammary cell growth and carcinogenesis.