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

Andrei M. Mikheev, Svetlana A. Mikheev, Yanni Zhang, Ruedi Aebersold, Helmut Zarb

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3762-3770
Number of pages9
JournalNucleic Acids Research
Volume28
Issue number19
DOIs
StatePublished - Oct 1 2000

ASJC Scopus subject areas

  • Genetics

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