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Nucleic Acids Research, 2000, Vol. 28, No. 19 3762-3770
© 2000 Oxford University Press

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

Andrei M. Mikheev, Svetlana A. Mikheev1, Yanni Zhang2, Ruedi Aebersold2 and Helmut Zarbl*

Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Mailstop C1-015, PO Box 19024, Seattle, WA 98104-2092, USA and 1Department of Microbiology and 2Department of Molecular Biotechnology, University of Washington, Seattle, WA, USA

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.

* To whom correspondence should be addressed. Tel: +1 206 667 4107; Fax: +1 206 667 5815; Email: hzarbl@fhcrc.org Present address: Yanni Zhang, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Mailstop C1-015, PO Box 19024, Seattle, WA 98109, USA


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