CArG binding factor A (CBF-A) is involved in transcriptional regulation of the rat Ha-ras promoter.
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.
Mikheev
A M
AM
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Mailstop C1-015, PO Box 19024, Seattle, WA 98104-2092, USA.
Mikheev
S A
SA
Zhang
Y
Y
Aebersold
R
R
Zarbl
H
H
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D011905:Genes, ras / Q000235:genetics*; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D006801:Humans; D013058:Mass Spectrometry; D008898:Mimosine / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012097:Repressor Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D012261:Ribonucleoproteins; D013816:Thermodynamics; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; C121363:Hnrnpab protein, rat; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger; D012097:Repressor Proteins; D012261:Ribonucleoproteins; D014157:Transcription Factors; D008898:Mimosine; D004247:DNA