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

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.

SUBMITTER: Mikheev AM

PROVIDER: S-EPMC110773 | biostudies-literature | 2000 Oct

REPOSITORIES: biostudies-literature

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

Mikheev A M AM Mikheev S A SA Zhang Y Y Aebersold R R Zarbl H H

Nucleic acids research 20001001 19

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 ...[more]

PMID: 11000268

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Project description:BackgroundRadiogenetic therapy is a novel approach in the treatment of cancer, which employs genetic modification to alter the sensitivity of tumor cells to the effect of applied radiation.AimTo select a potent radiation inducible promoter in the context of brain tumors and to investigate if CArG radio responsive motifs or other elements in the promoter nucleotide sequences can correlate to its response to radiation.MethodsTo select initial candidates for promoter inducible elements, the levels of mRNA expression of six different promoters were assessed using Quantitative RTPCR in D54 MG cells before and after radiation exposure. Recombinant Ad/reporter genes driven by five different promoters; CMV, VEGF, FLT-1, DR5 and survivin were constructed. Glioma cell lines were infected with different multiplicity of infection of the (promoter) Ad or CMV Ad. Cells were then exposed to a range of radiation (0-12 Gy) at single fraction. Fluorescent microscopy, Luc assay and X-gal staining was used to detect the level of expression of related genes. Different glioma cell lines and normal astrocytes were infected with Ad survivin and exposed to radiation. The promoters were analyzed for presence of CArG radio-responsive motifs and CCAAT box consensus using NCBI blast bioinformatics software.ResultsRadiotherapy increases the expression of gene expression by 1.25-2.5 fold in different promoters other than survivin after 2 h of radiation. RNA analysis was done and has shown an increase in copy number of tenfold for survivin. Most importantly cells treated with RT and Ad Luc driven by survivin promoter showed a fivefold increase in expression after 2 Gy of radiation in comparison to non-irradiated cells. Presence or absence of CArG motifs did not correlate with promoter response to radiation. Survivin with the best response to radiation had the lowest number of CCAAT box.ConclusionSurvivin is a selective potent radiation inducible promoter for glioblastoma viral gene therapy and this response to radiation could be independent of CArG motifs.

Dataset Information

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

Publications

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

Similar Datasets

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